Environmental Engineering and Management Journal ...

Environmental Engineering and Management Journal

Aims and Scope Environmental Engineering and Management Journal is an international journal that publishes reviewed original research papers of both experimental and theoretical nature in the following areas:                    

environmental impact assessment; environmental integrated management; risk assessment and management; chemical and biological process engineering; environmental chemistry; environmental protection technologies (water, air, soil); sustainable industrial production pollution reduction at source and waste minimization; clean technologies; sensors in environment control; sources of radiation and protection technologies; waste valorization technologies; biotechnology in environmental protection; energy and environment; modelling, simulation and optimization for environmental protection; technologies for drinking and industrial water; life cycle assessment of products; environmental strategies and policies; cost-benefit analysis in environmental protection; education for sustainable development.

Environmental Engineering and Management Journal publishes:    

original communications describing important new discoveries or further developments in the above-mentioned topics; reviews, mainly of new rapidly developing areas of environmental protection; special, theme issues on relevant topics; advertising.

All manuscripts as well as all correspondence for the Environmental Engineering and Management Journal should be addressed to the Managing Editor, Department of Environmental Engineering and Management, 73 Prof.Dr.docent Dimitrie Mangeron Street, 700050 – IASI, Romania, e-mail: [email protected], [email protected]

March 2012 Vol.11 No. 3 Supplement

ISSN 1843-3707

Environmental Engineering and Management Journal An International Journal Editor-in-Chief:

Matei Macoveanu

Environmental Microbiology and Biotechnology in the frame of the Knowledge‐Based Bio and Green Economy Bologna, April 10‐12, 2012 Conference Abstracts

“Gheorghe Asachi” Technical University of Iasi

Environmental Engineering and Management Journal

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Environmental Engineering and Management Journal

EDITORIAL BOARD Editor-in-Chief: Matei Macoveanu Gheorghe Asachi Technical University of Iasi, Romania

Managing Editor: Maria Gavrilescu Gheorghe Asachi Technical University of Iasi, Romania

EDITORIAL ADVISORY BOARD Ahmet Aktaş Akdeniz University, Antalya Turkey

Eugenio Campos Ferreira University of Minho, Braga, Portugal

Abdelnaser Omran University Sains Malaysia, Penang Malaysia

Maria Madalena dos Santos Alves University of Minho Portugal

Cristian Fosalau Gheorghe Asachi Technical University of Iasi Romania

Alexandru Ozunu Babes-Bolyai University of Cluj-Napoca Romania

Ecaterina Andronescu Polytechnica University of Bucharest Romania

Anton Friedl Vienna University of Technology Austria

Yannis A. Phillis Technical University of Crete, Chania Greece

Robert Armon Technion-Israel Institute of Technology Haifa Israel

Anne Giroir Fendler University Claude Bernard Lyon 1 France

Marcel Ionel Popa Gheorghe Asachi Technical University of Iasi Romania

Adisa Azapagic The University of Manchester United Kingdom

Ion Giurma Gheorghe Asachi Technical University of Iasi Romania

Marcel Popa Gheorghe Asachi Technical University of Iasi Romania

Hamidi Abdul Aziz Universiti Sains Malaysia, Penang Malaysia

Aurelian Gulea State University of Moldavia, Kishinew Republic of Moldavia

Valentin I. Popa Gheorghe Asachi Technical University of Iasi Romania

Ion Balasanian Gheorghe Asachi Technical University of Iasi Romania

Yuh-Shan Ho Peking University People's Republic of China

Gabriel-Lucian Radu Polytechnica University of Bucharest Romania

Pranas Baltrenas Vilnius Gediminas Technical University Lithuania

Arjen Y. Hoekstra University of Twente, Enschede The Netherlands

Raj Rajagopalan National University of Singapore, The Republic of Singapore

Hans Bressers University of Twente, Enschede The Netherlands

Nicolae Hurduc Gheorghe Asachi Technical University of Iasi Romania

Ákos Rédey Pannon University, Veszprém Hungary

Han Brezet Delft University of Technology The Netherlands

Ralf Isenmann Fraunhofer Institute for Systems and Innovation Research (ISI), Karlsruhe, Germany

Joop Schoonman Delft University of Technology The Netherlands

Dan Cascaval Gheorghe Asachi Technical University of Iasi Romania

Marcel Istrate Gheorghe Asachi Technical University of Iasi Romania

Dan Scutaru Gheorghe Asachi Technical University of Iasi Romania

Alexandru Cecal Al.I. Cuza University of Iasi Romania

Ravi Jain University of Pacific, Baun Hall Stockton United States of America

Ilie Siminiceanu Gheorghe Asachi Technical University of Iasi Romania

Aleg Cherp Central European University Hungary

Michael Søgaard Jørgensen Technical University of Denmark, Lyngby Denmark

Bogdan C. Simionescu Gheorghe Asachi Technical University of Iasi Romania

Yusuf Chisti University of Massey New Zealand

Gheorghe Lazaroiu Polytechnica University of Bucharest Romania

Florian Statescu Gheorghe Asachi Technical University of Iasi Romania

Valeriu David Gheorghe Asachi Technical University of Iasi Romania

Thomas Lindhqvist International Institute for Industrial Environmental Economics, Lund University, Sweden

Vyacheslav N. Stepanov Academy of Sciences, Institute of Market and Economic-Ecological Research, Odessa, Ukraine

Gheorghe Duca State University of Moldavia, Kishinew Republic of Moldavia

Andreas Paul Loibner University of Natural Resources and Life Sciences, Vienna, Austria

Carmen Teodosiu Gheorghe Asachi Technical University of Iasi Romania

Ioan Dediu Academy of Sciences, Institute of Ecology and Geography, Kishinev, Republic of Moldavia

Tudor Lupascu Academy of Sciences, Institute of Chemistry, Kishinev, Republic of Moldavia

Saulius Vasarevicius Vilnius Gediminas Technical University Lithuania

Ioan Dumitrache Polytechnica University of Bucharest Romania

Antonio Marzocchella University of Naples Federico II, Naples, Italy

Angheluta Vadineanu The University of Bucharest Romania

Emil Dumitriu Gheorghe Asachi Technical University of Iasi Romania

Shin' ichi Nakatsuji University of Hyogo Japan

Colin Webb The University of Manchester United Kingdom

Jurek Duszczyk Delft University of Technology The Netherlands

Valentin Nedeff Vasile Alecsandri University of Bacau Romania

Scott Weber State University of New York at Buffalo United States of America

Anca Duta Capra Transilvania University of Brasov Romania

Vadim I. Nedostup Academy of Science, Physico-Chemical Institute, Odessa, Ukraine

Peter Wilderer Technical University Munich Germany

Fabio Fava Alma Mater Studiorum University of Bologna Italy

Phylip Nyden Loyola University, Chicago, IL United States of America

Petra Winzer Bergische University Wuppertal Germany

Environmental Engineering and Management Journal Environmental Engineering and Management Journal is included and indexed in CABI Chemical Abstracts Service/SciFinder (ACS) (since 2002) EBSCO Database (since 2002) EVISA ICAAP (International Consortium for Advancement of Academic Publications) Index Copernicus Journal Master List (ICV/2010=14.00) Journal Citation Reports® (IF=1.435) MedSci ProQuest (since 2002) The National University Research Council (RO) Science Citation Index Expanded™ (Thomson ISI) SJR (SCImago Journal&Country Rank) (H=10, SJR index/2011 = 0.038, SNIP index/2011 = 0.45) SCOPUS (since 2008) Thomson ISI Master Journal List Web of Science® (Thomson ISI) (H=12)

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Editor-in-Chief: Matei Macoveanu, Iasi (RO) Managing Editor: Maria Gavrilescu, Iasi (RO) “Gheorghe Asachi” Technical University of Iasi, Faculty of Chemical Engineering and Environmental Protection Department of Environmental Engineering and Management 73 Prof.Dr.docent Dimitrie Mangeron Street, 700050 Iasi, Romania Phone: +40-232-278680, ext. 2240 Fax: +40-232-271759 e-mail: eemjournal.at.yahoo.com, eem_journal.at.yahoo.com, eemjeditor.at.yahoo.com, eemj_editor.at.yahoo.com, eemjournal.at.gmail.com, eemjeditor.at.gmail.com Editorial production and secretariat: Camelia Smaranda Laura Carmen Apostol Raluca-Maria Hlihor Petronela Cozma Cristina Ghinea Madalina Petraru Isabela Simion Maria Fortuna Dana Luminiţa Sobariu Published 12 issues per year, under the aegis of the “Gheorghe Asachi” Technical University of Iasi, Romania by EcoZone Publishing House of the Academic Organization for Environmental Engineering and Sustainable Development (OAIMDD), http://www.ecozone.ro Annual subscription rate 2012 (12 issues) Print only: EUR 350 per volume EUR 40 per issue Order directly to the Editorial Office 73 Prof.Dr.docent Dimitrie Mangeron Street, 700050 Iasi, Romania Phone/Fax: Fax: +40-232-271759 e-mail: mmac.at.ch.tuiasi.ro eemjournal.at.yahoo.com Electronic, full text: Order or purchase on-line at: www.ecozone.ro Bank account (EURO): Romanian Bank for Development, Groupe Societé Generale, Bucharest, Romania SWIFT Code: BRDEROBU Beneficiary: Iasi, Romania, RO44BRDE240SV09790262400 All rights reserved, including those of translation into foreign languages. No part of each issue may be reproduced in any form (photoprint, microfilm, or any other means) nor transmitted or translated without written permission from the publishers. Only single copies of contributions, or parts thereof, may be made for personal use. This journal was carefully produced in all its parts. Even so, authors, editors and publisher do not guarantee the information contained there to be free of errors. Registered names, trademarks etc. used in this journal, even when not marked as such, are not be considered unprotected by law.

  Environmental Microbiology and Biotechnology  in the frame of the Knowledge‐Based  Bio and Green Economy   

  Bologna, April 10‐12, 2012    Faculty of Engineering, Alma Mater Studiorum ‐ University of Bologna 

Via Terracini, 28. I‐ 40131 Bologna, Italy 

Environmental Engineering and Management Journal

March 2012, Vol.11, No. 3, Supplement, S1-S170

http://omicron.ch.tuiasi.ro/EEMJ/

“Gheorghe Asachi” Technical University of Iasi, Romania

CONTENTS ____________________________________________________________________________________________

Environmental Microbiology and Biotechnology in the frame of the Knowledge-Based Bio and Green Economy Bologna, April 10‐12, 2012 Session 1 ADVANCES IN BIODEGRADATION OF ORGANICS AND INORGANICS, PHYTOREMEDIATION AND MYCOREMEDIATION Bacteria with an unusual appetite for the Fuel Oxygenate Methyl Tert-Butyl Ether (MTBE) Keynote lecture Max M. Haggblom…………………………………………………………………………………

S1

Sub-session 1.1. Bacterial biodegradation of organics and inorganics Oral presentations Biodegradation of sodium laureth sulfate by facultative anaerobic bacteria Ana Maria da Silva Paulo, Caroline M. Plugge, Pedro A. García-Encina, Alfons J.M.Stams ....................................................................................................................

S2

Acclimation of a complex microbial community to degrade a combination of organochlorine herbicides in a biofilm reactor I. Nava-Arena, O.A. Ramos-Monro, N. Ruiz-Ordaz, C.J. Galindez-Mayer, L. Rocha-Martínez, C. Juárez-Ramírez, H.M. Poggi-Varaldo ……………............................

S2

Induction mechanism of biphenyl/PCB‐degradation pathway in a Rhodococcus degrader M. Fukuda, J. Shimodaira, K. Miyauchi, H. Takeda, D. Kasai, E. Masai …………………..

S3

Kinetics of reductive dechlorination of chlorinated ethenes: dynamics, abundance and activity of dechlorinating populations B. Matturro, M. Majone, F. Aulenta, V. Tandoi, S. Rossetti .................................................

S3

Synergistic action of azoreductase and laccase leads to maximal decolourization and detoxification of model dye‐containing wastewaters Sónia Mendes, Ana Farinha, Christian G. Ramos, Jorge H. Leitão, Cristina A. Viegas, Lígia O. Martins……………………………………………………………………………………

S4

Evolutionary insights into nickel tolerance in bacteria. Where do nre genes come from? F. Pini, M. Migliore, A. Florio, A. Benedetti, M. Galardini, G. Spini, M. Bazzicalupo, A. Mengoni ..................................................................................................

S4

Metabolism of sulfonated aromatic compounds in Novosphingobium subarcticum sa1 Strain G. Rákhely, B. Hegedűs, M. Magony, K. Laczi, A. Tóth, G. Maróti, F.K. Medzihradszky, K.L. Kovács, K. Perei ..........................................................................

S5

Ability of alkalophilic cyanobacterial strains to degrade phosphonate xenobiotics Hanna Studnik, Giuseppe Forlani, Paweł Kafarski, Jacek Lipok ........................................

S5

Sediment‐free consortia anaerobically dechlorinating Aroclor 1260 Vlasta Dudková, Kateřina Demnerová .................................................................................

S6

Posters Evolution of bacterial community in isopropanol-degrading biotrickling filters by Fluorescence In Situ Hybridization (FISH) M.C. Pérez, F.J. Álvarez-Hornos, P. San-Valero, C. Gabaldón............................................

S6

Degradation of sulfamethoxazole by pure strains isolated from an acclimated membrane bioreactor Boris A. Kolvenbach, Benjamin Ricken, Hélène Bouju, Philippe F.X. Corvini.....................

S7

Anaerobic treatment of mezcal vinasses in fluidized bed bioreactors V. Robles-González, J. Galíndez-Mayer, N. Ruiz-Ordaz, E. López-López, F. Martínez-Jerónimo, A. Ortega-Clemente, N. Rinderknecht-Seijas, Héctor M. Poggi-Varaldo ......................................................................................................

S7

Carboxylation is the initial enzyme reaction in the anaerobic degradation of the polycyclic aromatic hydrocarbon naphthalene H. Mouttaki, R.U. Meckenstock .............................................................................................

S8

Regulation of styrene catabolism in Pseudomonas fluorescens ST: functional characterization of the StyS sensor kinase Francesco Massai, Giordano Rampioni, Paolo Ascenzi, Livia Leoni, Elisabetta Zennaro..................................................................................................................

S8

Investigation of nanoparticles as potential activators for the optimization of PAH biodegradation T. Masy, W. Wannoussa, S. Hiligsmann, P. Thonart…………………………………………….

S9

Bioaugmentation potential of Novosphingobium sp. PP1Y in natural and artificial soils contaminated by PAHs and heavy metals V. Cafaro, E. Notomista, V. Izzo, L.Troncone, G.Donadio, P. Tedesco, A. Di Donato.........

S9

An extensive analysis of the hydrocarbon degrading abilities of bacteria belonging to the species Acinetobacter venetianus R. Fani, M. Fondi, I. Maida, E. Perrin, M.C. Papaleo, G. Emiliani, M. Galardini, K. Mara, F. Decorosi, C. Viti, L. Giovannetti, F. Baldi, A. Mengoni, V. Orlandini..............

S10

Biodegradation of ETBE and BTEX by a bacterial consortium V. Gunasekaran, L. Stam, M. Constantí.................................................................................

S10

Microcoms study of anaerobic bioconversion of hexachlorocyclohexane in heavily contaminated soils Roberta Verdini, Carmela Riccardi, Lucia Pierro, Marco Petrangeli Papini, Mauro Majone........................................................................................................................

S11

Characterization of n-hexadecane-degrading biosurfactant-producing Acinetobacter spp. isolated from petroleum hydrocarbon polluted soil P. Aguila, S. Fuentes, C. Bravo, H. Palma, M. Seeger...........................................................

S11

Isolation and characterization of biosurfactant-producing bacteria A.L. Olivas Tarango, G.V. Nevárez Moorillón, M.L. Ballinas Casarrubias, B.E. Rivera Chavira, E. Orrantia Borunda ...........................................................................

S12

Utilization of chitinous substances for the optimized bioproduction of antifungal chitinase by Paenibacillus tylopili Maria Swiontek Brzezinska, Urszula Jankiewicz...................................................................

S12

Electricity production and nitrogen removal from digestate by microbial fuel cells E.G. Di Domenico, G. Petroni, D. Mancini, L. Di Palma, A. Geri, F. Ascenzioni................

S13

Application of an electro‐biochemical slurry reactor for the treatment of a soil contaminated with lindane B. Camacho-Pérez, A.L. Vazquez-Larios, E. Ríos-Leal, J. Garcia-Mena, O. Solorza-Feria, N. Rinderknecht-Seijas, H.M. Poggi-Varaldo...........................................

S13

Remediation of oils spill impacted soil by bioaugmentation with free living nitrogen fixing bacteria Josefina Pérez Vargas, Sergio Vigueras Carmona, Noemí Araceli Rivera Casado, Graciano Calva Calva............................................................................................................

S14

Application of nitrifying bacteria as an indirect approach to reduce stress of transported zebrafish Anusha K. S. Dhanasiri, Jorge M.O. Fernandes, Viswanath Kiron.......................................

S14

Effect of redox mediators and various media on the decolourisation of the azo dye Methyl Red and its biodegradation by Providencia rettgeri strain ODO Olukanni Olumide David, Adekola Adejoke Adebimpe, Sunmola Damilola Banke, Osunmuyiwa Olufolabi................................................................

S15

Bioremediation of aquifers polluted by chlorinated aliphatic hydrocarbons: selection and characterization of an indigenous microbial consortium for a packed bed reactor on-site process G. Bucchi, R. Ciavarelli, F. Doria, R. Salviulo, G. Zanaroli, S. Fraraccio, D. Frascari, D. Pinelli, F. Fava.............................................................................................

S15

Bioslurry treatment of a clayish soil polluted with lindane by sequential methanogenic‐sulfate reducing bioreactors B. Camacho-Pérez, E. Ríos-Leal, P. A. Vazquez-Landaverde, J. Garcia-Mena, J. Barrera-Cortés, F. Fava, N. Rinderknecht-Seijas, Héctor M. Poggi-Varaldo...................

S16

Predictive evaluation of bioremediation potential of chlorinated solvents contaminated sites B. Matturro, S. Rossetti ..........................................................................................................

S16

Metabolisation of chlorobenzoic acids by plant‐bacteria associations Blanka Vrchotová, Martina Macková, Tomáš Macek ...........................................................

S17

Uranium reduction by bacteria M. Baiget, M. Constantí, F. Medina, M.T. Lopez...................................................................

S17

Isolation, selection and improvement of a Methylobacterium spp. strain for the bioremediation of anthropogenic organic compounds V. Ventorino, F. Sannino, A. Piccolo, O. Pepe.......................................................................

S17

Bioremediation with probiotics in shrimp farming E. Mayer, E.M. Gössl, G.A. Santos, M. Mohnl..............................................................................

S18

Comparative growth studies of the hyperthermophilic archaeon Sulfolobus solfataricus P2 on sulfur compounds found in fossil fuels Gokhan Gun, Yuda Yurum, Gizem Dinler…………………………………………………………

S18

Bioremediation of harbour sediments contaminated with organic compounds: microbial response and biodegradation efficiency L. Rocchetti, F. Beolchini, P. Renzi, M. Gabellini, A. Dell’Anno..........................................

S19

Utilization of a respirometric technique for the aerobic treatment of industrial wastewater I. Colussi, A. Cortesi, V. Gallo, R. Vitanza ............................................................................

S19

Metabolic and protein-protein interactions of sulfanilic acid catabolism in Novosphingobium subarcticum SA1 B. Hegedűs, K. Perei, M. Magony, K. Laczi, A. Tóth, K.L. Kovács, G. Rákhely ........................

S20

Bacterial biosynthesis of selenium nanoparticles by environmental isolates of Stenotrophomonas maltophilia Silvia Lampis, Emanuele Zonaro, Chiara Santi, Anita Ferrari, Giovanni Vallini.................

S20

Sub-session 1.2. Microbial biodegradation assessment and improvement Engineering environmental catalysts: from trial-and-error to Synthetic Biology Main lecture Victor de Lorenzo...................................................................................................................

S21

Oral presentations Microbial community structure shifts in response to different biotic and abiotic conditions Martina Mackova, Ondrej Uhlik, Lucie Musilova, Michal Strejcek, Petra Prouzova, Petr Stursa, Eva Hoskovcová, Petra Lovecka, Cestmir Vlcek, Jakub Ridl, Miluse Hroudova,Katerina Demnerova, Jan Paces, Tomas Macek....................

S22

GenoRem: improving bioremediation of polluted soils through environmental genomics G. Burger, F. Courchesne, C. Greer, M. Hijri, S. Jabaji, S. Joly, M. Labrecque, B.F. Lang, F. Pitre, M. St-Arnaud, E. Yergeau .....................................................................

S22

Gene for degradation of organomercurial compounds and its application to bioremediation of mercury contamination G. Endo, M.F. Chien, M. Narita, C.C. Huang........................................................................

S23

Bioremediation assessment on linear alkylbenzene-polluted aquifer E. Martínez, A. Burniol, G. Vidal, A.M. Solanas, M. Viñas…………………………………….

S23

Biodegradation and sorption of 17α-ethinylestradiol in submerged membrane bioreactor: effect of initial ammonium concentration Liza Bautista-Patacsil, Analiza P. Rollon, Aileen P. Huelgas, Jiangyong Hu......................

S24

Insights in the bioremediation of aromatic compounds by C. necator Nathalie Berezina, Bopha Yada.............................................................................................

S24

Operation of hybrid fluidized bioreactors for on site bioremediation of water polluted with high concentration of PCE Luz de M. Bretón-Deval, J. Galindez-Mayer, C.U. Moreno-Medina, E. Ríos-Leal, N. Rinderknecht-SeijaS, Héctor M. Poggi-Varaldo

S25

Posters Oil product degradation in the polluted soil Elena Bocharnikova…………………………………………………………………………………………

S25

Development of a biological biobarrier for in-situ treatment of gasoline-contaminated groundwater Matteo Daghio, Andrea Franzetti, Isabella Gandolfi, Giuseppina Bestetti, Alessandro Careghini, Laura Romele, Sabrina Saponaro, Elena Sezenna............................

S26

The use of Biolog EcoPlatesTM to measure the effects of temperature and moisture on microbial communities in Roadside Gully Pot contents K.M. Scott, T.J. Coulthard, J.D.W. Adams..............................................................................

S26

Genotoxicity of 4-nonylphenol and nonylphenol ethoxylate mixtures with the use of Saccharomyces cerevisiae D7 mutation assay and use of this text to evaluate the efficiency of biodegradation treatments Stefania Frassinetti, Leonardo Caltavuturo, Fabio Fava, Diana Di Gioia............................

S27

Effect of pre-treatment and biosurfactants on the enzymatic hydrolysis of lignocellulose R. Khvedelidze, G. Kvesitadze, T. Urushadze, L. Kutateladze, V. Potschishvili……………...

S27

Aminophosphonate xenobiotics as a nutritive phosphorus source for mycelial fungi capable for C-P bond biodegradation Dorota Wieczorek, Paweł Kafarski, Jacek Lipok....................................................................

S28

Role of the H-NS family proteins in cooperative function of carbazole degradative plasmid pCAR1 and host chromosome C. Suzuki, C.S. Yun, S. Horita, T. Terada, M. Tanokura, H. Yamane, H. Nojiri……………...

S29

Microbial populations during bioremediation of PAHs in wood sleepers treated with creosote oil B. Slavik, K. Cajthamlová, M. Čvančarová, Z. Křesinová, T. Cajtham..................................

S29

A multivariate statistical analysis of the performance of fluidized bed bioreactors used for ad situ remediation of water polluted with PCE Cuauhtémoc U. Moreno-Medina, Luz M. Bretón-Deval, Favio Fava, María T. Ponce-Noyola,Josefina Barrera Cortés, N. Rinderknecht-Seijas, Juvencio Galíndez-Mayer, Héctor M. Poggi-Varaldo…………………………………………..

S29

Comparison of ozonation and fungal treatment for the depuration of an anaerobically pretreated mezcal vinasse Vania Robles-González, Nora Ruiz-Ordaz, Juvencio Galíndez-Mayer, Noemí Rinderknecht-Seijas, Héctor M. Poggi-Varaldo..........................................................

S30

A designed and tailored compost for different applications: innovative procedure applied to different compost property characterization Alessandra Bonoli, Alice Dall’Ara, Silvia Serranti................................................................

S30

Hormonal activities of novel brominated flame retardants and their biodegradation by white rot fungi M. Ezechiáš, K. Svobodová, T. Cajtham.................................................................................

S31

Removal of polycyclic aromatic hydrocarbons (PAHs) by selected microbial strains isolated from highly contaminated soil F. Sannino, V. Ventorino, O. Pepe, A. Piccolo.......................................................................

S31

Interaction between degradative plasmid and host chromosome differ between three pseudomonas hosts Y. Takahashi, M. Shintani, N. Takase, Y. Kazou, F. Kawamura, H. Hara, H. Nishida, H. Yamane, H. Nojiri...........................................................................................

S32

Identification of cultivable and non-cultivable rhizosphere bacteria from long term contaminated soil by PCBs using molecular genetic Petr Stursa, Petra Prouzova, Tomas Macek, Martina Mackova.............................................

S32

Level of environmental pollution triggers colonizing Miscanthus sinensis by endobacteria possessing genes coding for 2,3-dioxygenase S.W. Gawronski, A.Kacprzyk..................................................................................................

S33

Transgenic plants in rhizoremediation of PCB-contaminated soil V. Kurzawova, M. Novakova, M. Chovancova, T. Macek, M. Mackova……………………….

S33

Stimulation of bacterial chlorobiphenyl metabolism through the addition of plant secondary metabolites Lucie Musilova, Ondrej Uhlik, Michal Strejcek, Petra Lovecka, Vlasta Dudkova, Cestmir Vlcek, Jakub Ridl, Miluse Hroudova, Tomas Macek, Martina Mackova, Katerina Demnerova....................................................................................................................

S34

Looking in the environment for new enzymes as a tools for biocatalysis and bioremediation Adam Sobczak, Paweł Krawczyk, Dorota Tokarska, Dorota Adamska, Michał Kamiński, Marta Błaszkiewicz, Marcin Ostajewski, Marcin Gołębiewski, Leszek Lipiński, Andrzej Dziembowski....................................................................................

S34

Sub-session 1.3. Phytoremediation and mycoremediation Oral presentations The poplar endophyte Pseudomonas putida w619 as a key to a successful phytoremediation of volatile organic contaminants: from the lab to the field Nele Weyens, Sofie Thijs, Panos Gkorezis, Daniel van der Lelie, Safiyh Taghavi, Jaco Vang…………………………………………………………………………..

S35

GM plants expressing bacterial dioxygenases for enhanced phytoremediation of organic pollutants M. Novakova, M. Mackova, Z. Antosova, J. Viktorova, L. Trbolova, M. Sylvestre, T. Macek............................................................................................................

S36

Spatial and temporal characterization of bacterial communities in a phytoremediation Pilot plant aimed at decontaminating polluted sediments dredged from Leghorn harbor area Carolina Chiellini, Renato Iannelli, Veronica Bianchi, Giulio Petroni.................................

S36

Removal of phenolic compounds in constructed wetlands mesocosms treating sugar cane stillage at high surface organic load rates E.J. Olguín, R.E. González-Portela, G. Sánchez-Galván, K.L. Tapia-Fierro………………..

S37

Assessing the ability to treat nitrogen compounds in domestic wastewater of a constructed wetland with different aquatic plant species Nguyễn Thị Loan....................................................................................................................

S37

Anaerobic digestion of corn stover as sustainable soil management concept for metal contaminated agricultural land A. Peene, B. Mattheeuws, Jan Smis........................................................................................

S38

Mycoremediation of contaminated soil in field scale M. Tuomela, K. Jørgensen, E. Winquist, K. Björklöf, E. Schultz, F. Anasonye, L. Häkkinen,M. Räsänen, J. Sorvari, E.S. Hartikainen, K. Steffen, L. Valentin.....................

S38

Inoculation of poplar with arbuscular mycorrhizal fungi exerts a protective role on heavy metal stress by transcriptome modulation A. Cicatelli, V. Todeschini, G. Lingua, P. Torrigiani, S. Biondi, S. Castiglione....................

S39

Posters Effect of combined pollution of chromium and benzo (a) pyrene on seed growth of Lolium perenne Chigbo Chibuike Onyema, L.C. Batty………………………………………………………………

S39

Using biosurfactants in phytoremediation of soil polluted with petroleum hydrocarbons G. Kvesitadze, E. Karpenko, G. Khatisashvili, R. Vildanova, T. Sadunishvili, N. Gagelidze, G. Adamia, L. Amiranashvili, M. Pruidze, N. Kuprava ………………………..

S40

Molecular biodiversity of arbuscular mycorrhizal fungi (AMF) in trace metals contaminated soils and their role in soil phytoremediation S.E. Hassan, M. St-Arnaud, M. Hijri……………………………………………………………….

S40

Biodegradation of 17α-ethinylestradiol by edible white rot fungus - a mechanistical study Zdena Kresinova, Martin Ezechias, Tomas Cajthaml.............................................................

S41

Utilization of arbuscular mycorrhizal fungi for the protection of tomato plants (Solanum lycopersicum) of Cr(VI) toxic concentrations Yazmín Carreón-Abud, Miguel Martínez-Trujillo………………………………………………..

S41

Using arbuscular mycorrhizal fungi to enhance plant growth in maize (Zea mays) in soils with high concentrations of aluminum María del Rocío Madrigal-Pedraza, Mayra E. Gavito, Miguel Martínez-Trujillo, Yazmín Carreón-Abud………………………………………………………………………………..

S42

Bioreactor optimization for the treatment of industrial wastewaters by means of a fungal strain Federica Spina, Antonella Anastasi, Alice Romagnolo, Valeria Tigini, Valeria Prigione,Giovanna Cristina Varese...........................................................................

S42

New insight into fungal degradation of polychlorinated biphenyls Tomáš Cajthaml, Monika Čvančarová, Zdena Křesinová, Alena Filipová, Tatiana Stella, Stefano Covino................................................................................................

S43

Mycoremediation of PCBs dead-end metabolites: in vivo and in vitro degradation of chlorobenzoic acids by the white rot fungus Lentinus tigrinus T. Stella, S. Covino, Z. Křesinová, A. D’Annibale, M. Petruccioli, T. Cajthaml....................

S43

Selection of microfungi with high lipolytic activity and their lipase characterization S. Chinaglia, L.R. Chiarelli, G. Valentini, A.M. Picco...........................................................

S44

Degradation of endocrine disrupting chemicals and removal of estrogenic activity by Lentinus tigrinus and its extracellular enzymes S. Covino, T. Stella, Z. Křesinová, A. D’Annibale, M. Petruccioli, T. Cajthaml....................

S44

Eco-efficiency analysis of a fungal bioremediation method Markus Räsänen, Erika Winquist, Marja Tuomela, Matti Leisola, Jaana Sorvari ................

S45

Evaluation of mycrocistin biodegradation by wild yeasts for application in water treatment Gisele Maria de Andrade de Nobrega, T.A. Miguel, D.D. Lopes, C.L. Yokoyama, F.C. Pagnocca, E.K. Kuroda, K. Tsuji, A.R. Coelho, E.H. Hashimoto, S.Garcia, L.D. Paccola-Meirelles, O. Kawamura, K-I. Harada, E.Y. Hirooka…………………………...

S45

Enzyme activities and respiration as bioindicators of the biological quality of Pb-Cd-contaminated soil under aided phytostabilization Jacek Krzyżak, Grażyna Płaza, Rosa Margesin, Daniel Wasilkowski, Agnieszka Mrozik....................................................................................................................

S46

Laser stimulation of native Trichophyton mentagrophytes var. granulosum as a new method for the enhancement biodegradation of petrochemical hydrocarbons Jan W. Dobrowolski, A. Budak, D. Trojanowska, M. Rymarczyk, J. Macuda .......................

S46

Phenolic metabolites, oxidative enzymes and microbial activity in the rizosphere of Cyperus laxus in phytoremediation oil spill impacted-sites process Noemí Araceli Rivera Casado, María del Carmen Montes Horcasitas, Refugio Rodríguez Vázquez, Fernando José Esparza García, Armando Ariza Castolo, Josefina Pérez Vargas, Octavio Gómez Guzmán, Graciano Calva Calva………………………………………………………………………………..

S47

Phyto-mycoremediation: morphological and molecular characterization of arbuscular mycorrhizal fungi from a heavy metal polluted ash dump downtown Venice Alessandra Turrini, Stefano Bedini, Emanuele Argese, Manuela Giovannetti.......................

S47

Halophytes present new opportunities in heavy metal phytoremediation E. Manousaki, N. Kalogerakis………………………………………………………………………………

S48

Connections between rhizobacterial communities of reed sediments and land-use in Zhangye City area (Gansu Province, China) L. Borruso, J. Köbbing, L.L. Yu, K. Ott, N. Thevs, H. Ping, S. Zerbe, L. Brusetti……………

S48

Combined biological and physicochemical processes for treatment of baker’s yeast wastewater Sahand Iman Shayan, Maryam Hosseini, Sabra Rostami, Sirous Ebrahimi..........................

S49

High concentration of copper and zinc influences microbial biodiversity of Zea mays L. rhizosphere and selects multimetal resistant bacteria strains S. Matrella, G. Vigliotta, A. Cicatelli, S. Castiglione.............................................................

S49

Anti cancer drug from algae and wastewater treatment: a double winner Faraz M. Harsini, Milad Dehghani, Mohammadreza Mehrnia, Mohammad H. Sarrafzadeh, S. Shahab Edin Farazandeh mehr, A. Sadeghian Haghighi………………………………………...

S50

nirK-type denitrifier community composition and denitrification activity in a buffer strip B. Boz M. Bottegal, Md.M. Rahman, F. Fontana, M. Basaglia, B. Gumiero, S. Casella.................................................................................................................

S50

Preparation of transgenic plants with enhanced heavy metal accumulation J. Fiser, E. Neumannova, J. Viktorova, M. Novakova, M. Vrbova , P. Kotrba, M. Mackova, T. Macek ………………………………………………………………………….......

S51

Plant - microbe interactions in PCB contaminated soil Petra Prouzova, Eva Hoskovcova, Eva Bedrlikova, Petr Stursa, Katerina Demnerova, Martina Mackova................................................................................

S51

Enhancement of phytoremediation ability of Medicago sativa by the mycorrhizal fungus Glomus intraradices Stefano Bedini, Emanuele Argese, Manuela Giovannetti.......................................................

S52

Session 2 ORGANIC WASTE MULTIPURPOSE BIOREFINERIES FOR THE CONVERSION OF HIGH ENVIRONMENTALLY IMPACTING MATRICES AND EFFLUENTS INTO BIO-BASED CHEMICALS, MATERIALS AND FUELS Zerowastewater: short-cycling of wastewater resources for sustainable factories and cities of the future Keynote lecture Willy Verstraete……………………………………………………………………………………….

S53

Sub-session 2.1. Food processing and agro-industrial waste biorefinery Oral presentations Exploitation of starch industry by-product to produce bioactive peptides from rice protein Hydrolysates Lucilla Dei Più, Annalisa Tassoni, Diana Isabella Serrazanetti, Maura Ferri, Elena Babini, Andrea Gianotti ……………………………………………………………………

S53

Systems for biohydrogen and bioelectricity generation: a crucial component of biorefineries P. Robledo-Narváez, E. Rios-Leal, N. Rinderknecht-Seijas, A. Ortega-Clemente, M.T. Ponce-Noyola, H.M. Poggi-Varaldo………………………………………………………

S54

Engineering amino acid producing Corynebacterium glutamicum for access to alternative carbon sources C. Matano, J.W. Youn, J. Lindner, T. Meiswinkel, V.F. Wendisch.........................................

S55

Improved orange peel waste pretreatments for bioethanol production G. Santi, S. Crognale, M. Moresi, M. Petruccioli, A. D’Annibale ........................................

S55

The use of a vegetable waste-derived fermentation product for H2 production by anoxygenic phototrophic bacteria Alessandra Adessi, Giovanni Colica, Roberto De Philippis..................................................

S56

Upgrade of waste glycerol to short-chain polyhydroxyalkanoates co-polymers M.M. Da Fonseca, J.M. Cavalheito, R. Raposo, M.C.D. De Almeida, M.T. Cesãrio, E. Pollet, H. Diogo, C.C. De Carvalho............................................................

S56

Posters Commercialization of new value chains in the bioeconomy Manfred Kircher, Achim Marx................................................................................................

S57

Process intensification in biotechnology: towards an integrated biorefinery Ludo Diels, Wouter Van Hecke, Heleen De Wever, Bert Lemmens, Luc Van Ginneken, Karolien Vanbroekhoven.........................................................................

S57

New advances in the integrated management of food processing by-products in India and Europe: sustainable exploitation of fruit and cereal processing by-products with the production of new foods and feeds (NAMASTE EU) Fabio Fava, Keith Waldron, Carlos Bald, András Sebők, Jan Broeze, Victor M. Garijo, Hans-Georg Brendle..................................................................................

S58

Production of polyhhydroxyalkanoates (PHAs) from used frying oils and polymer recovery using different strategies M. Scandola, L. Martino, A. Scoma, M. Cruz, F. Freitas, A.R. Gouveia, M.A.M. Reis......................................................................................................

S58

Influence of carbon sources on the production and characterization of the exopolysaccharide (EPS) by Bacillus sphaericus 7055 strain M. Yilmaz, G.Y. Celik, B. Aslim, D. Onbasili..........................................................................

S59

Metabolic engineering in Enterococcus faecalis for its use in bio-ethanol production from whey N.F. Rana, S. Gente, J.M. Laplace, Y. Auffray........................................................................

S59

A new approach on the biohydrogen production process via methane generated from effluents J.O.B Carioca, O. Araújo, C. Morais, S.Macambira, S. Galdino, A. Siqueira, E. Lima, J. Furlan............................................................................

S60

Bio-hydrogen production by Escherichia coli WDHL and Bacillus sp. using wheat straw hydrolysate as substrate Zazil Donaxí Alvarado Cuevas, Arturo Sánchez, Leandro G Ordoñez, José Tomás Ornelas Salas, Antonio De León Rodríguez........................................................

S60

Cheese whey waste as raw material for the bio-hydrogen production by Escherichia coli WDHL: importance of amino acids availability Zazil Donaxí Alvarado Cuevas, Leandro G. Ordónez, José Tomás Ornelas Salas, Antonio De León Rodríguez........................................................

S61

Biochemical methane potential (BMP) test of residual biomass from agro-food industry Mariangela Soldano, Nicola Labartino, Claudio Fabbri, Sergio Piccinini.......................................................................................................................

S61

Bioethanol production from dairy effluents: improvement of the process efficiency Francesca Zoppellari, Laura Bardi........................................................................................

S62

Batch tests of biological hydrogen production from food industry wastes by four thermotoga thermophilic strains in 0.12-L microcosms and in a 19-L fermentor A. Alberini, S.J. Mendes, G. Bucchi, C. Manfreda, M. Cappelletti, D. Pinelli, S. Fedi, F. Fava, D. Frascari.................................................................................

S63

Comparison of biohydrogen production in fluidized bed bioreactor at room temperature and 35°C K.M. Muñoz-Páez, J. García-Mena, M.T. Ponce-Noyola, A. Ramos-Valdivia., I.V. Robles González, N. Ruiz-Ordáz, L. Villa- Tanaca, N. Rinderknecht-Seijas, R. De Philippis, H.M. Poggi-Varaldo.....................................................................................

S63

Synthesis of biodiesel from hydrolysates of arundo donax Domenico Pirozzi, Abu Yousuf, Gaetano Zuccaro, Rosaria Aruta, Filomena Sannino...................................................................................................................

S63

Evaluation of carob pulp as fermentation substrate for biohydrogen and organic acids production: sugars richness vs toxicity potential M. Lima, J. Ortigueira, L. Alves, S.M. Paixão, P. Moura.......................................................

S64

Robust yeasts for the conversion of lignocellulosic hydrolysates into ethanol Lorenzo Favaro, Alberto Trento, Marina Basaglia, Sergio Casella.........................................................................................................................

S64

Bioenergy from lignocellulosic wastes: preatreatment, enzymatic hydrolysis and ethanol productivity B. Ribeiro, S. Alves, L. Baeta-Hall, L. Duarte, F. Carvalheiro, José C. Duarte.........................................................................................................................

S65

Computer-based artificial intelligence strategies in modeling and optimization of microbial bioprocesses optimization case of biogas generation from wastes E.B. Gueguim Kana.................................................................................................................

S65

Bioethanol production from mixed sugars using Scheffersomyces stipitis Isabella De Bari, Paola De Canio, Daniela Cuna, Federico Liuzzi, Patrizia Romano, Angela Capece................................................................

S66

Biomethane production from tobacco plants with modified cell wall Marianna Villano, Fedra Francocci, Felice Cervone, Giulia De Lorenzo, Mauro Majone.........................................................................................

S66

Si-microbial symbiotic effect on the root formation of the cultivated plants Vladimir Matichenkov.............................................................................................................

S67

Optimization of production conditions for Trichoderma sp. P25 as a biocontrol agent by using solid state fermentation S. Sözer, S. Sargın,R. Eltem, F. Vardar Sukan.......................................................................

S67

A high-added value product from tomato pomace conversion: -L-arabinofuranosidase from Pleurotus ostreatus for lignocellulose conversion Antonella Amore, Angela Amoresano, Leila Birolo, Vincenza Faraco..................................

S68

Vanillin production from wheat bran with Pseudomonas fluorescens BF13-1p Elena Dal Bello, Stefano Rebecchi, Andrea Negroni, Giulio Zanaroli, Diana Di Gioia, Maurizio Ruzzi, Fabio Fava.........................................................................

S68

Increasing the feed value of olive oil cake by solid state cultivation of the white-rot fungus Fomes fomentarius Mohamed Neifar, Amany Ayari, Abdellatif Boudabous, Ameur Cherif, Atef Jaouani............

S69

Biotransformation of agricultural wastes with innovative enzymes A. Morana, S.M. Paixão, S.A. Ladeira, L. Alves, E. Ionata, F. La Cara................................

S69

Screening of novel yeast inulinases and further application to bioprocesses S.M. Paixão, P.D. Teixeira, T. Silva, A.V. Teixeira, L. Alves.................................................

S70

Recent advances in the biorefinery of olive mill wastewater Alberto Scoma, Lorenzo Bertin, Matilde Monti, Fabio Fava.................................................

S70

Olive-mill waste as potting substrate for olive tree cultivation: effects on the microbiota of soil and rhizosphere L. Fidati, S. Scargetta, L. Nasini, G. Gigliotti, P. Proietti, G. Cenci, E. Federici............................................................................................

S71

Morphological analysis of yeasts bioprospected from the Brazilian savannah Igor Chiarelli Perdomo, Janina Zanoni Camargo, Gustavo Graciano Fonseca.....................................................................................................

S71

Growth kinetics of yeast strains isolated from the Brazilian savannah Janina Zanoni Camargo, Igor Chiarelli Perdomo, Rosimeire Oenning da Silva, Marney Pascoli Cereda, Gustavo Graciano Fonseca.............................................................

S72

Evaluation of the capacities of assimilation and fermentation of several yeasts strains isolated from exotic fruits from the Brazilian savannah Zanoni Camargo J., Avelino Gonã§alves F., Chiarelli Perdomo I., Graciano Fonseca G............................................................................

S72

Comparison of bacteriocins production from Enterococcus faecium strains in cheese whey and optimised commercial MRS medium Lorenzo Favaro, Stefano Schirru, Roberta Comunian, Marina Basaglia, Sergio Casella, Antonio Paba, Elisabetta Daga, Bernadette Dora Gombossy de Melo Franco, Ricardo Pinheiro de Souza Oliveira, Svetoslav Dimitrov Todorov......................................................

S73

Production of enzymes from grape stalks and wheat bran in solid state fermentation D.C. Masutti, A. Borgognone, L. Setti.....................................................................................

S73

Sub-session 2.2. Organic waste and effluent biorefinery Biotechnology for sustainalbe supply and use of phosphorus Main lecture Hisao Ohtake..........................................................................................................................

S75

Oral presentations Bio-hydrogen and bio-methane co-production by sequential two-phases dark fermentation from agro-industrial wastes (IMERA) Giuseppe Lustrato, Gabriele Alfano, Giancarlo Ranalli.......................................................

S76

Investigation of the microbial community in biogas plants by metaproteome analysis R. Heyer, D. Benndorf, F.Kohrs, A. Hanreich, M. Klocke, E. Rapp, U. Reichl.....................

S76

Enhanced anaerobic digestion performances: effect of sludge ultrasound pre-treatment and role of the microbial population dynamics C.M. Braguglia, M.C. Gagliano, A. Gianico, S. Rossetti.......................................................

S77

Industrial by-products as a source of volatile fatty acids by anaerobic digestion Fátima Sousa, Susana R.S. Pereira, Ana M.R.B. Xavier, Dmitry V. Evtuguin, Luísa S. Serafim....................................................................................

S77

Analysis of microbial diversity of inocula used in a microbial fuel cell A.C. Ortega Martínez, K. Juarez-López, O. Solorza-Feria, N. Rinderknecht-Seijas, H.M. Poggi-Varaldo.........................................................................

S78

Biotechnological process for the valorisation of residual glycerol from the biodiesel industry. broadening the biorefinery Xavier Turon, Sergi Abad, Francesc Padrès.........................................................................

S78

Coupling wastewater treatment to methane generation in bioelectrochemical systems Marianna Villano, Federico Aulenta, Stefano Scardala, Mario Beccari, Mauro Majone...............................................................................................

S79

Posters Mutagenic activity of biochars obtained from pyrolysis biorefinery processes in Salmonella typhimurium TA100 and TA 98 tester strains with and without metabolic S9 mix activation Anna V. Piterina, Kevin Chipman, J.J. Leahy, J. Tony Pembroke, Michael H. Hayes....................................................................................................................

S79

Chlorella vulgaris growth on digested urban sludge Alessandro A. Casazza, Bahar Aliakbarian, Patrizia Perego, Attilio Converti......................

S80

Isolation and characterization of potential biosurfactants produced by Bacillus strains growing on agroindustrial wastes Anna Poliwoda, Ewa Krzosok, Piotr P. Wieczorek, Paweł Kafarski, Grażyna Płaza............

S80

Selection of cellulase producer micromycetes of the south caucasus for the production of high technology sweeteners from agrarian wastes E. Kvesitadze, T. Urushadze, R. Khvedelidze, L. Kutateladze………………………………

S81

Influence of the type of alkali used for pH correction of acid growth media on methane production by acetoclastic methanogens C. Vasmara, L. Sghedoni, V. Faeti, R. Marchetti....................................................................

S81

Effect of total solids content of the feedstock, temperature and mass retention time on the performance of biohydrogenic solid substrate fermentation of organic waste C. Escamilla-Alvarado, M.T. Ponce-Noyola, E. Ríos-Leal, Héctor M. Poggi-Varaldo.........

S82

A study on the influence of activated sludge as nitrogen source on hydrogenic batch dark fermentation of the organic solid waste C. Escamilla-Alvarado, M.T. Ponce-Noyola, H.M. Poggi-Varaldo.......................................

S82

Series hydrogenesis-methanogenesis of ofmsw: operation and energetic feasibility of the process C. Escamilla-Alvarado, M.T. Ponce-Noyola, E. Ríos-Leal, H.M. Poggi-Varaldo.................

S83

Biomethane production from co-digestion of sewage sludge and crude glycerol from biodiesel production C. Salomoni, A. Caputo, M. Bonoli, O. Francioso, M.T. Rodriguez-Estrada, D. Palenzona..................................................................................

S83

Effect of glycerol on biomethane production and volatile organic compounds in anaerobic digestion of urban sewage sludge O. Francioso, M.T. Rodriguez-Estrada, A. Pisi, S. Savioli, C. Salomoni, A. Caputo, M. Bonoli, D. Palenzona.......................................................................................

S84

Characterization of a five-face parallelepiped microbial fuel cell equipped with sandwich electrodes A.C. Ortega Martínez, O. Solorza-Feria, M.T. Ponce-Noyola, N. Rinderknecht-Seijas, H.M. Poggi-Varaldo.........................................................................

S84

Application of RuxMoySez for oxygen reduction reaction in a microbial fuel cell A.L. Vázquez-Larios, O. Solorza-Feria., E. Ríos-Leal, N. Rinderknecht- Seijas, R. de G. González-Huerta, H.M. Poggi-Varaldo............................

S85

Effect of anodic material on the performance of a single chamber microbial fuel cell A.L. Vázquez-Larios, M.T. Ponce-Noyola, J. Barrera-Cortés, R. de G. González-Huerta, O. Solorza-Feria, H.M. Poggi-Varaldo.......................................

S85

Effect of anodic material on the internal resistance of a single chamber microbial fuel cell G. Hernández-Flores, O. Solorza-Feria, M.T. Ponce Noyola, N. Rinderknecht-Seijas, H.M. Poggi-Varaldo................................................................................................................

S86

Production of oxidative enzymes by trametes ochracea on the high-molecular weight fraction of olive-mill wastewater M. Petruccioli, T. Stella, E. Carota, A. D’Annibale………………………………………....

S86

Acidogenic digestion of deproteinized cheese whey Alberto Scoma, Lorenzo Bertin, Fabio Fava ……………………………………………….

S87

Improvement of butyric acid production in Clostridium tyrobutyricum by surfactant for detoxification of lignocellulose hydrolysates Kyung Min Lee, Ki-Yeon Kim, Sung Ok Han, Byoung-In Sang, Youngsoon Um....................

S87

Effect of different carbon sources on exopolysaccharide production by Enterobacter A47 Filomena Freitas, Ana Rosa Gouveia, Cristiana A.V. Torres, Maria A.M. Reis....................

S88

Session 3 BIOLOGICAL TRANSFORMATI ON OF CONVENTI ONAL AND BIOBASED POLYMERS IN THE ENVIRONMENT Bioplastics science from a policy vantage point Main lecture J.C. Philp………………………………………………………………………………….

S89

Oral presentations Novel eco-friendly multiblock copolymers poly(butylene/Triethylene succinate): effect of block length on mechanical properties and biodegradability M. Gigli, N. Lotti, A. Munari, A. Negroni, G. Zanaroli, F. Fava...........................................

S90

Biobased feedstock valorisation through polyhydroxyalkanoate production: from excess cheese whey to eco-efficient bioplastics Maria G.E. Albuquerque, Rocco Adiutori, Nuno M.P. Trindade, Inês T.D. Carmo, Catarina S.S. Oliveira, Filipa Pardelha, Maria A.M. Reis.......................

S90

Innovative value chain development for sustainable plastics in Central Europe (PLASTICE) M. Scandola, I. Voevodina......................................................................................................

S91

Posters Thermophilic microbial communities degrading selected synthetic polymers Lucie Husarova, Petr Stloukal, Sophie Commereuc, Vincent Verney, Marek Koutny...............................................................................................

S91

Synthesis of textile dyes by laccase biotransformations Daniele Spinelli, Andrea Martorana, Maria Camilla Baratto, Riccardo Basosi, Rebecca Pogni.............................................................................................

S92

Antimicrobial activity of phb based polymeric compositions Svetlana Gonta, Ludmila Savenkova, Irina Krallish, Elena Kirilova.....................................

S92

Biosynthesis of biotin and selenobiotin in biotin- independent S. cerevisiae strain cultivated in molasses medium Piotr Patelski, Katarzyna Pielech-Przybylska, Maria Balcerek, Anna Diowksz, Agnieszka Nowak, Urszula Dziekonska...............................................................

S93

Photodegradation of aliphatic polyesters and their composites with TiO2 S. Sullalti, G. Totaro, H. Askanian, A. Celli, P. Marchese, V. Verney, S. Commereuci.......................................................................................................

S93

Production of hollocellulolytic enzymes using agro-industrial residues: selection of type of pretreatment and substrate A.G. López-Nevarez, H.M. Poggi-Varaldo, E. Cristiani-Urbina, M.T. Ponce-Noyola.................................................................................................................

S94

Enzymatic processing of chitinaceous wastes for N-acetyl-D-glucosamine production Sancharini Das, Ramkrishna Sen, Debasis Roy......................................................................

S94

Bio-oil as feedstock for the production of biopolymers by aerobic mixed cultures Rita Moita, P.C. Lemos...........................................................................................................

S95

Hyaluronic acid from biofermentation–molecular weightB2 (TCE-enriched)>B3 (cis-DCE-enriched). Dechlorinating bacteria were quantified by combining in situ hybridization techniques and PCR-based approaches. Fluorescence in situ hybridization (FISH and CARD-FISH) allowed the cell numbers estimation of active and not active i) dechlorinating bacteria involved in the partial degradation of PCE or TCE to cis-DCE (Desulfitobacterium spp., Dehalobacter spp., spp., Geobacter spp., Sulfurospirillum spp.) as well as ii) “Dehalococcoides” spp. known to be able to completely dechlorinate such compounds to harmless ethene. Gene expression profile of reductive dehalogenase genes (tceA, bvcA, vcrA) were also estimated and correlated to kinetic performances and dechlorinating bacteria abundances. The potential for field application of the outputs of this study will be discussed.

S3

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S1-S20

SYNERGISTIC ACTION OF AZOREDUCTASE AND LACCASE LEADS TO MAXIMAL DECOLOURIZATION AND DETOXIFICATION OF MODEL DYE-CONTAINING WASTEWATERS Sónia Mendes1, Ana Farinha1, Christian G. Ramos2,3, Jorge H. Leitão2,3, Cristina A. Viegas2,3, Lígia O. Martins1 2

1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal Department of Bioengineering, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; 3IBB-Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia Biológica e Química, IST, Lisboa, Portugal

Abstract Many of the azo dyes used in the textile, paper, leather or plastic industries, and/or their breakdown products are toxic, potentially carcinogenic and can lead to the formation of bladder cancer in humans, tumours, allergies, nuclear anomalies in experimental animals, and chromosomal aberrations in mammalian cells. Among the few redox-active enzymes showing azo dyes degradative activity, azoreductases are particularly effective since they act on the reduction of the azo linkage, however, azoreductases require the addition of expensive cofactors such as NAD(P)H as electron donors for the reductive reaction and the products released are aromatic amines which are potentially toxic. In contrast, laccases are oxidoreductases that have a great potential for various biotechnological processes mainly due to their high non-specific oxidation, the lack of a requirement for cofactors, and the use of readily available oxygen as an electron acceptor capacity. In the present study, the enzymatic degradation of an array of 18 azo dyes and of three model dye baths was tested with two enzymes with proven ability to degrade synthetic dyes, recombinant FMN-dependent NADPH azoreductase (PpAzoR) from Pseudomonas putida MET94 and recombinant CotA-laccase from the bacterium Bacillus subtilis. The PpAzoR showed a broader specificity for decolourisation of azo dyes than CotA-laccase. However, the final products of PpAzoR activity exhibited in most cases a 2 to 3-fold higher toxicity than intact dyes themselves. We showed that addition of CotA-laccase to PpAzoR treated reaction mixtures lead to a significant drop in the final toxicity. An Escherichia coli strain co-expressing ppAzoR and cotA genes was constructed where the sequential action of PpAzoR and CotA enzymes could be tuned by aeration conditions. Whole-cell assays of recombinant strain for the treatment of model dye wastewater resulted in decolourisation levels above 80% and detoxification levels up to 50%. The high attributes of this strain, make it a promising candidate for the biological treatment of industrial dye containing effluents.

EVOLUTIONARY INSIGHTS INTO NICKEL TOLERANCE IN BACTERIA. WHERE DO NRE GENES COME FROM? F. Pini1*, M. Migliore2, A. Florio2, A. Benedetti2, M. Galardini1, G. Spini1, M. Bazzicalupo1, A. Mengoni1 1 Department of Evolutionary Biology, University of Florence, via Romana 17, I-50125 Firenze, Italy; 2C.R.A.—Centro di Ricerca per lo Studio delle Relazioni tra Pianta e Suolo, Via della Navicella 2-4, 00184 Rome, Italy; *present address: Institut de Recherche Interdisciplinaire-IRI CNRS USR3078 Parc de la Haute Borne 50 avenue de Halley F-59658 Villeneuve d'Ascq Cedex France

Abstract Heavy-metal tolerance in bacteria is a widespread phenotype, particularly occurring in strains isolated from heavy-metal contaminated sites, which often are of recent origins. Due to its rapid appearance, it is likely a few genes may confer metal tolerance via a limited number of mutations, and that these genetic determinants conferring heavy-metal resistance could have orthologs present in most of nonresistant strains, which may contributed to some other cellular functions. Concerning nickel tolerance several genetic determinants have been characterized and the nre system, which employs a Ni/H+ antiporter (NreB), has been found as one of the most diffused, being present in both strains isolated from polluted sites and in soils naturally rich in Ni (serpentine soils). Interestingly, an ortholog of nreB gene is also present in the nonresistant legume symbiont strain Sinorhizobium meliloti Rm1021 and recent genome sequencing efforts have shown this gene to be present in other S. meliloti strains, all of them nonresistant to Ni. Here, we report a comprehensive phylogenetic analysis of nreB genes in all completely sequenced bacterial genomes, showing that this gene was subjected to strong horizontal gene transfer events, possibly mediated via mobile genetic elements, often present in the proximity of its genomic locations. Moreover, a deletion mutant for nreB was obtained in S. meliloti Rm1021 and shown to be more sensitive to Ni than the parental strain, this deletion mutant was also characterized by Phenotype Microarray analysis and symbiosis with legumes. A strain overexpressing nreB under a lac inducible promoter was also constructed and compared with a strain overexpressing all the operon nreAB. Moreover two Escherichia coli strains overexpressing nreB of S.meliloti and the ortholog from the model heavy-metal resistant strains Cupriavidus metallidurans CH34 were also compared, in order to elucidate differences between these two orthologs. In conclusion nreB is probably involved also in other cellular functions than Ni tolerance. Ni-tolerant strains may consequently arise via a facilitated variation evolutionary pathway, explaining their independent appearance in many different heavy-metal rich environments.

S4

Sub-session 1.1 - Bacterial biodegradation of organics and inorganics

METABOLISM OF SULFONATED AROMATIC COMPOUNDS IN NOVOSPHINGOBIUM SUBARCTICUM SA1 STRAIN G. Rákhely 1,2, B. Hegedűs1, M. Magony1, K. Laczi1, A. Tóth2, G. Maróti2, F.K. Medzihradszky 3, K.L. Kovács1,2, K. Perei1 1

Department of Biotechnology, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary; 2Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary; 3Mass Spectrometry Laboratory, Biological Research Center, Hungarian Academy of Sciences

Abstract Novosphingobium subarcticum SA1 (Sphingomonas subarctica SA1, formerly identified as P. paucimobilis) contains all enzymes necessary for biodegradation of sulfanilic acid. Screening the substrate specificity of the strain disclosed its ability to degrade five analogue aromatic compounds: sulfanilic acid, protocatechuate, p-aminobenzoic acid, 4-sulfocatechuate, 4-hydroxybenzoate, 3,5dihydroxybenzoate and oil contaminations. S. subarctica seemed to use distinct enzyme cascades to utilize sulfonated and nonsulfonated molecules. The genome of the strain was sequenced by new generation genome sequencers and a proteomic approach was used to identify the components involved in sulfanilic acid assimilation. A genomic region was identified, which contained genes coding for sulfocatechol dioxygenase, sulfomuconate cycloisomerase (scaA), sulfomuconolactone hydrolase (scaB), oxidoreductase (scaC), sulfocatechol dioxygenase (scaD, scaE), permease (orf1) in a single gene cluster. These enzymes were overexpressed in E. coli and the sulfocatechol degradation pathway was reconstituted by active recombinant proteins. Other proteins appearing in cells grown on sulfanilic acid were also identified. These were membrane-bound enzymes and participated in the transfer of the amino group and ring hydroxylation. Proteinprotein interaction assay indicated a membrane associated complex converting the toxic sulfanilic acid into the less poisonous sulfocatechol which was further oxidized in the cytoplasm. Genes likely involved in the first catalytic step were identified in distinct loci. Whole genome transcript analysis of cells grown under distinct conditions revealed several operons of which expression was upregulated by sulfanilic acid. From the genomic and functional genomic data, a complex picture of sulfanilic acid assimilation is outlined.

ABILITY OF ALKALOPHILIC CYANOBACTERIAL STRAINS TO DEGRADE PHOSPHONATE XENOBIOTICS Hanna Studnik1, Giuseppe Forlani2, Paweł Kafarski1, Jacek Lipok1 1

Faculty of Chemistry, Opole University, 45-052 Opole, Oleska 48, Poland; 2Department of Biology & Evolution, University of Ferrara, via L. Borsari 46, 44100 Ferrara, Italy

Abstract Organophosphonates, compounds characterized by the presence of a direct, carbon to phosphorus (C-P) bond, are endowed with a striking stability even at extreme pH values and in a broad range of temperatures. Nowadays, synthetic phosphonates are widely used in various fields of human activity as pesticides, antibiotics, polymer additives, flame extinguishers and corrosion inhibitors. As a consequence of an increasing range of applications, thousand tons of phosphonate xenobiotics are released annually into the environment. The lack of information on their environmental fate, linked to analytical problems that hamper the determination of trace concentrations of these substances, led to an indiscriminate and to date unregulated use in most EU countries. This could exert undesirable environmental effects, particularly in aquatic ecosystems. Moreover, the biodegradation of organophosphonates has been thoroughly studied only in the case of a few substances, and it was generally investigated with soilborne microorganisms. If the effect of these compounds on terrestrial ecosystems seems limited by the ability of soil bacteria to cleave the C-P bond, mainly under conditions of phosphorus starvation, an increasing amount of data suggests that the release of phosphonate xenobiotics may induce adverse effects on water ecosystems. Besides a consequent enrichment of nutrients, especially phosphorus and nitrogen, possibly leading to water eutrophication, because of their inherent ability to chelate divalent cations phosphonates were reported to interfere with mineral nutrition and inhibit algal growth. Results of our studies indicated that some cyanobacterial strains, when grown in media containing phosphonates, possess a remarkable ability to degrade the C-P bond. As a consequence, pollution by organophosphonate could represent the basis for cyanobacterial blooming that often occurs in lakes and ponds, with severe adverse consequences on water quality. Additionally, we confirmed a relatively low toxicity of some compounds used on industrial scale, mainly polyphosphonates, towards various species of cyanobacteria. Metabolization of some aminophosphonates by Spirulina platensis and Arthrospira fusiformis was followed by non destructive 31P NMR analysis, showing the ability to completely degrade millimolar levels of these compounds. This unusual capability was confirmed in a labscale pilot plant for bioremediation of polyphosphonates from polluted waters.

S5

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S1-S20

SEDIMENT-FREE CONSORTIA ANAEROBICALLY DECHLORINATING AROCLOR 1260 Vlasta Dudková, Kateřina Demnerová Institute of Chemical Technology, Prague; Faculty of Food and Biochemical Technology, Department of Biochemistry and Microbiology, Technická 3, 166 28 Prague 6; Phone: (00420) 220 443 021, Czech Republic, e-mail: [email protected]

Abstract Polychlorinated biphenyls (PCBs) are ubiquitous manmade contaminants that pose a continuing risk to the health of humans and ecosystems. Bacteria that live in PCB-contaminated sediments have adapted to dechlorinate and detoxify PCBs, but very little is known about these organisms. Learning how to effectively stimulate and grow these dechlorinators offers the best chance for developing safe, effective, and economic in situ remediation techniques in the future. By a series of sequential transfers we developed enriched sediment-free consortia with stable dechlorinating activity against Aroclor 1260 from sediment of the river Strážský Sewer (Slovakia). Nona-, octa-, and heptachlorobiphenyls were dechlorinated to tri-, tetra-, penta- and hexachlorobiphenyls when pyruvate or lactate was added as a carbon source. The chlorophenyl rings targeted were: 23456-, 2346-, 2345-, 234- and 245-. Our consortia removed flanked meta- and para- chlorines. Under laboratory conditions, we accelerated the rate of dechlorination (maximal value of 0.020 chlorines removed per biphenyl per day) and reduced the period necessary for microorganisms to adapt by adding known haloprimers: 4-4-dibromobiphenyl or 26-dibromobiphenyl. We also demonstrated the presence of PCB dechlorination activity in sediment not treated with primers. We explored the onset addition of 2-bromethanesulphonate, molybdate and selected antibiotics (ampicillin, kanamycin and vancomycin) on PCB dechlorination. We enriched completely new dechlorinating consortia which do not correspond to any of the known microbial PCB dechlorination processes or their combination. The two different haloprimers did not influence the pattern of dechlorination observed in these consortia. The key role in our isolated consortia was played by sulphate-reducing bacteria. Acknowledgements This work was supported by grants GAČR GA525/09/058, MŠMT ME09024. We thank Donna L. Bedard for helpful discussion.

Posters P1 EVOLUTION OF BACTERIAL COMMUNITY IN ISOPROPANOLDEGRADING BIOTRICKLING FILTERS BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH) M.C. Pérez*, F.J. Álvarez-Hornos, P. San-Valero, C. Gabaldón University of Valencia, Dep. Chemical Engineering, Av. de la Universidad s/n, 46100 Burjassot, Spain, Phone: +34963543169, Fax: +34963544898, e-mail: [email protected]

Abstract One of the most important industrial pollutant sources is the emission of volatile organic compounds (VOCs) from the use of solvents. Biological treatment technologies, such as biofilters (BFs) and biotrickling filters (BTFs), for controlling VOC emissions are attractive techniques because of their efficiency and cost-effectiveness. BFTs use a specified inert packing material and involve a liquid phase, which trickles through the bed providing nutrients. The biofilm is developed on the packing surface. Removal efficiency in BFTs depends on multiple parameters, being the microbial community a key parameter in the performance of this process. In this study, the bacterial population of two biotrickling filters (BFTs) treating isopropanol by using fluorescence in situ hybridization (FISH) is analyzed. The experimental system consists in two identical laboratory-scale BFTs named as BFT1 and BFT2. The two bioreactors were operated in parallel during an experimental period of one year working under intermittent feeding conditions Operating conditions and maintenance were identical in both BFT. The FISH technique was carried out following the procedure described in literature with a Cy5-labelled EUBmix probe for most bacteria and Cy3-labelled specific probes (Thermo Fisher Scientific, Germany). Specific probes were quantified as a proportion of EUBmix labeled bacteria using image analysis with the methodology developed by Jubany et al (2009). The FISH technique was applied in order to study the changes of the microbial population within the different operational conditions applied in the BFTs during days 64 to 276. For example, the relative abundance of the general groups of bacteria in BTF1 is shown; in particular, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria (high G+C Gram-positive bacteria) and Firmicutes (Low G+C gram-positive bacteria) are expressed as percentage of EUB338mix stained cells. It can be observed a variation in the composition of the bacterial community with time of operation. Acknowledgments The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grant agreement nº 284949. Finnancial by Ministerio Ciencia e Innovación (Spain, research Project CTM2010-15031) is also acknowledged. M.C.Pérez has a FPU grant from Ministerio de Ciencia e Innovación, Spain.

S6

Sub-session 1.1 - Bacterial biodegradation of organics and inorganics

P2 DEGRADATION OF SULFAMETHOXAZOLE BY PURE STRAINS ISOLATED FROM AN ACCLIMATED MEMBRANE BIOREACTOR Boris A. Kolvenbach1, Benjamin Ricken1, Hélène Bouju1, Philippe F. X. Corvini1,2 1

Institute for Ecopreneurship, Life Sciences Schoool, University of Applied Sciences Northwestern Switzerland, Muttenz, Switzerland; 2School of the Environment, Nanjing University, Nanjing, China

Abstract Sulfonamide antibiotics such as sulfamethoxazole are readily found in surface waters worldwide in concentrations of up to several micrograms per liter. Due to the risk of spreading antibiotic resistances, their ubiquitous presence is of great concern. Especially sulfamethoxazole has been shown to be difficult to remove in biological wastewater treatment plants, and results can be highly variable. Currently, only energy-intensive processes such as ozonation and methods based on it are able to completely transform sulfamethoxazole. Nevertheless, possible by-products of incomplete treatments have not yet been tested for negative impacts on the environment. Until now, only two studies reported the removal of sulfamethoxazole from the aqueous phase by pure strains. However, the apparent biodegradation remained below 30% in both cases. This evidences the need to isolate and identify bacterial strains able to biodegrade sulfamethoxazole to higher extent. A total number of five strains able to grow on sulfamethoxazole as a carbon and energy source were isolated from a membrane bioreactor continuously fed with synthetic wastewater spiked with sulfamethoxazole. Single isolates and a consortium were tested for degradation of 14 C-sulfamethoxazole. Mineralization was detected by trapping the formed 14CO2 in NaOH. Within few days, significant amounts of the total applied radioactivity could be recovered in the CO2 traps, indicating a fast mineralization. A mixed consortium revealed even higher rates of mineralization than those observed in the resepective axenic cultures. Analysis of the culture media after incubation revealed the presence of a peak in one axenic culture which was not present in other ones nor in the mixed culture. Currently we are investigating the nature of the metabolite. A better understanding of sulfamethoxazole degradation by bacteria may contribute to the optimization of biological wastewater treatment plants for the efficient removal of pharmaceuticals.

P3 ANAEROBIC TREATMENT OF MEZCAL VINASSES IN FLUIDIZED BED BIOREACTORS V. Robles-González1, J. Galíndez-Mayer2, N. Ruiz-Ordaz2, E. López-López2, F. Martínez-Jerónimo2, A. Ortega-Clemente3, N. Rinderknecht-Seijas4, Héctor M. Poggi-Varaldo*5 1

3

Universidad Tecnológica de la Mixteca, Agroindustr. Inst., Huajuapan de León, Oax., México; 2ENCB del IPN, Mexico ITBR, Ver., México; 4ESIQIE del IPN, Div. Ciencias Básicas, México; 5Environmental Biotechnology and Renewable Energies Group, Dept. Biotechnology and Bioengineering, CINVESTAV del IPN, México D.F., 07320, Mexico, e-mail: [email protected]

Abstract Mezcal distilleries are one of the most polluting industries in Mexico generating large volumes of A very aggressive wastewater called mezcal vinasse (MV). Anaerobic digestion has been one of the most employed system for vinasses treatment because of low operational costs, aeration savings, low sludge production and the generation of by-products as methane gas. In the present work the anaerobic digestion of mezcal vinasse in fluidized bed bioreactors was evaluated. Mesophilic lab scale fluidized bed bioreactors (AFBBR) were used. MV (pH adjusted to 7.0) was fed to the AFBBR with five steady states with decreasing hydraulic retention times (or increasing organic loads, i.e., 1.96, 2.73, 5.70, 10.70 and 30.40 gCOD/LFB*d). Wastewater analyses were performed according to Standard Methods (APHA, 1998); total aromatics compounds (TAC) and total phenolic content were determined according to literature. Organic matter removal was in the range 62-85%. As expected, it decreased with increasing loading rates. At an organic load of 10.70 gCOD/LLF*d the methanogenic regime in the AFFBR began to deteriorate (decrease in COD removal efficiency (60-70%) and increase in the parameter alpha (from 0.4 to 0.6-0.7). Also, methane content in biogas diminished to 48.9 from 82.9%. Removal efficiencies of total aromatics compounds and phenols were within the ranges 67.8% and 76.3% respectively. The anaerobic digestion achieved moderate-to-high contaminant removal efficiencies of COD as well as recalcitrant and toxic substances reductions such as TAC and phenols. However, it still remained a fraction of undigested compounds, since the COD removals should be compared to 85% degradability of raw mezcal vinasses. Microorganisms sometimes lack the enzymes necessary for complete biodegradation of recalcitrant compounds in conventional biological treatment. High values of alpha parameter correlated well with poor performance of the AFBBR at the highest loading rates; the alpha parameter could be a valuable warning tool of process (methanogenic) deterioration. Key words: anaerobic digestion, fluidized bed reactor, mezcal vinasses

S7

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S1-S20

P4 CARBOXYLATION IS THE INITIAL ENZYME REACTION IN THE ANAEROBIC DEGRADATION OF THE POLYCYCLIC AROMATIC HYDROCARBON NAPHTHALENE H. Mouttaki, R.U. Meckenstock Helmholtz Zentrum München, Institute for Groundwater Ecology, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany

Abstract Polycyclic aromatic hydrocarbons such as naphthalene are highly recalcitrant environmental pollutants that are only slowly metabolized by microorganisms under anoxic conditions. The anaerobic biochemical attack on such extremely stable molecules is unknown but based on metabolite analyses of supernatants of enriched and pure anaerobic cultures, carboxylation or methylation have been proposed as initial naphthalene reactions. However, the extremely tedious growth of such cultures has prevented in vitro studies and the identification of the initial activation reaction, so far. Here, we provide biochemical evidence that anaerobic naphthalene degradation is initiated via direct carboxylation. Crude cell extracts of a sulfate-reducing enrichment culture N47 converted naphthalene and 13C-labelled bicarbonate to 2[carboxyl-13C]naphthoic acid at a rate of 0.12 nmol min-1 mg-1 protein. Divalent metal ions or ATP were not required nor did avidin or EDTA inhibited the carboxylase activity. The enzyme, namely naphthalene carboxylase, catalysed a much faster exchange of 13C-labelled bicarbonate with the carboxyl group of [12C]2-naphthoic acid at a rate of 3.2 nmol min-1 mg-1 protein, indicating that the rate limiting step of the carboxylation reaction is the activation of the naphthalene molecule rather than the carboxylation itself. The new carboxylation reaction is unprecedented in biochemistry and opens the door to understand the anaerobic degradation of polycyclic aromatic hydrocarbons which are among the most hazardous environmental contaminants.

P5 REGULATION OF STYRENE CATABOLISM IN Pseudomonas fluorescens ST: FUNCTIONAL CHARACTERIZATION OF THE styS SENSOR KINASE Francesco Massai, Giordano Rampioni, Paolo Ascenzi , Livia Leoni, Elisabetta Zennaro Department of Biology, University “Roma Tre”, Rome, Italy

Abstract The regulation of styrene catabolism in Pseudomonas fluorescens ST represents a model to understand how the expression of a peripheral catabolic pathway for the degradation of a toxic compound could be integrated into the central metabolism. In P. fluorescens ST the expression of the styrene degradation operon, styABCDE, is induced in the presence of styrene and repressed by the simultaneous presence of preferential carbon sources, such as glucose. The promoter of styABCDE, PstyA, is under the control of a two-component system consisting of the hybrid histidine kinase (HK), StyS, and its cognate response regulator (RR), StyR. According to the current model, styABCDE styrene-induced transcription is repressed when cells accumulate high levels of phosphorylated StyR (StyRP), as it occurs in the presence of glucose. The levels of StyR-P are modulated by StyS. StyS contains two putative PAS sensing domains and two complete HK modules. This structural complexity leads to the hypothesis that StyS could integrate two different stimuli, one related to the presence of styrene and a second one representative of the energetic state of the cell, thus determining adequate levels of StyR-P and, ultimately, PstyA activity. This study represents a first step toward the structural and functional characterization of StyS and of its interaction with StyR. In vitro autophosphorylation assays have been performed on the full-length StyS protein and on each isolated HK domains (N-terminal HK1D and C-terminal DHK2, respectively). Results showed that styrene exerts a positive effect on the phosphorylation activity of StyS and HK1D by decreasing the dephosphorylation rate of the kinase, and pointed out that styrene is directly perceived by the N-terminal PAS-1 domain. Consistently with the experimental failure of DHK2 to autophosphorylate in vitro, we propose that the phosphoryl group is provided to the C-terminal domain by the N-terminal histidine kinase, through an internal phosphorelay mechanism. Transphosphorylation assays demonstrated that StyS is able to transfer a phosphoryl group to StyR, most likely through the C-terminal kinase domain. Therefore, the reduced dephosphorylation rate observed for HK1D, with respect to the full-lenght StyS, strongly suggests that the Cterminal kinase domain may play a main role in modulating the phosphorelay rate, hence in determining the levels of StyR-P. In this view, the activity of StyS could be determined by a still unknown stimulus, perceived by the PAS domain of HK2 and probably related to the redox status of the cell, in accordance with our current model of styrene-catabolism regulation.

S8

Sub-session 1.1 - Bacterial biodegradation of organics and inorganics

P6 INVESTIGATION OF NANOPARTICLES AS POTENTIAL ACTIVATORS FOR THE OPTIMIZATION OF PAH BIODEGRADATION T. Masy, W. Wannoussa, S. Hiligsmann, P. Thonart Bio-industries, Gxabt-ulg, Gembloux, Belgium

Abstract These last decades, through the industrial development and its imperfect waste management, the contamination by hydrocarbons in water and soils has led to a progressive deterioration of environmental quality, which is more and more considered nowadays. Amongst all the existing cleaning-up techniques, the bioremediation appears as the best compromise between treatment costs and effectiveness. However, this bioprocess remains time-consuming, especially for persistent pollutants as PAHs (Polycyclic Aromatic Hydrocarbons). A first way to improve the biodegradation consists in adding a consortium of efficient microorganisms in the polluted area (bioaugmentation). A further way to improve the bioremediation rate is based on the use of nutrients to assist the microbial metabolism (biostimulation). Our project gathers these two methods, specifically for the PAH biodegradation of polluted soils. Firstly, different suitable strains from our lab will be compared together in terms of PAH-degrading rate, in order to select the best microorganisms. As all these strains were selected from a long-term oil-polluted dried soil, they should be able to compete against the endogenous microflora, even if they are injected in the soil in a powdered starter. Secondly, trace elements in the nanoparticulate form, with concentrations of about 10-5M, will be added to catalyze the bacterial metabolism. First results already showed a sharp increase (2 to 3 fold) in the biodegradation kinetics, which is promising for the further scaling-up stages. In addition, this PhD project attempts to understand the mechanism of interaction between bacteria and nanoparticulate catalysts.

P7 BIOAUGMENTATION POTENTIAL OF Novosphingobium SP. PP1Y IN NATURAL AND ARTIFICIAL SOILS CONTAMINATED BY PAHS AND HEAVY METALS V. Cafaro, E. Notomista, V. Izzo, L.Troncone, G. Donadio, P. Tedesco, A. Di Donato Université de Caen Basse-Normandie - UR ABTE - F-14032 Caen, France

Abstract Polycyclic Aromatic Hydrocarbons (PAHs) are among the most widespread organic pollutants in the environment. PAHs are of great concern because some of them show strong mutagenicity and carcinogenity. Bioremediation, which involves the use of microorganisms to reduce the concentration of various chemical pollutants, has become nowadays one of the most promising approach for environmental clean-up. Unfortunately, the presence of heavy metals in environments contaminated by organic compounds often hamper the environmental restoration. Several species of bacteria have been isolated from polluted environments able to degrade recalcitrant compounds. We have recently characterized a novel Sphingomonadales, Novosphingobium sp. PP1Y, isolated from surface seawater samples collected inside the harbour of Pozzuoli (Naples, Italy). The strain PP1Y not only uses a surprisingly large number of mono and polycyclic aromatic compounds as the sole carbon and energy sources but it shows a very interesting and effective adaptation to grow on complex mixtures of aromatic compounds dissolved in oil phases like diesel-oil and gasoline. Novosphingobium sp. PP1Y showed the ability to form “biofilm” on several types of hydrophobic surfaces and, in water/oil systems. It is well known that biofilm formation make bacteria able to adhere to growing substrates and to confer the resistance towards heavy metals and organic pollutants. We investigated the biodegradation abilities of Novosphingobium sp. PP1Y in natural soil and compost, artificially contaminated by PAHs and heavy metals such as nickel, copper and zinc, in small-scale experiments. N. sp. PP1Y showed the ability to quickly remove phenanthrene from the compost after addition of the organic compound in crystal form or dissolved in oil phase. The bacterium was also able to metabolize phenanthrene in the presence of heavy metals, showing the most significant delay in phenanthrene removal only at high nickel concentration. When a mixture of PAHs in paraffin containing naphthalene, phenanthrene and pyrene, was added to the compost, the bacterium was able to remove all the three compounds, although with different rates. Furthermore, the addition of N. sp. PP1Y to a natural soil contaminated by heavy metals and PAHs, already endowed with a microbial community adapted to use PAHs in the presence of heavy metals, halved the time of natural self-purifying of the soil, thus suggesting the ability of N. sp. PP1Y to compete with the adapted endogenous microbial community.In conclusion Novosphingobium sp. PP1Y is a good candidate for the bioremediation of organic compound contaminated soils in the presence of heavy metals.

S9

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S1-S20

P8 AN EXTENSIVE ANALYSIS OF THE HYDROCARBON DEGRADING ABILITIES OF BACTERIA BELONGING TO THE SPECIES Acinetobacter venetianus R. Fani1, M. Fondi1, I. Maida1, E. Perrin1, M.C. Papaleo1, G. Emiliani2, M. Galardini1, K. Mara1, F. Decorosi3, C. Viti3, L. Giovannetti3, F. Baldi4, A. Mengoni1, V. Orlandini1 1

Department of Evolutionary Biology, University of florence, Florence, Italy; 2Tree and Timber Institute, National Research Council, Florence, Italy; 3 Department of Agricultural Biotechnology, University of Florence, Florence, Italy; 4Department of Environmental Sciences, Cã foscari University, Venezia, Italy

Abstract In recent decades, accidental oil spills are one of the major sources of environmental pollution. Biodegradation is an efficient, economic and versatile method to detoxify a polluted environment. Indeed, the efforts of researchers are focused on oil bioremediation and, in particular, on the characterization of those microbial consortia able to growth in oil-contaminated soil and on their possible utilization in the treatment of such polluted environments. The aim of the present study was to characterize six Acinetobacter venetianus strains able to use diesel fuel oil as sole carbon source by integrating data coming from physiological experiments and whole genome sequencing. Data obtained showed that all the strains are hydrophobic both in LB and in MMV exception for the A. venetianus VE-C3 strain that resulted hydrophilic only in LB, suggesting the use of different strategies to adhere to pollutants by the representatives of this species. The production of emulsifying molecules was also tested, revealing that the two strains, out of the six strains tested in this work display an emulsifying activity. To gain deeper insights in the phenotypic traits of these strains, their utilization of different carbon and nitrogen sources was checked by phenotype microarray analysis, revealing (in some cases) deep differences and suggesting an unexpected metabolic heterogeneity. These physiological properties led us to choose these strains as candidates for the determination of their genome sequences in order to gain more insights into those genetic traits involved in the degradation of n-alkanes and the generic features representative for the species A. venetianus. Genome sequencing was performed with both Illumina and Roche 454 technologies and preliminary analysis of genome assemblies allowed us to identify important differences among all A. venetianus strains thus revealing the possible genetic basis of observed different phenotypic traits. Furthermore, the analysis of the emulsan gene cluster (embedding the genes responsible of the bioemulsifying activity) it was shown that the two strains RAG-1T and LUH7437 share a similar array of genes, thus explaining their similar emulsifying activity. The whole body of data obtained showed that these strains afford an opportunity for analysis and manipulation of genes and gene products related to bioremediaton, opening new possibilities for their large scale industrial application.

P9 BIODEGRADATION OF ETBE AND BTEX BY A BACTERIAL CONSORTIUM V. Gunasekaran, L. Stam, M. Constantí University Rovira i Virgili, Tarragona, Spain

Abstract Ethyl tert-butyl ether (ETBE) is one of the commonly used fuel oxygenate in Europe to increase the octane number in order to achieve complete combustion of automobile fuel to control air pollution. Accidental spillage of this compound can pollute soil and water as ETBE is a highly water soluble compound leaving an unpleasant odour and taste to drinking water. Although ETBE is not extensively studied for its potential risks, Methyl tert-butyl ether (MTBE) which has similar properties to ETBE has been labeled as a potential human carcinogen. Therefore there is an urgent need to develop methods to eliminate ETBE from the environment. Bacterial degradation can be a promising option to achieve complete mineralization of this compound. Two microbial consortia namely A and B have been isolated and enriched from water samples highly contaminated with fuel oxygenate. Both consortium A and B were found to degrade 100 mg/l of ETBE to about 38% and 40% in 6 days. Consortium A is found to contain Microbacterium sp., Xanthomonas sp. through 16srDNA PCR and sequencing. Herbaspirillum sp., Pseudacidovorax sp., Arthrobacter sp., Pseudomonas sp., Xanthomonas sp. were found in consortium B. Apart from this, consortium A was tested for co-metabolic degradation together with BTEX compounds. Degradation studies showed that consortium A follows an order of preference with BTEX and ETBE together, with toluene recording the highest degradation of about 86% in 11 days. This is followed by Xylene at 80%, Benzene at 61%, and ETBE at 9%. This also implies that the presence of BTEX in the environment can inhibit the degradation of ETBE. Further investigations will be focused on versatile degradation capabilities of xenobiotic compounds for both of the consortium as well as for the enzymes which were highly expressed during degradation through proteomic studies.

S10

Sub-session 1.1 - Bacterial biodegradation of organics and inorganics

P10 MICROCOMS STUDY OF ANAEROBIC BIOCONVERSION OF HEXACHLOROCYCLOHEXANE IN HEAVILY CONTAMINATED SOILS Roberta Verdini1, Carmela Riccardi2, Lucia Pierro1, Marco Petrangeli Papini1, Mauro Majone1 1

Department of Chemistry, Sapienza University of Rome; 2INAIL, Research sector, Monte Porzio Catone, Roma

Abstract Hexachlorocyclohexane (HCH) is a highly chlorinated compound, mainly composed of four isomers (α, β, γ, and δ). It is classified as a priority organic pollutant due to its persistence and adverse effects on ecosystem and human beings. HCH has been heavily used as an organochlorine insecticide since 1940s, being commercially available in two grades: technical HCH and lindane. The technical grade HCH is composed of α-HCH (55-80%), β-HCH (5-14%), γ-HCH (8-15%), and δ-HCH (2-16%). Lindane contains more than 90% of the γ-HCH isomer and trace levels of other isomers. HCH low aqueous solubility, relative high stability, lipophilicity and chlorinated nature contribute to its environmental persistence and resistance to degradation. Here, the anaerobic biodegradation of HCH in heavily contaminated soils has been investigated by microcosm experiments under “natural” or enhanced conditions. The study was performed on two soil samples with a different concentration of HCH: The study was performed on two soil samples with a different initial concentration of HCH, about 1% w/w or 10% w/w, respectively. In both case, the α-HCH was the isomer present at the highest percentage In the microcosm experiments, several soil samples were put in contact with a liquid phase (mineral medium) inside of serum bottles, either containing a mineral medium only (the control test simulating “natural conditions”) or an electron donor too. As for the latter, either acetate, yeast extract or a solid phase biopolymer (polyhydroxybutyrate) were investigated. Each experiment was replied by adding an exogenous microbial consortium capable of anaerobic reductive dechloration of aliphatic chlorinated solvents (bioaugmentation). Overall, a set of over 50 microcosms was started up and monitored, by analysing both soil and liquid phases by extraction and GC/MS analysis, after 1, 2 and 6 months of incubation time (a sub set of microcosm experiments is going on for final analysis at 12 months). Preliminary results show that main metabolites of HCH degradation as PCCH, TCCH, TCB, DCB and CB were formed in all the samples, that indicates the presence of anaerobic bioconversion. On the other hand, the degradation of HCH was very slow, probably due to its very high initial concentration. For this reason, HCH concentration in the solid phase did not show a significant decrease after 6 month incubation. Furthermore, no significant differences were observed among microcosms, showing that the observed bioconversion was not enhanced by adding different electron donors o an exogenous microbial consortium. This suggests that the observed bioconversion (even limited) was due to microorganisms that were already present in the soils, probably due to their long-term acclimation to the HCH carbon sources and to the thereby available carbon sources.

P11 CHARACTERIZATION OF N-HEXADECANE-DEGRADING BIOSURFACTANT-PRODUCING- Acinetobacter SPP. ISOLATED FROM PETROLEUM HYDROCARBON POLLUTED SOIL P. Aguila1, S. Fuentes 1, C. Bravo2, H. Palma2, M. Seeger1 1

Laboratorio Microbiología Molecular y Biotecnología Ambiental, Universidad Técnica Federico Santa María, Valparaíso, Chile 2 Laboratorio de Química Orgánica y Forénsica Ambiental, Universidad Austral de Chile, Valdivia, Chile e-mail: [email protected]

Abstract Soil contamination by petroleum hydrocarbons is a worldwide problem, becoming a major environmental threat and a potential human health risk. In contaminated sites, bioremediation is an attractive alternative for decontaminating soils. In this study, 11 strains of Acinetobacter spp. were characterized. These strains were isolated from chronically oil-polluted soil at the Aconcagua river estuary bank (Región de Valparaíso, Chile). Low generation time, high growth rate, and the ability to grow in several hydrocarbons as sole carbon source, were the main selection criteria for further characterization. Degradation of n-hexadecane by strains codenamed 53, and 64 were tested. At 55 hours, strain 64 degraded 100% of 0,1% v/v n-hexadecane, whereas strain 53 degraded 75% at the same time. Strains 75, 78 and 80 had the highest emulsification index (E24): 70.0%, 72.4% and 64.7%, respectively. Additionally, in strains 75 and 78 were detected alnA gene, which encodes an outer membrane protein part of the biosurfactant called “alasan” described in Acinetobacter radioresistens KA53. Moreover, strain 80 showed properties similar to those described for “emulsan” like as an emulsion stabilizer and bioemulsifier power, this one biosurfactant was described in Acinetobacter venetianus RAG-1. As surface tension reduction and emulsifying power increase bioavavailability of hydrophobic compounds, these strains are potential candidates for use in bioremediation. This characteristic should act synergistically with catabolic abilities, thus increasing biodegradation in contaminated environments.

S11

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S1-S20

P12 ISOLATION AND CHARACTERIZATION OF BIOSURFACTANT-PRODUCING BACTERIA A.L. Olivas Tarango1, G.V. Nevárez Moorillón2, M.L. Ballinas Casarrubias2, B.E. Rivera Chavira 2, E. Orrantia Borunda 1 1

Research Center in Advanced Materials, México; 2University of Chihuahua, Chihuahua, México

Abstract Contaminants such as petroleum-derived hydrocarbons have been heavily released to the environment. Natural mechanisms of contaminant removal can take several years and bioremediation strategies have been proposed as alternative treatments. Biosurfactants are amphipatic molecules than have the ability to reduce the superficial and interfacial tension between immiscible liquids or liquid-air phase. The isolation of biosurfactant producing microorganisms from soil obtained form contaminated and uncontaminated sites, will allow the discovery of novel biosurfactants. The strategy for isolation of microorganisms can help on the isolation of bacterial strains that are not commonly associated with biosurfactant production. Therefore, the aim of this study was the isolation of bacterial strains able to produce biosurfactants from contaminated and uncontaminated soil, using different media for isolation. For primary isolation, samples were plated in agar plates with nutrient agar (NA) or tryptic soy agar (TSA) at different pH (5, 7, 9) and different temperatures (8, 32, 45°C). Also, samples were inoculated in EMB Agar, and M9 salt medium with 0.1% sodium benzoate or 1% cellulose as carbon source. Pure cultures were obtained from the inoculated plates, and were preliminary identified by Gram stain, catalase and oxidase. Biosurfactant production was identified by the drop collapse method. A total of 802 bacterial strains were isolated; those initially identified as belonging to the Pseudomonas group or Bacillus genus were not selected, because of previous and current studies in our research group including those bacteria. From the remaining bacterial strains, 40 were positive for the drop collapse test. Regarding the strategy for isolation, most of the strains (68%) were isolated from contaminated sites (automobile workshop and mine waste). Most of the strains were isolated at basic and neutral pH at 8 and 32°C and the isolation was higher on TSA (42%) than on NA (26%). Micrococcus, Rhodococcus and Enterobacter were the most common genus found. The strains belonging to the genus Micrococcus had the best results in emulsion index (60-70%) and surface tension reduction (48-61 Din/cm2). Three strains were isolated in EMB at 32°C. Further studies will be undertaken to describe the biosurfactants that each microorganism produce.

P13 UTILIZATION OF CHITINOUS SUBSTANCES FOR THE OPTIMIZED BIOPRODUCTION OF ANTIFUNGAL CHITINASE BY Paenibacillus tylopili Maria Swiontek Brzezinska1, Urszula Jankiewicz2 1

Department of Environmental Microbiology and Biotechnology, Institute of Ecology and Environmental Protection, Nicolaus Copernicus University, Gagarina 9, Torun, Poland; 2Department of Biochemistry, Warsaw University of Life Sciences, Nowoursynowska 159, Warsaw, Poland

Abstract Chitin is the major source of natural organic compounds. This long biopolymer chain contains N-acetyl-D-glucosamine (GLcNAc) monomers forming covalent β-1,4 linkages. It is widely dispersed in the structural components of numerous organisms including crustacean and mollusc shells, arthropod exoskeletons as well as fungal cell walls. Chitin is created from marine food production waste, e.g. shrimp and crab shells or krill. Approximately 75% of the total weight of shellfish such as shrimp, crab and krill is considered waste, and chitin represents from 20% to 58% of the dry weight of this waste. Both degradation and recycling of chitin constitute an important phase in maintaining the global circulation of carbon and nitrogen. Chitinases play an important role in the decomposition of chitin and potentially in the utilization of chitin as a renewable resource, this is why they are widely applied. Four Bacillus strains were screened from soil and rhizosphere in the centre of Poland on chitin medium. They were identified as Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis and Paenibacillus tylopili. The identification was based on standard biochemical tests and analysis of the 16S rRNA gene sequence. The chitinase activity was detected after the second day: it increased gradually and reached its maximum after six days of cultivation. Shrimp shell waste was the finest inducer for the synthesis of chitinases. Under the experimental conditions tested, Paenibacillus tylopil was selected as the best enzyme producer. Chitinase was purified from a culture medium by fractionation with ammonium sulphate and chitin affinity chromatography. Purified proteins produced were subjected to identification by mass spectrometry. The molecular weight of the purified enzyme as determined by SDS-PAGE was approximately 55 kDa. The optimum temperature of the chitinase proved to be 45C. The enzyme was characterised by thermostability at 40C and 45C during 180 minutes of preincubation. The activity of the enzyme was strongly inhibited in the presence of Hg2+ and Pb2+ and stabilized by the ions Mg2+. Purified chitinase inhibited growth of fungal phytopathogenes: Alternaria alternata. Additionally, the crude chitinase inhibited the growth of potential phytopathogenes Penicillium purpurogenum and Penillium sp.

S12

Sub-session 1.1 - Bacterial biodegradation of organics and inorganics

P14 ELECTRICITY PRODUCTION AND NITROGEN REMOVAL FROM DIGESTATE BY MICROBIAL FUEL CELLS E.G. Di Domenico1, G. Petroni3, D. Mancini2, L. Di Palma2, A. Geri3, F. Ascenzioni1 1

Biology and Biotechnology “Charles Darwin” Department, via dei Sardi 70, 0018, Italy; 2Chemical Engineering Materials and Environment Department, via Eudossiana 18, 00184, Roma, Italy; 3Astronautic, Electric and Energetic Department, via delle Sette Sale 12/b, 00184, Roma, Italy; Sapienza University of Rome

Abstract The final product (digestate) from biogas power plant is a nutrient-rich sludge produced by anaerobic digestion and commonly used as a fertiliser. According to the EU Directive 91/676/CE, treatment of digestate may be mandatory prior its sparging over soil in order to reduce the impact that nitrogen has on the natural environment. Biological nitrogen removal can be achieved in two steps: nitrification (aerobic oxidation of ammonia to nitrite and nitrate) and denitrification (reduction of nitrate to nitrogen gas). More recently, the concept of nitrogen removal has been changed since the confirmation of the anaerobic ammonium oxidation (anammox), where autotrophic oxidation process converts ammonia to N2 using nitrite as the electron acceptor. It has been shown that nitrification and denitrification can occur into the microbial fuel cell (MFC) cathodic compartment leading to simultaneous nitrogen and organic carbon removal. The goal of our study was the simultaneous removal of carbon and nitrogen in MFC reactors fed with digestate produced in the anaerobic codigestion of an undefined mixture of agricultural wastes and cow manure (Azienda Agrozootecnica Bruni Sutri - VT). We assembled two-chamber reactors separated by cation exchange membrane; the anodic chamber was fed with the liquid phase of the digestate whereas catholite was inoculated in the cathodic compartment. Two reactors have been assembled: I) pre-conditioned MFC, containing a Geobacter sulfurreducens bioanode; II) un-conditioned MFC, containing a sterile anode. The anodic and cathodic electrodes were connected through an external resistor. The reactors have been operated under batch feeding for two months during which they were monitored daily. Following feeding of the anodic chamber of the pre-conditioned reactor, there was a rapid increase of electricity production (up to a maximum power transfer of 0.6 mW–240.2 mW/m2-at 346.8 mV). At the same time, the unconditioned reactor did not show appreciable electrical activity. Nonetheless, following a 28 days lag phase (most probably the time required for electrode colonization by anode-respiring bacteria) the un-conditioned reactor showed electricity generation as well (the maximum power transfer reached 0.4 mW–172.2 mW/m2-at 359.4 mV). The electrochemical activity of the anodic biofilms and the redox potentials were established by cyclic voltammetry. Additionally, chemical oxygen demand and nitrogen removal efficiencies in both reactors were also determined.

P15 APPLICATION OF AN ELECTROBIOCHEMICAL SLURRY REACTOR FOR THE TREATMENT OF A SOIL CONTAMINATED WITH LINDANE B. Camacho-Pérez1, A.L. Vazquez-Larios1, E. Ríos-Leal2, J. Garcia-Mena3, O. Solorza-Feria4, N. Rinderknecht-Seijas5, H.M. Poggi-Varaldo1,* 1

Environmental Biotechnology and Renewable Energies R&D Group, Dept. of Biotechnology and Bioengineering, CINVESTAV del IPN, P.O. Box 14-740, 07000 México D.F., México; 2Central Analítica, ibídem; 3Dept. of Genetics and Molecular Biology, ibídem; 4Dept. of Chemistry, ibídem; 5ESIQIE del IPN, Mexico

Abstract Lindane is a chlorinated pesticide known for its toxicity persistence in the environment. Recently, it has been proposed that soil microbial fuel cell technology (SMFC) could be applied to enhance the removal of organic matter, phenol, and petroleum hydrocarbon in contaminated soil and simultaneous electricity output. The purpose of this research was to enhance biodegradation of lindane and simultaneously electricity generation on electrobiochemical slurry reactor (EBCR). The electrobiochemical slurry reactor consisted of a Plexiglass cylinder approximately 6 cm in diameter and 8 cm in height (308 mL capacity) and was inoculated with a sulfate reducing inoculum acclimated to lindane. The internal resistance (Rint) of EBCR was calculated as a function of cell voltage using electrochemical impedance spectroscopy (EIS). The EBCTR was batch-operated for 30 day at room temperature. Results from EIS showed that the equivalent circuit obtained from the Nyquist plot had anodic resistance R1=2064Ω, cathodic resistance R3 = 192 Ω; and electrolyte/membrane resistance R2 = 7Ω; so the total internal resistance Rint was 2263Ω. The EBCR showed a 30% lindane removal efficiency along with a maximm volumetric power volumetric of 165 mWm-3. Organic matter removal was 72% . After 30 d operation lindane metabolites were not detected in the EBCR. It was reported a removal of 90% of initial 80 mg/L phenol (10 d operation) from a rice pad soil using a fuel cell. The latter developed a voltage of 530mV and a power density of 29.5 mW/m2 cathode surface. Our results were relatively lower, although it has to be considered that the log of octanol water partition coefficient of phenol is 1.46 whereas that of lindane is 3.6, that is, lindane is less bioavailable, more toxic, and much less soluble than phenol. Also, a great variety of bacteria has been reported to use phenol as carbon and energy source, whereas lindane is less biodegradable. Finally, it can be concluded that our EBCR showed a low-to-moderate lindane removal capability.

S13

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S1-S20

P16 REMEDIATION OF OILS SPILL IMPACTED SOIL BY BIOAUGMENTATION WITH FREE LIVING NITROGEN FIXING BACTERIA Josefina Pérez Vargas1, Sergio Vigueras Carmona1, Noemí Araceli Rivera Casado2, Graciano Calva Calva2 1

Tecnológico de Estudios Superiores de Ecatepec, División de Ingeniería Bioquímica. Av. Tecnológico s/n. Col. Valle de Anáhuac, Ecatepec de Morelos, Edo. México; 2CINVESTAV-IPN, Biotecnología, Av. IPN 2508, México D. F.; e-mail: [email protected]; [email protected]

Abstract The oil industry in the state of Tabasco Mexico causes a yearly average of 114 environmental contingencies due to oil spills or leaks. Also faces risks of oil and oily water spilling, leaking and filtrations derived from operational accidents, insufficient maintenance or pipeline corrosion due to rains and prolonged floods. The presence of petroleum hydrocarbons in oil spill impacted sites has important impact in the environment and human health. Oil spills and leaks, drilling wastes and oily water alter the chemical, physical and biological properties of soil. Bioaugmentation is a bioremediation technology useful for the treatment of these impacted sites by increasing the microbiota present in the contaminated soil by supplying exogenous or increasing the native microorganism. The objective of this work was to investigate the effect of augmentation the native free living nitrogen fixing bacteria (FLNFB) in combination with the addition of a biosurfactant produced by two of these bacteria over the bioremediation of soil contaminated with 120,000 ppm of total oil hydrocarbons in a biopile system. The total microbial account (UFC) previous to the inoculation with the FLNFB was 13 X 104 UFC, and after the inoculation was 200 X 107 UFC. Curiously, during the first two months of treatment, the UFC gradually decreased close to the initial value. Therefore more bacterial biomass was added to the system. After 6 months the UFC showed continuous augmentation until 22 X 1011 UFC after 16 months. By three months of treatment in the biopiles added with the FLNFB the hydrocarbons content was almost unchanged, however after 6 months the concentration decreased notoriously, and by 16 months the total hydrocarbon removal was 80%. This is the first experiment performed in real condition for these bacteria native from the contaminated sites. Interestingly, these bacteria showed three important abilities: to fix nitrogen, to remove hydrocarbons, and to produce biosurfactant compounds. These results offer the opportunity to study the physical, chemical and biological interaction between the soil and microorganisms, beside the possibility to propose a bioremediation technology for oil spill impacted sites.

P17 APPLICATION OF NITRIFYING BACTERIA AS AN INDIRECT APPROACH TO REDUCE STRESS OF TRANSPORTED ZEBRAFISH Anusha K. S. Dhanasiri, Jorge M.O. Fernandes, Viswanath Kiron Faculty of Biosciences and Aquaculture, University of Nordland, Bodø, Norway

Abstract Deterioration of water quality mainly due to the accumulation of fish metabolic wastes, including ammonia, during transport in closed containers can cause severe stress to fish. This is a major welfare issue, particularly for ornamental fish that are traded in large numbers. It is therefore important to keep the water quality in check, so as to reduce the stress associated with transport. During the present study we have examined if the nitrifying bacteria can function as novel bioremediation agents to lessen the harmful effects of ammonia accumulated during transport. For this purpose firstly we applied two commercial nitrifying bacterial consortia in the transport systems to understand their capacities to reduce the accumulating ammonia. The efficacy of nitrification process was evaluated based on the water quality parameters and the community structures of ammonia oxidizing bacteria (AOB). Secondly we applied the most efficient nitrifying bacterial consortia from above in the transport systems and study its effect on reducing the stress of transporting fish. Primary (cortisol) and secondary (glucose) stress responses as well as representative molecular markers of stress- genes involved in hypothalamic-pituitaryinterrenal (HPI) stress axis (crf, star and 11β hydroxylase) and corticosteroid signaling (gr) of fish prior to and after transport were assessed. Application of nitrifying bacterial consortia significantly improved the nitrifying activity that facilitated the removal of the ammonia accumulated in live fish transport systems. The diverse AOB populations observed were related to the difference in nitrifying activity. At the end of the 72h transport, the primary and secondary stress indicators were lower in fish that were transported with nitrifying bacterial consortia, demonstrating the efficacy of these microorganisms in indirectly reducing transport-associated stress. However, the expression of key genes involved in the HPI stress axis (crf, star and 11β hydroxylase) did not correlate with the stress indicators. Our data suggests that improved water quality achieved through bioremediation could alleviate the stress of zebrafish occurring from transport.

S14

Sub-session 1.1 - Bacterial biodegradation of organics and inorganics

P18 EFFECT OF REDOX MEDIATORS AND VARIOUS MEDIA ON THE DECOLOURISATION OF THE AZO DYE METHYL RED; AND ITS BIODEGRADATION BY Providencia rettgeri STRAIN ODO Olukanni Olumide David, Adekola Adejoke Adebimpe, Sunmola Damilola Banke, Osunmuyiwa Olufolabi Environmental Biotechnology Laboratory, Department of Chemical Sciences, College of Natural Sciences, Redeemer’s University, P.M.B. 3005 Redemption City, Ogun State, Nigeria

Abstract The discharge of coloured wastewaters into water-bodies is a serious environmental and public health problem. Developing efficient, effective and low cost procedure for wastewater treatment thus becomes expedient. A bacterium isolated from textile dye was identified using 16S rDNA as Providencia rettgeri. The organism decolourised various textile and non-textile dyes within 6 h at 100 mg/L. The prospect of redox mediators: quinol, nicotinamide adenine dinucleotide (NAD), reduced NAD (NADH), nicotinamide adenine dinucleotide phosphate (NADP) and reduced NADP (NADPH) to influence the decolourisation of the azo dye Methyl Red was investigated. Effects of various media on the decolourisation were also examined. The possible biodegradation of the model dye was investigated by subjecting the decolourised dye to UV-visible, high performance liquid chromatography (HPLC) and fourier transform infrared (FTIR) analyses. The organism preferred quinol to other redox mediators and nutrient broth to yeast, glucose, starch and peptone as media. The decolourisation time was reduced to 5 h. The results of the UV-visible and HPLC analyses suggested the biodegradation of the dye. This was confirmed by the removal of signature peaks of aromatic C-H bending (617, 704 and 834 cm -1) and the N=N peak at 1621 cm-1 observed when the FTIR of the dye was compared to that of its metabolite. The high Methyl Red decolourisation and biodegradation ability of Providencia rettgeri would enable this bacterium to be used in the biological treatment of industrial effluents containing azo dyes. Key words: biodegradation, decolourisation, Methyl Red, Providencia rettgeri, redox mediators

P19 BIOREMEDIATION OF AQUIFERS POLLUTED BY CHLORINATED ALIPHATIC HYDROCARBONS: SELECTION AND CHARACTERIZATION OF AN INDIGENOUS ICROBIAL CONSORTIUM FOR A PACKED BED REACTOR ON-SITE PROCESS G. Bucchi1, R. Ciavarelli1, F. Doria1, R. Salviulo1, G. Zanaroli2, S. Fraraccio2, D. Frascari1, D. Pinelli1, F. Fava2 1

Department of Chemical, Mining and Environmental Engineering, University of Bologna, Via Terracini 28, 40131 Bologna, Italy 2 Department of Civil, Environmental and Materials Engineering, University of Bologna, Via Terracini 28, 40131 Bologna, Italy

Abstract The aim of this work was to develop a high-performing microbial consortium capable to degrade trichloroethylene (TCE) and 1,1,2,2tetrachloroethane (TeCA) via aerobic co-metabolism in packed bed reactors (PBRs). To this goal, five groundwaters were sampled from different monitoring wells in an aquifer contaminated by TCE and TeCA, and 25 119mL batch tests were set up to test five different growth substrates: methane, propane, butane, pentane and phenol. Substrate and oxygen were periodically re-added. TCE and TeCA were spiked at increasing concentrations in order to select microorganisms able to rapidly degrade TCE and TeCA. Preliminary DGGE analysis showed appreciable differences in the microbial populations enriched. Butane was identified as the best-performing growth substrate, and a high-performing butane-growing consortium (B4) was selected for the subsequent continuous-flow tests. The selection was made on the basis of the substrate, TCE and TeCA specific biodegradation rates. Consortium B4 showed a specific rate of 129 L/gprotein/day for TCE and 2.6 L/gprotein/day for TeCA. The first value is comparable to the rates attainable in active carbons adsorption processes, with the great advantage of the total mineralization of the chlorinated hydrocarbons. The highest co-metabolized concentrations were 10 mg/L for TCE and 3 mg/L for TeCA. Specific tests aimed at developing and calibrating a kinetic model of substrate consumption and TCE/TeCA degradation are in progress. Attached-cell preliminary tests showed the ability of consortium B4 to form a stable biofilm on porous carriers. The attached biomass was able to utilize butane as carbon source and to biodegrade TCE and TeCA. In order to select the most suitable biofilm carrier for TCE/TeCA aerobic cometabolism by B4 in a PBR, 4 1-L glass columns were filled with 4 types of porous biofilm carriers. Fluid dynamics tests were performed with oxygen, TCE and TeCA in order to estimate the longitudinal dispersivity, the effective porosity and the TCE/TeCA retardation factor of each tested carrier. Four liters of a suspension of consortium B4 were produced in a fermentor in order to colonize the 4 glass columns. Continuous-flow tests of TCE and TeCA cometabolism by biofilms of consortium B4 are in progress, and will be utilized to select the best-performing carrier. Acknowledgment Project co-funding by the European Commission within the Seventh Framework Programme under Grant Agreement No. 265946 (Minotaurus project) is acknowledged.

S15

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S1-S20

P20 BIOSLURRY TREATMENT OF A CLAYISH SOIL POLLUTED WITH LINDANE BY SEQUENTIAL METHANOGENIC-SULFATE REDUCING BIOREACTORS B. Camacho-Pérez1, E. Ríos-Leal2, P. A. Vazquez-Landaverde3, J. Garcia-Mena4, J. Barrera-Cortés2, F. Fava5, N. Rinderknecht-Seijas6, Héctor M. Poggi-Varaldo1* 1

Environmental Biotechnology and Renewable Energies R&D Group, Dept. of Biotechnology and Bioengineering, CINVESTAV del IPN, P.O. Box 14-740, 07000 México D.F., México, e-mail: [email protected]; 2Dept. of Biotechnology and Bioengineering, Ibídem; 3CICATA-IPN, Qro., México; 4 Dept. of Genetics and Molecular Biology, CINVESTAV del IPN; 5DICAM, Unit of Environmental Biotechnology and Biorefineries, Faculty of Engineering, Universita di Bologna, Italy, 6ESIQIE del IPN, Mexico

Abstract Lindane is a recalcitrant organochlorine pesticide widely found in soils of Mexico. Slurry bioreactors (SBs) are successfully used for ex situ and on site treatment of contaminated soils and sludges (Robles-González et al., 2008). The goal of this research was to assess the effect of solvent (silicone oil, 0 and 20% v/v) on lindane removal from an agricultural soil with high contents of clay and organic matter, using lab scale batch sequential methanogenic-sulfate reducing SBs. Our matrix was a clayish soil with high contents of organic matter (8%), contaminated with 100 mg lindane/kg. Reactors were operated in a sequential mode (15 d methanogenic followed by 15 sulfate reducing, herein after sequential M-SR). Some units received 20% v/v silicone oil (letter S in the abbreviation) and were operated as triphasic SBs (soil-aqueous phase-solvent). All SBs were supplemented with sucrose (1g/L), bioaugmented with lindane-acclimated inocula, incubated, sampled, and analyzed as described in literature. The M-SR reactor without silicone oil showed 66% lindane removal efficiency. Unexpectedly, units added with solvent exhibited a lower removal (54%). The second stage (the SR) contributed the most to lindane reduction. Only 4.5% of lindane was removed in the 15-d M stage whereas the SR stage was responsible for 59% disappearance of the pollutant. After 30 d operation chlorobenzene and 1-2 dichlorobenzene were detected in the slurry of control III and M-SR-S. Abiotic removal of lindane was very low (Control I). Sequential M-SR-S reactors in our work showed performances superior to those reported for partially aerated methanogenic SBs treating the same soil (9.5 and 18% lindane reductions) and only methanogenic SBs (41%). In literature it was found a similar 62% removal of lindane after 60 d treatment of a sandy soil in batch methanogenic SBs, biostimulated with starch. Unexpectedly, our results were lower than those reported for sequential M-SR SB without silicone oil and without sucrose. Since the first stage methanogenic of SB operation contributed very little to lindane removal, it seems that the inclusion of a methanogenic stage in the process is not relevant or required. Lindane removal in control II (live soil and sterile lindane-clastic inoculum) suggested that the native soil microflora had a low-tomoderate capability to degrade lindane. Finally, we found there was no distinct, beneficial effect of silicone oil (solvent) on lindane removal from soil in sequential methanogenic-sulfate reducing SBs.

P21 PREDICTIVE EVALUATION OF BIOREMEDIATION POTENTIAL OF CHLORINATED SOLVENTS CONTAMINATED SITES B. Matturro, S. Rossetti IRSA-CNR, Via Salaria km 29.300, Monterotondo (RM) 00015, Italy

Abstract The extent of chlorinated solvents biodegradation in contaminated aquifers is critically dependent upon several factors, namely, the presence of contaminant degrading bacteria and the creation of optimal environmental conditions to stimulate biodegradative activity. The early estimation of the occurrence and abundance of contaminant key-degrading bacteria at field scale can therefore strongly assist the definition of the bioremediation potential as well as the decision making process prior to initiating bioremediation activities and the detailed site characterization. In this study, a wide screening was made on groundwater samples taken from several Italian chlorinated solvents contaminated sites to estimate the dechlorinators cell densities and activity. In particular, the analysis was made by means of a combination of biomolecular tools including in situ detection methods (CARD-FISH) for the accurate estimation of dechlorinators abundances and RT-qPCR for the expression analysis of reductive dehalogenase. Here, the outputs obtained by the application of the biomolecular tools were further compared with the results obtained by conventional treatability approach (i.e. microcosm study).

S16

Sub-session 1.1 - Bacterial biodegradation of organics and inorganics

P22 METABOLISATION OF CHLOROBENZOIC ACIDS BY PLANT-BACTERIA ASSOCIATIONS Blanka Vrchotová, Martina Macková, Tomáš Macek Institute of Chemical Technology, Prague, Faculty of Food and Biochemical Technology, Department of Biochemistry and Microbiology, Technicka 3, 166 28 Prague, Czech Republic

Abstract Chlorobenzoic acids, like other xenobiotics present in the environment, can be detoxified by biological systems, plants or microorganisms or consortia of both. In this work we tested degradation potential of plant-bacteria associations on soil contaminated by mixture of chlorobenzoic acids and compared this potential with degradation activity of plants or bacterial strains alone. Plants of black nightshade (Solanum nigrum) and strain A18 (Pseudomonas pseudoalcaligenes) or UH82 (Arthrobacter sp.) were used for testing. After half a year experiment, the amount of bacteria, concentration of individual chlorobenzoic acids in plant biomass and in soil was analyzed. In plant biomass all chlorobenzoic acids initially present in soil were determined in trace amounts. Amounts of bacteria were in all cases comparable. In soil seven from eleven added chlorobenzoic acids were in the end of experiment below detection limit. Detected were 2,3,5-tri; 2,3-di; 2,6-di a 2,4,6-trichlorobenzoic acid in the ascend order. Positive effect of bacterial strains addition on chlorobenzoic acids removal from soil was not unequivocally proved. On the other site presence of black nightshade plants had positive effect on chlorobenzoic acids disappearance from soil. Acknowledgements The work was sponsored by the grants ME 09024 and GAČR 525/09/1058.

P23 URANIUM REDUCTION BY BACTERIA M. Baiget1, M. Constantí1, F. Medina1, M.T. Lopez2 1

University Rovira i Virgili. Tarragona, Spain; 2Enresa, Spain

Abstract The use of microorganisms in bioremediation is a clean and environmental friendly method to decontaminate toxic compounds from different habitats. In this study, uranium (VI) dissolved in water has been reduced to uranium (IV) which precipitates, in order to decontaminate an aquifer from an inactive mining area. The bioreduccion has been studied through two approaches. On one hand, Shewanella was used and it reduced 50 mg/L uranium in 3h hours in synthetic contaminated water. However it does not reduce uranium in real contaminated water. On the other hand, the autochthonous microorganisms with reduction capacity were stimulated. Several electron donors were tried and lactate showed the best results. The combination of active coal and lactate increased the reduction of uranium. Further, the addition of iron nanoparticles to the abovementioned combination reduced uranium in 30 min.

P24 ISOLATION, SELECTION AND IMPROVEMENT OF A Methylobacterium spp. STRAIN FOR THE BIOREMEDIATION OF ANTHROPOGENIC ORGANIC COMPOUNDS V. Ventorino 1, F. Sannino 2, A. Piccolo 2, O. Pepe 1 1 DSA-Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy; 2DISSPAPA-Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy

Abstract The widespread environmental contamination due to the placing of large amounts of persistent chemicals in soil resulting from urban, industrial and agricultural activities, is a problem that is becoming increasingly important in the world. The technologies based on the use of microorganisms able to degrade the pollutant charge, allow to accelerate the natural detoxification processes in the soil environment and represent an alternative method of environmental remediation compared to the harmful traditional methods (incineration and chemical treatment). For this aim we isolated, characterized and selected 14 indigenous bacteria strains obtained from soil of the industrial site exACNA (Associate National Chemical Companies) in Cengio (Savona, Italy) contaminated by different classes of organic compounds (PAHs). The isolation was carried out using a minimal selective solid medium containing 0.5% of Contaminated Soil Aqueous Extract (CSAE) extracted from the soil mentioned above, with or without addition of natural nutrients (1% of soil extract obtained from freshly collected meadow soil). Further biotechnological selection allowed to detect a bacterial strain able to grow in solid medium containing 1.5% of CSAE as sole nutrient source. This strain was characterized and identified by phenotypic (morphological and biochemical tests) and molecular methods (16S rDNAs sequence analysis of ribosomal genes) as belonging to Methylobacterium spp. (97%). The ability of Methylobacterium spp. strain to grow in the highly contaminated habitat was enhanced using serially enrichment strategy in liquid media containing increasing concentrations of CSAE (up to 40%), with or without addition of nutrients (1% soil extract obtained from meadow soil). During enrichment experiments, the growth of the bacteria in the liquid culture was determined by spread plate count method using minimal solid media containing the same amount of CSAE. Moreover, to demonstrate the degradative capacities of Methylobacterium spp. strain, GC-MS analysis of the centrifuged broth cultures supernatant containing 40% of CSAE, was performed. Methylobacterium spp. was able to remove partially or completely some pollutants from liquid medium containing CSAE. These results demonstrate the effectiveness of selective ecological strategy that employs indigenous strains naturally present in highly contaminated soils, able to express their potential biodegradation of xenobiotic organic compounds of industrial origin and their potential use to remediate contaminated soil.

S17

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S1-S20

P25 BIOREMEDIATION WITH PROBIOTICS IN SHRIMP FARMING E. Mayer1, E.M. Gössl2, G.A. Santos1, M. Mohnl1 1

BIOMIN Holding GmbH, Industriestrasse 21, A-3130 Herzogenburg; 2BIOMIN Research Center, Technopark 1, A-3430 Tulln

Abstract Intensive aquaculture produces large amounts of organic wastes. Oxidation of these waste compounds facilitates the formation of toxic metabolites (H2S, NH3, NO2-) causing deteriorated water quality. This also changes bacterial composition in water and soil of ponds, increasing the presence of pathogenic bacteria, thus contributing to the occurrence of diseases in shrimp. The application of beneficial bacteria, probiotics and biodegrading microorganisms, to the pond water and soil (bioremediation) is a sustainable approach to minimize the environmental impact of aquaculture. NH4+- or NO3--removal processes (nitrification and denitrification) are essential for the pond water quality and can be carried out by nitrifying or denitrifying bacteria such as Nitrobacter, Thiobacillus and Paracoccus. The aim of the studies was to show that the strain Paracoccus pantotrophus (PP) 768 is able to reduce undesirable waste compounds and has a positive impact on pond soil quality. An in vitro study was conducted to test the biodegrading effect of PP 768 in two media: an aerobic buffer with 1 g/L yeast extract and 1 g/L sodium acetate as an additional carbon source, and without sodium acetate. The results showed that the heterotrophic PP 768 strain impaired the nitrogen cycle by using NO3- and other oxidized nitrogen compounds instead of oxygen and drastically reduced the amount of nitrogen compounds. The strain was able to degrade almost the total amount of 60 mg/L NO3- in the test solutions within 24 hours. Furthermore, sodium acetate in the medium led to decrease of 16 mg/L NH4+ to 7 mg/L within 48 hours. A field study using a commercial probiotic product (2 x 109 CFU/g) containing the strain PP 768 was conducted during intensive farming of white shrimp (Litopenaeus vannamei) to test the effects on soil quality under practical pond conditions. The trial was carried out for a period of 57 days with a dosage of 600 g/ha of product applied every 5 days. It has been confirmed that the ponds using bacterial strains showed better soil conditions (yellow soil) compared to the control ponds (black soil) and enhanced shrimp performance parameters. Average daily growth of shrimp in the probiotic treatment was improved by 36 % and FCR by 9 % when compared to the control. It has been shown that Paracoccus is able to control the above mentioned processes.

P26 COMPARATIVE GROWTH STUDIES OF THE HYPERTHERMOPHILIC ARCHAEON SULFOLOBUS SOLFATARICUS P2 ON SULFUR COMPOUNDS FOUND IN FOSSIL FUELS Gokhan Gun1, Yuda Yurum2, Gizem Dinler1 1

Istanbul Technical University, Molecular Biology and Genetics Department, Istanbul, Turkey; 2Sabanci University, Faculty of Engineering and Natural Sciences, Istanbul Turkey

Abstract All fossil fuels contain at least 200 kinds of organic sulfur compounds (benzothiophene, dibenzothiophene, their derivatives and variety of complex molecules) and inorganic sulfur compounds. To remove the sulfur compounds from petroleum or coal, hydrodesulfurization (HDS) carried out with chemical catalysts is not effective to completely remove heterocyclic organosulfur compounds such as dibenzothiophene (DBT), benzothiophene (BT) and their derivatives. Biodesulfurization, the use of microorganisms and/or enzymes for lowering the sulfur content of petroleum products, is an alternative process to HDS. It requires relatively low temperature and pressure. The process is also contributes both organic and inorganic sulfur removal in fossil fuels. In our study, an aerobic thermoacidophilic archaeon Sulfolobus solfataricus P2 that grows optimally at 80°C and pH 3.0 was used for biodesulfurization of organosulfur compounds. First, a sulfur free mineral (SFM) medium was developed and supplemented with various carbon sources containing arabinose, ethanol, glucose, mannose and mannitol to a final concentration of 0,2% w/v to find the most suitable carbon source for optimal archeal growth. After determining glucose as a carbon source in our SFM medium, we found the most effective glucose concentrations 2% w/v. Later, the ability of the S. solfataricus to grow on organic and inorganic sulfur sources such as DBT, dibenzothiophene sulfone, BT, 4,6-dimethyldibenzothiophene, sodium sulfite, sodium sulfate, potassium disulfite, and potassium persulfate was investigated under SFM and yeast-supplemented mineral medium (Brock’s medium). Addition of organic sulfur sources, such as 0,3 mM of dibenzothiophene sulfone, 4,6-dimethyldibenzothiophene and BT, to SFM after reaching to a moderate optical density (OD600nm=~0,4) caused an increase on the growth rate but DBT at this concentration ceased the S. solfataricus growth. Further investigations revealed the maximum DBT tolerance of S. solfataricus as 0,1 mM. We also investigated the growth behavior of S. solfataricus when inorganic sulfur compounds were supplemented both to SFM and yeast-supplemented media. We showed that 0,3 mM of sodium sulfite, sodium sulfate, potassium disulfite, and potassium persulfate caused a significant increase in the growth rate of S. solfataricus. Current studies are underway to determine the consumption rates of DBT and BT by S. solfataricus in a minimal and enriched growth media. The results of this work will enable us to determine the optimal growth and biodesulfurization conditions while studying coal samples. Acknowledgements This work is supported partially by the TUBITAK grant, 110M001, and Istanbul Technical University internal funds.

S18

Sub-session 1.1 - Bacterial biodegradation of organics and inorganics

P27 BIOREMEDIATION OF HARBOUR SEDIMENTS CONTAMINATED WITH ORGANIC COMPOUNDS: MICROBIAL RESPONSE AND BIODEGRADATION EFFICIENCY L. Rocchetti 1, F. Beolchini 1, P. Renzi 2, M. Gabellini 2, A. Dell’Anno 1 1

Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy; 2Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA), Rome, Italy

Abstract The management of contaminated dredged sediments is a widespread problem and there is a urgent need to develop low cost and environmental friendly strategies able to reduce the concentrations of pollutants at levels which can allow the sediment re-use. Such a need is exacerbated for contaminated harbour sediments periodically dredged to maintain the navigable depths. In this study we carried out different bioremediation experiments on dredged harbour sediments contaminated with hydrocarbons and organotin compounds (mainly tributyltin). In particular we investigated the effects of nutrient amendments (inorganic vs organic compounds), temperature (20°C vs 35°C) and redox conditions (aerobic vs anaerobic) on the metabolism, diversity and composition of bacterial assemblages in relation with their biodegradation performance. The nutrient amendments to the sediments significantly stimulated the growth rate of the microbial assemblages leading to an enhanced biodegradation of hydrocarbons. Similarly a higher temperature determined a significant increase of bacterial growth rates both in aerobic and anaerobic conditions. However, whereas in aerobic conditions this was associated with a significant increase of the degradation rates of hydrocarbons, in anaerobic conditions a significant decrease of hydrocarbon degradation rates in nutrient-enriched systems was observed. Although tributyltin (TBT) was more refractory to biodegradation than hydrocarbons, an increase of the concentrations of its congeners (i.e. DBT and MBT) was observed in a relatively short timescale of bio-treatment (few weeks). Independently by the experimental conditions significant changes of the biodiversity and composition of the bacterial assemblages (determined by different molecular tools) were found together with higher biodegradation performance associated with higher biodiversity levels. Overall our findings suggest that bioremediation strategies able to maintain over time high metabolic rates and high diversity levels of the bacterial assemblages can improve the effectiveness of the bio-treatments on dredged sediments contaminated with different types of organic pollutants.

P28 UTILIZATION OF A RESPIROMETRIC TECHNIQUE FOR THE AEROBIC TREATMENT OF INDUSTRIAL WASTEWATER I. Colussi, A. Cortesi, V. Gallo, R. Vitanza DI3, Dipartimento di Ingegneria Industriale e dell’Informazione, Università di Trieste, Via Valerio, 10–34127 Trieste, Italy

Abstract The aerobic biodegradation of different types of industrial wastewaters in an industrial activated sludge WWTP with preanoxic denitrification has been the aim of the present study. The considered wastewaters were produced from textile industries, dairy farming, landfill leachate, and composting. Aerobic biodegradation was studied by respirometric tests, being the respirometry a widely used technique for the characterisation of wastewater and activated sludge. Respirometry is the measurement and interpretation of the biological oxygen consumption rate under well-defined experimental conditions. Because respiration rate is directly linked to substrate removal and biomass growth, respirometry is an effective tool in the control of the activated sludge process. Respirometric test were carried out continuously measuring the dissolved oxygen concentration and pH and ORP values inside the reactor. Measured data were recorded by a data acquisition unit. A mathematical model based on the Activated Sludge Model No.1 was developed and calibrated using the experimental results from the lab-scale plant. Non linear differential equations of the model were implemented in a self-made software in order to obtain the biomass kinetic parameters and the COD fractionation in classes of biodegradability. The respirometric technique applied was helpful for the description of the microbial activity of the considered (heterogeneous complex) wastewaters/WWTP activated sludge systems. Consequently, useful suggestions about operating conditions were obtained, by which the obtained purification efficiencies for COD, ammonia and nitrates were about 60%.

S19

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S1-S20

P29 METABOLIC AND PROTEIN-PROTEIN INTERACTIONS OF SULFANILIC ACID CATABOLISM IN Novosphingobium subarcticum SA1 B. Hegedűs1, K. Perei1, M. Magony1, K. Laczi1, A. Tóth2, K.L. Kovács1,2, Rákhely G.1,2 1

Department of Biotechnology, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary; 2Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary

Abstract Novosphingobium subarcticum SA1 (Sphingomonas subarctica SA1, formerly identified as P. paucimobilis) alone is able to degrade sulfanilic acid. It was shown that the strain could catabolize five analogue aromatic compounds including protocatechuate, p-aminobenzoic acid, 4-hydroxybenzoate. The protein patterns of the strains grown on sulfonated and nonsulfonated molecules were distinct indicating alternative routes for the assimilation of these compounds. The genome of the strain was sequenced by new generation genome sequencers and revealed numerous genes of enzymes potentially catalyzing the oxidation of aromatic compounds. A genomic region containing the genes coding for sulfocatechol and protocatechuate dioxygenase were identified in distinct gene clusters. The analysis of enzymatic activity of cells grown on sulfo- or protocatechuate revealed, that the protocatechol and sulfocatechol pathways overlapped at the ring cleaving reaction, but the next step required different specific cycloisomerase enzymes. Investigation of the proteins appearing upon sulfanilic acid induction disclosed proteins likely involved in the sulfanilic acid transport, conversion as well as the iron transport. The oxidation of sulfanilic acid requires iron containing enzymes, therefore this would be reasonable that the increased iron demand of the dioxygenase enzymes would be provided by the induction of an iron transport pathway. A localization study of the proteins likely involved in the transport of amino group and ring hydroxylation pointed out that these enzymes were membrane associated and they appeared simultaneously. Recombinant enzymes were produced in homologous host and the proteins copurifying with either of these proteins were identified. The results suggest a membrane associated complex which is responsible for coupled uptake and conversion of sulfanilic acid.

P30 BACTERIAL BIOSYNTHESIS OF SELENIUM NANOPARTICLES BY ENVIRONMENTAL ISOLATES OF Stenotrophomonas maltophilia Silvia Lampis, Emanuele Zonaro, Chiara Santi, Anita Ferrari, Giovanni Vallini Department of Biotechnology, University of Verona, Verona, Italy

Abstract Five bacterial strains identified in soil samples collected at three dismissed industrial sites contaminated by heavy metals and metalloids such as lead, arsenic, and selenium were analysed for their capability to reduce the toxic oxyanion selenite (SeO32-) to the non-toxic zerovalent selenium. All isolates resulted taxonomically related to the Stenotrophomonas maltophilia species and capable of forming Senanopaticles (SeNPs). The strains labelled as A16, AW, B, T, and SeITE02 were obtained by means of enrichment cultures added with organic lead (A16), arsenite (AW, B, T), and selenite (SeITE02) respectively. Each isolate was investigated for the degree of resistance to selenite, the rate of selenite reduction to elemental selenium, as well as the formation of SeNPs and their localization within the bacterial cell. Strains SeITE02 and AW, able to reduce 0.5 mM SeO32- within 48 hours of incubation in rich medium, evidenced the highest efficiency in selenite reduction. Strain A16 was also efficacious in selenite reduction, showing the greatest reduction rate in early exponential growth. Nevertheless, it did not effect the whole reduction of the SeO32- amount initially added to the culture medium. Moreover, a pre-treatment with SeO32- at sub lethal concentrations (0.2 mM) positively affected both the rate and the efficiency of selenite reduction. This was particularly true for the strains SeITE02 and A16. All bacterial isolates were then found capable of producing selenium nanospheres. Localization of these SeNPs varied either with the strain or with the age of the bacterial cultures. In general, once reached the stationary phase, bacterial culture specimens showed extracellular SeNPs embedded in complex aggregates probably due to the concurrent production of EPS substances. In a biotechnological perspective, the strains S. maltophila A16, AW, and SeITE02 may thus be considered either for detoxification purposes in the treatment of seleniferous environmental matrices (e.g. soils, surface waters, and wastewaters) or for the controlled biosynthesis of nanostructured particles of interesting physic-chemical characteristics.

S20

Environmental Engineering and Management Journal

March 2012, Vol.11, No. 3, Supplement, S21-S34

http://omicron.ch.tuiasi.ro/EEMJ/

“Gheorghe Asachi” Technical University of Iasi, Romania

Sub-session 1.2. Microbial biodegradation assessment and improvement Main lecture ENGINEERING ENVIRONMENTAL CATALYSTS: FROM TRIAL-AND-ERROR TO SYNTHETIC BIOLOGY Victor de Lorenzo Systems Biology Program, Centro Nacional de Biotecnología CSIC, Madrid 28049, Spain

Abstract One key objective of Environmental Biotechnology is the pursuit of biocatalysts for conversion of industrial waste into value and –if no other choice is available, mineralization, detoxification or immobilization of man-made pollutants. Traditional Genetic Engineering based on one-at-a-time modifications of the DNA complement of an existing organism (i.e., deletions, mutations, cloning and expression of one or few genes in a heterologous host) is quickly giving way to the systemic refactoring of entire functional blocks in heavily engineered bacteria. Still, our current capacity of DNA synthesis á la carte (> 1 Mb) is much more than our competence to engineer complex phenotypes, which hardly goes beyond handling 10-20 genes (~10-20 Kb). To mine desired activities out of the existing catalytic landscape of the metagenome we have developed a number of unidirectional transducers for coupling biotransformations to transcriptional activation of a selectable property. With these tools, desired reactions can be reclaimed directly from environmental DNA based on their biological activity instead of DNA sequence similarity. The next step to exploit biological properties for biotechnological applications is framed in what is growingly denominated Synthetic Biology (SB). This increasingly inclusive concept [i] encompasses new theoretical frameworks that address biological systems with the conceptual tools and the descriptive language of Engineering, [ii] addresses old questions and challenges with fresh approaches inspired in electric circuitry and mechanical manufacturing and [iii] pursues the creation of new materials with á la carte properties based on the rational combination of standardized biological parts decoupled from their natural context. In fact, standardization and detailed description of minimal biological parts and their interfaces, to the degree of reliability of the components of modern electronic circuits is one of the trademarks of the whole field. The basic notion behind SB is that any biological system can be seen as a complex combination of functional, stand-alone elements not unlike those found in man-made devices, and can thus be-deconstructed in a limited number of components and reconstructed in an entirely different configuration for the sake of modifying existing properties or creating altogether new ones. In this context, Engineering as a discipline transits from being an analogy of the rational combination of genes made possible by modern Molecular Biology and Biotechnology to being a veritable methodology to construct complex systems and novel properties based on formatted biological components. Some applications of these concepts in the design of superior whole-cell catalysts will be discussed.

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S21-S34

Oral presentations MICROBIAL COMMUNITY STRUCTURE SHIFTS IN RESPONSE TO DIFFERENT BIOTIC AND ABIOTIC CONDITIONS Martina Mackova1*, Ondrej Uhlik1, Lucie Musilova1, Michal Strejcek1, Petra Prouzova1, Petr Stursa1, Eva Hoskovcová1, Petra Lovecka1, Cestmir Vlcek2, Jakub Ridl2, Miluse Hroudova2, Katerina Demnerova1, Jan Paces2, Tomas Macek1 1

Department Biochemistry and Microbiology, Fac. of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic; 2Department of Genomics and Bioinformatics,Institute of Molecular Genetics, Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic

Abstract Wide-spread use, improper handling, and disposal of certain synthetic organic chemicals have resulted in contamination of soils, waters, and sediments. Among the compounds released, polychlorinated biphenyls (PCBs) are of major concern due to their toxicity for certain organisms. In addition, PCBs volatilize and migrate to places far from the original release and pose a threat to human health not only in the neighbourhood of the contaminated area. One of the most ecologically-sound and inexpensive ways of removing PCBs from contaminated sites is bioremediation, which takes advantage of enzymatic capabilities of certain microorganisms (microbial bioremediation) and plants (phytoremediation) and/or their consortia (rhizoremediation) to transform these compounds. In this study, microbial community structure shifts were monitored in relation to different biotic and abiotic conditions. These included vegetation by different plant species (horseradish /Armoracia rusticana/, black nightshade /Solanum nigrum/, and tobacco /Nicotiana tabacum/), addition of fertilizers (N-P-K), and addition of allochtonous bacterial degraders isolated previously from a PCB-contaminated sediment. Several approaches were used to assess the changes in microbial community structure, including direct plate counting (using plate count agar and mineral medium with biphenyl as a sole carbon and energy source), pyrosequencing of 16S rRNA gene amplicons, and stable isotope probing. Our results show that the vegetation itself influences microbial diversity and metabolic degradation activity to a much larger extent than fertilization, which can be documented at the level of cultured bacteria, total communities, and metabolically active populations (as determined, respectively, by direct plate counting, pyrosequencing analyses, and stable isotope probing with 13C-labeled biphenyl as a substrate). This trend was especially obvious with horseradish and tobacco. At the same time, our experiments pointed out to a poor survival of allochtonous bacteria added into soil, either bulk or vegetated. In summary, obtained data can be beneficial for more effective designing of engineered bioremediation strategies based, for instance, on intrinsic microflora stimulation. Acknowledgement This work was supported by EU project MINOTAURUS within 7th Framework Programme (project no: 265946), the Ministry of Education, Youth and Sports of the Czech Republic (project ME 10041), and Czech Science Foundation No 525/09/1058).

GENOREM: IMPROVING BIOREMEDIATION OF POLLUTED SOILS THROUGH ENVIRONMENTAL GENOMICS G. Burger, F. Courchesne, C. Greer, M. Hijri, S. Jabaji, S. Joly, M. Labrecque, B.F. Lang, F. Pitre, M. St-Arnaud, E. Yergeau Sciences Biologiques, University of Montreal, Canada

Abstract Mining activities, oil and gas extraction, industrial processes, and most forms of modern agriculture all contribute to soil contamination, creating a significant problem world-wide. Efforts are underway to reduce the production of industrial and agricultural pollutants at their source, but this does not address the enormous legacy sites containing trace metals and organic pollutants that can remain in the soil. Pollution is not only threatening the quality of life in terms of human and environmental health, but also the future availability of essential resources, such as agricultural land and clean water. If we do not intervene promptly, repercussions on the ecological balance on our planet may be either irreversible or most costly to remediate. GenoRem project proposes an innovative phytoremediation approach to decontaminate polluted sites, employing scientifically controlled, most effective associations of plants, fungi and bacteria. Yet, the decontamination process itself is due to complex interactions of plants with soil microbes. Major objectives of the GenoRem project are therefore: (i) isolation of soil microbes that are most effective in decontamination, either by degradation of organic materials such crude oil, or in case of toxic trace metals, take-up and concentration by plants; (ii) developing combinations of the most effective plants fungal and bacterial symbionts, and other microbes, which will be outperform current phytoremediation practices without these selected microbes. A large-scale (0.5 h) phytoremediation trial was set up in summer 2011, in the field of an old oil refinery in Montreal region (Quebec, Canada), to submit 11 willow cultivars to a soil contaminated with a mixture of organic and inorganic pollutants. In total, about 10 000 trees were planted using split-plot design. We collected soil and root samples in May, August and October 2011 and isolated a broad rang of free living fungi and bacteria, arbuscular mycorrhizal fungi (AMF), endophytes and rhizosphere microorganisms. We used novel cultivation strategies such as high-throughput dilution-to-extinction on low nutrient content media amended with crude oil and other organic pollutants. Fungal and bacterial isolates have been molecularly identified using the ITS and 16S rRNA genes, respectively. Endophytes and AMF were trapped using indigenous plants. Metagenomics approaches were also used to investigate microbial diversity and community structures associated with willow cultivars and their analyses are in progress.

S22

Sub-session 1.2 - Microbial biodegradation assessment and improvement

GENE FOR DEGRADATION OF ORGANOMERCURIAL COMPOUNDS AND ITS APPLICATION TO BIOREMEDIATION OF MERCURY CONTAMINATION G. Endo1, M.F. Chien 2, M. Narita 3, C.C. Huang4 1

Engineering, Tohoku Gakuin University, Tagajo, Japan; 2Engineering, Tohoku Gakuin University, Tagajo, Japan 3 Technology Development, Tohoku Forestration and Environmental Protection, Sengdai, Japan; 4Life science, Chung hsing University, Taichung, Taiwan

Abstract Organomercury lyases (MerBs) are key enzymes in bacterial degradation and detoxification of organomercurial contaminants, and essential for bioremediation of environmental pollution caused by several types of organomercurials. MerBs are encoded by merB-genes in mer operons of environmental mercury-resistant bacteria. Each bacterial mer operon contains none to three merB genes. In this study, first, organomercury removal capabilities and specificities of Bacillus megaterium MB1, Bacillus megaterium MK64-1, Bacillus cereus RC607, Staphylococcus aureus RN23, and Pseudomonas sp. K-62 that are possessing merB gens were tested as wild-type organomercurial-resistant bacterial strains. Second, to investigate the substrates specificities of MerB enzymes, the different merB genes, i.e., merB1, merB2, and merB3 genes from B. megaterium MB1, merB1 and merB2 genes from Pseudomonas sp. K-62, and a merB gene from S. aureus RN23, were cloned into Escherichia coli with the mercuric ion reductase gene (merA) from B. cereus RC607 and the organomercury removal (volatilization) capabilities and specificities of the E. coli recombinants were investigated. Five chemical species of organomercurial compounds, i.e., methylmercury chloride (MMC), ethylmercury chloride (EMC), and phenylmercury acetate (PMA), thimerosal (TH) and p-chloromercuribenzoate (PCMB) were used as substrates for the removal by the wild-type mercury resistant bacterial isolates and the recombinant E. coli strains. The assay results showed that all Bacillus species used in the assay degraded and volatilized MMC, EMC, TH and PCMB rapidly, S. aureus RN23 volatilized MMC, EMC and TH but slightly volatilized PCMB, and Pseudomonas sp K-62 volatilized EMC, TH and PCMB but slightly volatilized MMC. MerBs cloning assay using E. coli strain showed that the merB1 gene from B. megaterium MB1 conferred the highest removal abilities against MMC, EMC, TH and PCMB, while the merB1 gene from Pseudomonas sp. K-62 conferred the highest removal ability against PMA. The recombinant E. coli strain that produced MerB1 protein of B. megaterium MB1 conferred the highest mercury volatilization abilities to MMC, EMC, and TH, while the recombinant strain that produced MerB3 protein of B. megaterium MB1 conferred the fastest mercury volatilization activity against PCMB. The recombinant strain that produced MerB2 protein of B. megaterium MB1 could slightly decompose EMC, TH, and PCMB but not MMC and PMA. These results suggested that there are organomercurial substrate specificities among these six MerB enzymes. The substrate specificities indicate importance of selection of the appropriate bacterial strains or merB genes to apply them in bioremediation engineering for cleaning up specific organomercurial pollution sites.

BIOREMEDIATION ASSESSMENT ON LINEAR ALKYLBENZENE-POLLUTED AQUIFER E. Martínez*1, A. Burniol2, G. Vidal3, A.M. Solanas1, M. Viñas2 1

Departament de Microbiologia, Universitat de Barcelona, Diagonal 645, E-08028 Barcelona; 2GIRO Centre Tecnològic. Rambla Pompeu Fabra, 1. E08100 Mollet del Vallès, Spain; 3D'ENGINY biorem S.L. Madrazo 68. E-08006 Barcelona

Abstract Linear alkylbenzenes (LABs) are used to manufacture alkylbenzene sulphonates (LASs), one of the most widely used anionic surfactants in domestic and industrial applications. An industrial site near Barcelona (Spain) whose groundwater was polluted following the rupture of an old storage tank was examined. The aim of the presentation is to offer an overview of the research related and the implementation of an in-situ bioremediation system. Phase I includes in vitro degradation tests under different redox conditions and in vitro chemical oxidation coupled to biodegradation. In phase II in-situ microbial community analysis using BioSep technology and in-situ bioremediation actuations were carried out. Oxygen and nitrate concentrations in groundwater suggested that denitrification processes may take place. Therefore, feasibility assays of bioremediation were carried out under aerobic, denitrifying and aerobic-denitrifying sequential conditions, at lab scale, with successful biodegradation of LABs. More than 40% and 90% of degradation were found under denitrifying and aerobic conditions, respectively. Additionally, in order to increase the degradation rates of LNAPL (hydrophobic and viscous), in vitro chemical oxidation assays were performed, coupled to bio stimulation. Sodium persulfate, potassium permanganate and a modified Fenton reaction were tested. In addition the capability of subsequent natural attenuation after chemical oxidation and the changes on the product bioavailability were also assessed. The results showed that chemical oxidation did not increase the degradation compared to enhanced natural attenuation (biostimulation). Anyway, the quantification of functional genes related to alkane and aromatic biodegradation pathways, such as alkB, tol, tod, bssA and phe was assessed to determine autochthonous microbial populations resilient to chemical oxidation for further biodegradation of LABs. To apply a bioremediation, it is necessary to demonstrate the in-situ occurrence of natural attenuation processes. Conventionally, monitored indicators of biodegradation include the distribution of contaminants, the occurrence of metabolic products, geochemical parameters and functional gene analysis. BioSep technology, a down-well aquifer microbial sampling system, was used to gain insight on microbial populations related to alkylbenzene biodegradation under in-situ groundwater environmental conditions, monitoring the groundwater microbial community dynamics during field bioremediation experiments. Main microbial populations and functional genes above described and denitrification-related-genes such nosZ gene were assessed by means of DGGE and quantitative PCR (qPCR). Lab tests and in-situ microbial communities exhibited changes significantly throughout biodegradation processes. Bearing in mind lab test and in-situ results, an in-situ actuation has been defined. A physical barrier has been placed to hold the LNAPL, whereas subsequent bioremediation will be applied downstream.

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Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S21-S34

BIODEGRADATION AND SORPTION OF 17-ETHINYLESTRADIOL IN SUBMERGED MEMBRANE BIOREACTOR: EFFECT OF INITIAL AMMONIUM CONCENTRATION Liza Bautista-Patacsil1,3*, Analiza P. Rollon1, Aileen P. Huelgas2, Jiangyong Hu3 1 College of Engineering, University of the Philippines Diliman, Quezon City 1101 Philippines; 2Department of Chemical Engineering, De Lasalle University Manila, Taft Ave, Manila, Philippines; 3Department of Civil and Environmental Engineering, National University of Singapore, 1 Engineering Drive 3,Singapore 117576

Abstract Batch experiments were done to assess the removal of 17-Ethinylestradiol (EE2) using sludge acclimated from a lab-scale submerged membrane bioreactor (SMBR). Though activated sludge (AS) was found to effectively remove EE2 from wastewater, membrane bioreactor showed a faster rate of removal. Results obtained revealed that EE2 was removed completely within 18hrs in SMBR while it took 96 hrs in AS. The removal of EE2 fits first-order rate reactions with a kinetic rate constant, k, of 0.96 d-1 and 6.96 d-1 for AS and SMBR, respectively. This showed that EE2 rate of removal using SMBR sludge is 7x faster than using AS. The role of nitrification in enhancing the removal of EE2 was investigated in terms of the effect of different initial ammonium concentration on both sorption and biodegradation. As the initial ammonium concentration is increased, biodegradation of EE2 is enhanced whereas sorption of EE2 onto the SMBR biomass is decreased. Sorption studies revealed that competition for sorption sites happened between ammonium ions and EE2. Sorption isotherms obtained revealed that physisorption is dominantly occurring with little chemisorption. The partitioning coefficients, KD were 0.31 L/gMLSS and 0.09 L/gMLSS for SMBR and AS, respectively. These results showed that EE2 adsorbed more to SMBR sludge than to AS. Results of this study suggest that MBR improves the biological removal of EE2.

INSIGHTS IN THE BIOREMEDIATION OF AROMATIC COMPOUNDS BY C. NECATOR Nathalie Berezina*, Bopha Yada Materia Nova R&D Centre, rue des Foudriers 1, 7822 Ghislenghien, Belgium, e-mail : [email protected]

Abstract Due to very important ecological problems induced by wild industrialisation during the XXth century, bioremediation is a very hot topic today. One of the interesting approaches to pollutants treatment can be their transformation to valuable molecules. Polyhydroxyalkanoates (PHA) in general and more particularly polyhydroxybutyrates (PHB) are well known chiral bio-products discovered by Lemoigne in 1925. PHB is a highly crystalline thermoplastic; it is highly biodegradable and sustainable product. Thus, the transformation of aromatic pollutants to aliphatic PHB seems to be a very attractive solution for this important issue. Some previous works showed that such a transformation can be accomplished by PHA producing microorganisms. We made a systematic study on the growth of C. necator on different aromatic compounds. Thus, we showed that the substitution degree of the aromatic compound is the most important point to be considered; indeed, only mono-substituted aromatic compounds are substrates for transformation by C. necator. Inside this family the transformation is easier when the oxidation degree is higher (benzoic acid transformation is faster than this of benzaldehyde and benzaldehyde transformation is more efficient than this of toluene). We have also established that two other different groups of compounds have to be considered: definitely toxic to the microorganism compounds and compounds, only toxic enough to create a stress situation which allows a more important PHA accumulation in cells. Finally we have also studied different aspects of this process. On a chosen model compound, benzoic acid, we made concentration studies in batch and continuous fermentations. Also we worked on the isolation of intermediate products to better understand what kinds of enzymes are involved in this process for their further possible application in biocatalysis.

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Sub-session 1.2 - Microbial biodegradation assessment and improvement

OPERATION OF HYBRID FLUIDIZED BIOREACTORS FOR ON SITE BIOREMEDIATION OF WATER POLLUTED WITH HIGH CONCENTRATION OF PCE Luz de M. Bretón-Deval1, J. Galindez-Mayer4, C.U. Moreno-Medina1, E. Ríos-Leal2, N. Rinderknecht-SeijaS3, Héctor M. Poggi-Varaldo1,* 1

Environmental Biotechnology and Renewable Energies Group, Department of Biotechnology and Bioengineering CINVESTAV-I.P.N; 2Department of Biotechnology and Bioengineering CINVESTAV-I.P.N; 3ESIQIE, I.P.N., México; 4Escuela Nacional de Ciencias Biologicas-I.P.N.; e-mail: [email protected]

Abstract Perchloroethylene (PCE) can be removed via biological processes like reductive dehalogenation and halorespiration, and by abiotic mechanisms such as zero-valent iron (ZVI) and zinc. Coupling these processes in bioreactors could be useful to treat effluents with high concentrations of PCE solubilized with Tween 80, for future applications to on site remediation of polluted groundwaters. Thus, the aim of this work was to evaluate the effect of increasing PCE concentrations (80, 165, 278 mg/L) on the performance of methanogenic and hybrid fluidized bed bioreactors. Two factors were tested, i.e., the PCE loading rate πv and coupling to ZVI filter. Four methanogenic fluidized bed bioreactors were implemented, two plain (MFBBR) and two bioreactors coupled to filters filled with particulate ZVI and sand (hybrid bioreactors or HFBBR). πv was set at 80, 165, 278 mg PCE/(L*d) (Periods 1, 2, and 3, respectively). Performance of bioreactors was evaluated as described elsewhere. Coupling to the ZVI filter in the HFBBR was associated to a higher PCE removal in Period 2 whereas in Period 3, PCE removal was poor for both types of bioreactors. There was either a negative effect of the increased πv or PCE plus increased Tween 80 concentration. Cconcentration of metabolites in the effluents, gas trap (volatilized PCE and metabolites) and sorbed onto bed bioparticles were lower in the HFBBR than in MFBBR in all periods. The main metabolite of PCE degradation found in the effluent of Periods 2 and 3 was vinyl chloride (VC). PCE and VC sorbed onto bed bioparticles increased with πv; this trend was consistent with the decrease of PCE removal at the highest πv. The HFBBR performance was satisfactory in the first two periods, highlighting the second period in which the PCE removal was significantly higher than that of MFBBR reactors, despite the high concentration of PCE (165 mg/L*d). These results are encouraging, since most research on bioreactors has been focused on lower concentrations of PCE. Performance of HFBBR deteriorated at the highest πv (278 mg/(L*d), although there is preliminary evidence that the bioreactors could recover with prolonged operation at that level. It can be concluded that HFBBR is a promising alternative for the on site treatment of waters contaminated high concentrations of PCE (up to 165 mg/L PCE). Key words: fluidized bed bioreactor, on site remediation, perchloroethylene, Tween 80

Posters P31 OIL PRODUCT DEGRADATION IN THE POLLUTED SOIL Elena Bocharnikova Phys-Chem Kab, Ipcbpss Ras, Pushchino, Russia

Abstract Petroleum hydrocarbon contamination is one of the main environmental concerns. At present, in situ oil- and oil product-remediation strategy is based on two main approaches: a) agricultural methods (addition of fertilizers and mechanical cultivation of polluted soil) and b) microbiological technologies (addition of hydrocarbon-degrading microorganisms). Our previous investigations and literature data showed that the application of the active forms of Si can benefit the soil remediation by the both methods. However, the mechanism by which active Si impacts oil hydrocarbon-polluted soil is not clearly understood. The greenhouse studies were conducted with Grey Forest Soil polluted by mixture of diesel and used motor oil at the concentration of 3%. The following treatments were applied: control with and without pollution, NPK, NPK+Si, Si, microbial commercial product, microbial product + Si. The diatomite mined in the Central Volga region was used as a source of active Si at the rate 10 t ha-1. Fertilizers and microbial product were applied at the rates accordingly recommendations. During 3 months, the dynamic of CO2 emission from soil surface and biomass of the barley was examined. The emission of the CO2 is correlated with soil breathing and microbial activity. In the end of the experiment, the total hydrocarbon content in the soil was analyzed. The obtained data showed that the application of all treatments enhanced the microbial activity and reduced the content of hydrocarbons. A maximum effect was examined for NPK+Si treatment and microbial product+Si. The content of hydrocarbons was decreased from 3% to 0.2 and 0.25%, accordingly for NPK+Si and microbial product+Si. The microbial activities at these treatments were increased 2.5 and 2.2 times as compared with contaminated control. The following mechanisms of the active Si action in hydrocarbon-polluted soil were supposed. Firstly, active Si can partly adsorb hydrocarbons thus reducing their negative influence on the soil-plant-microorganism system. The second mechanism is connected with increasing resistance of soil microorganisms and plants against stresses, including toxic impact of oil-products. By this means, the active Si forms can increase the efficiency of the technologies used for purification of oil- and oil product- contaminated soils.

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Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S21-S34

P32 DEVELOPMENT OF A BIOLOGICAL BIOBARRIER FOR IN SITU TREATMENT OF GASOLINE-CONTAMINATED GROUNDWATER Matteo Daghio1, Andrea Franzetti1, Isabella Gandolfi1, Giuseppina Bestetti1, Alessandro Careghini2, Laura Romele2, Sabrina Saponaro2, Elena Sezenna2 1

Department of Environmental Sciences, University of Milano, Bicocca, Milano, Italy; 2DIIAR-Environmental Section, Politecnico di Milano, Milano, Italy

Abstract New in situ remediation technologies are under development in order to treat groundwater contaminated by petroleum hydrocarbons. Among these technologies, permeable reactive barriers based on biological removal mechanisms (biobarriers - BBs) currently need labscale feasibility studies for optimal field applications. Among the different investigated filling materials, pumice showed the suitable physical-chemical properties affecting BB hydraulics and the highest sorption capacity of bacteria. Pumice was used in a lab-scale test aiming at simulating BB treatment in a wooden teflon-coated tank in which gasoline-contaminated water flew for 43 days of treatment. The concentration of the main contaminants in water and some chemical-physical parameters were monitored over time and at different distances from the water input in the BB. Denaturing Gradient Gel Electrophoresis (DGGE) analyses were also conducted on the filling pumice and at the end of the test to describe the development of the degrading microbial communities. The results of the chemical analyses showed a significant decrease in the steady-state concentrations of all compounds tested in the output column. DGGE indicated that the microbial communities were differentiated on the basis of their position along the BB. Particularly in the first part of the BB similar microbial communities developed and the main bacteria populations in this zone were Xanthomonadaceae and Comamonadaceae. Distinct, but similar among them, communities were found in the second part of the BB. In this part microorganisms belonging to Thauera sp. were dominant while Hydrogenophaga sp. microorganisms were evenly distributed along the BB.

P33

THE USE OF BIOLOG ECOPLATESTM TO MEASURE THE EFFECTS OF TEMPERATURE AND MOISTURE ON MICROBIAL COMMUNITIES IN ROADSIDE GULLY POT CONTENTS K.M. Scott 1*, T.J. Coulthard 1, J.D.W. Adams2

1

Department of Geography, University of Hull, Hull, HU6 7RX, U.K.; 2Environmental Technologies Centre for Industrial Collaboration, University of Hull, Hull, HU6 7RX, U.K.

Abstract Roadside gully pots are ubiquitous in urban drainage networks. They are primarily used to retain sediments from road runoff, leaves and organic litter, in order to avoid blockages or hydraulic restriction in the system, which may lead to flooding. Recent research investigating gully pot content activity ex situ indicated that they were relatively similar systems across space and time, allowing the contents to be evaluated universally. Although season did not prove to be a major issue when studying the activity of the contents ex situ, the microbial community of the contents in situ and how temperature and moisture impact upon it is yet unknown. This study is based on gully pots in the city of Kingston upon Hull, U.K. where blocked gullies were partially blamed for exacerbating the flooding in the city during the 2007 floods. Model gully pots were set up in a laboratory environment to replicate the activity of the contents in situ under controlled conditions, and were monitored over a six month period. Biolog EcoPlatesTM were used to evaluate the effects of different temperature (5°C, 16°C, 25°C and 30°C) and moisture levels (60% and 80%) on the microbial community of gully pot contents. The results showed over a six month period different moisture levels did not cause a change in the microbial communities. However, obvious differences in the microbial community were observed between temperatures, where increased temperatures showed an increase in the relative activity observed in the carbon utilisation profiles. These apparent temperature effects were observed throughout the six month observation period This study demonstrates that temperature has a more dominant effect on the microbial community of roadside gully pot contents in situ as opposed to the effects of moisture. These results are important when considering future research investigating more sustainable methods for managing urban drainage.

S26

Sub-session 1.2 - Microbial biodegradation assessment and improvement

P34 GENOTOXICITY OF 4-NONYLPHENOL AND NONYLPHENOL ETHOXYLATE MIXTURES WITH THE USE OF Saccharomyces cerevisiae D7 MUTATION ASSAY AND USE OF THIS TEXT TO EVALUATE THE EFFICIENCY OF BIODEGRADATION TREATMENTS Stefania Frassinetti1, Leonardo Caltavuturo1, Fabio Fava2, Diana Di Gioia3* 1 National Research Council, Institute of Biology and Agricultural Biotechnology (IBBA), Pisa Unit, Research Area of Pisa, Via Moruzzi 1, 56124, Pisa, Italy; 2DICAM, Unit of Environmental Biotechnology and Biorefineries, Faculty of Engineering, University of Bologna,Via Terracini 28, 40131, Bologna, Italy; 3Department of Agroenvironmental Sciences and Technologies, University of Bologna, Via Fanin 42, 40136, Bologna, Italy

Abstract Nonylphenol ethoxylates (NPnEOs, n is the number of ethoxylic units in the molecule) are non-ionic surfactants widely used in several industrial applications, such as textile and leather processing, paper industry, formulation of pesticides, paints and washing cleaners. 4Nonylphenol (4-NP), the main product of NPnEO biodegradation, is a toxic xenobiotic compound classified as endocrine disrupter. While numerous studies reported the toxicity and estrogenic activity of nonylphenols, little is known about the mutagenicity of these compounds. In this paper, the genotoxicity of 4-NP and NPnEO mixtures was evaluated by using the D7 strain of Saccharomyces cerevisiae as experimental model. The same tests were applied to effluents deriving from laboratory scale packed-bed bioreactors, developed for the treatment of NPnEO contaminated wastewater, in order to evaluate the residual genotoxic potential with respect to the influent waste. The target compounds fed to the bioreactors were 4-NP and NPnEO mixtures possessing an average of 5 or 1.5 ethoxylic units (Igepal CO-520 and Igepal CO-210, respectively). The results showed that 4-NP induced significant cytotoxic effect on S. cerevisiae cells at 60 mg/L, as well as mutagenic effects at 15 and 30 mg/L. 4-NP was the most genotoxic compound among those assayed, followed by Igepal CO-210, whereas Igepal CO-520 did not induce genotoxicity at any of the assayed concentrations. The genotoxic effects evidenced on yeast cells treated with 4-NP disappeared after the treatment in the bioreactor. This indicates that the biological treatment is capable of removing not only the pollutant, but also the toxicity associated to the compound and its degradation metabolites. This study represents, to the best of our knowledge, the first report that evaluates the genotoxicity of both 4-NP, NPnEOs and their potential aerobic degradation products on an eukaryotic organism. The obtained results suggest that the S. cerevisiae D7 strain is a very effective model to study the induction of genotoxic damage by the compounds under study. In addition, this method is much more simpler and easier to be applied than other tests described in the literature to study genotoxic effects. Moreover, the test described in this work has also proven to be effective in evaluating the toxicity of effluents deriving from laboratory scale biotreatment processes. It would be really interesting to apply the described tests on real wastewater treatment system in order to estimate the detoxification potential of the process applied, which is often not directly correlated with the removal of the target pollutant.

P35 EFFECT OF PRE-TREATMENT AND BIOSURFACTANTS ON THE ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSE R. Khvedelidze, G. Kvesitadze, T. Urushadze, L. Kutateladze, V. Potschishvili Biotechnology, Durmishidze institute of biochemistry and biotechn, Tbilisi, Georgia

Abstract Reactivity of the natural cellulose containing products is low due to the rough structure of cellulose and lignin content in the plant biomass. It is possible to increase the reactivity of the cellulose containing products by various methods of pretreatment of such substrates. Eecologycally safe method of cellulose pre treatment, which comprises the combination of mechanical granulation and freeze explosion, was used in experiment. By using the above mentioned methods, loosening of cellulose structure is achieved as well as partial break down of lignin.The effectiveness of pretreatment based on the final sugar content yield was assesed.By freeze freeze explosion pre treatment of the agricultural waste the degree of the enzymatic hydrolysis has increased by 12-15% on average. In order to intensify the process of hydrolysis, often low- molecular weight compounds are used. The influence of effect of biosurfactants on the synthesis of the cellulases, on their stability and on the process of hydrolysis of the agricultural wastes was studied. Biosurfactants enchance the emulsification of hydrocarbons, have the potential to solubilize hydrocarbon contaminants and increase their availability for microbial degradation. Addition of the biosurfactants (rhamnolipid, trehalose lipid and mixture of biosurfactants 0.1%) to the nutrient medium of the cellulase producing microorganisms resulted in the increase of the cellulases synthesis by about 60%. Direct addition of the biosurfactants to the incubation medium had no effect on cellulase activity, but thermostability has increased by 28%. Biosurfactants action prolongs the process of enzymatic hydrolysis of agricultural waste and glucose productivity by about 20-25%.

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Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S21-S34

P36 AMINOPHOSPHONATE XENOBIOTICS AS NUTRITIVE PHOSPHORUS SOURCE FOR MYCELIAL FUNGI CAPABLE FOR C-P BOND BIODEGRADATION Dorota Wieczorek, Paweł Kafarski, Jacek Lipok Faculty of Chemistry, Opole University, Oleska 48, 45-052 Opole, Poland

Abstract Due to their permanence/persistance and simultaneously a broad and increasing range of applications, aminophosphonic xenobiotics belong to the onerous compounds for numerous inhabitants of various environments. Scientific efforts dedicated to fate and biodegradation of aminophosphonic compounds in environment had begun in 80’s last century. Currently it is known that many microorganisms, mainly bacteria, are able to decompose the C-P bond. Interestingly, similar ability of filamentous fungi, which are known from their skills to adaptation to various nutritional conditions was studied hardly less intensive so far. Considering the increasing usage of aminophosphonates, our still fragmentary knowledge about the environmental fate of such substances, especially about their interaction with mycelial fungi, the study on ability of representatives of mentioned microorganisms for degradation of aminophosphonic compounds, were conducted. Nine strains of fungi: Alternaria alternata, Aspergillus niger, Aspergillus terreus, Fusarium dimerum, Fusarium oxysporum, Penicillium janthinellum, Penicillium simplicissimum, Paecilomyces variotti and Trichoderma koningii and seven phosphonic compounds were chosen. Two main aspects of abiotic interactions, namely fungal sensitivity towards phosphonates used in the range of concentrations from 1 to 10mM and the ability of fungi to transform these compounds, were studied. The experiments related to sensitivity of fungi towards phosphonates were conducted in classic Czapek media, which contained inorganic phosphate and phopshonates as additives, whereas the ability of these biota to degrade aminophosphonates was studied in two kinds of Czapek medium: the one, supplemented with phosphonates and the second containing phosphonates as the sole phosphorus sources. Toxicity of phosphonates were set by measurements of dry mass of mycelium as well as macro and microscopic examinations of the fungi, while biotransformation was investigated employing various technics of nuclear magnetic resonance spectroscopy, mainly 31P NMR. Other analytical methods such HPLC –UV and GC-MS were also applicated in few cases when some more detailed experiments regarding the metabolization of phosphonates were carried out. The results of presented study, show that tested fungal strains were substantially resistant towards action of examined aminophosphonate compounds, with some of the fungi being able not only to transform and/or degrade studied compounds, but also to utilise them as a nutritive phosphorus source for growth. Moreover, the process of biodegradation seemed to proceed independently on phosphorus status of fungal cells, being often accompanied by release of intermediate metabolites containing also C-P bonds.

P37 ROLE OF THE H-NS FAMILY PROTEINS IN COOPERATIVE FUNCTION OF CARBAZOLE DEGRADATIVE PLASMID PCAR1 AND HOST CHROMOSOME C. Suzuki, C.S. Yun, S. Horita, T. Terada, M. Tanokura, H. Yamane, H. Nojiri The University of Tokyo, Japan

Abstract Degradation of environmental pollutants using bacteria is a valuable method in bioremediation. Although many degradative genes are carried by plasmids, behaviours of degradative plasmids and effects of plasmid carriage for host cells have not been well-investigated. In order to regulate bacterial degradative ability, it is important to clarify the effects of plasmid carriage. To clarify this, we used IncP-7 carbazole degradative plasmid pCAR1 and Pseudomonas putida KT2440 as a model plasmid and host strain. pCAR1 contains the gene encoding an H-NS family protein Pmr, which is one of the nucleoid-associated proteins. Pmr regulates the transcription of many genes on both pCAR1 and host chromosome, and simultaneously minimizes the effect of pCAR1 carriage for host cell. Because KT2440 chromosome contains two mainly-transcribed genes encoding H-NS family proteins, TurA and TurB, Pmr and these two proteins could function cooperatively when KT2440 receives pCAR1 through conjugation. Previously, our transcriptome analyses showed that disruption of each of these genes had different effect, suggesting that functions of the three proteins are non-equivalent. However, the distributions of the binding sites of these three proteins were shown to be almost identical. Because H-NS family proteins form homo- and heterooligomers, this discrepancy may be originated from their oligomerization manner. In this study, we evaluated homo- and hetero-oligomerization functions of Pmr. We found that the first 61 residues (Pmr_nt61) were enough to form homo-oligomers. Charged 22 residues of Pmr_nt61 were individually substituted with alanine, and their oligomerization capacity was evaluated. Seven of them formed less amount of homo-oligomers compared with wild type, suggesting that such residues were important for homo-oligomerization. These residues are well conserved in TurA, whereas only three are conserved in TurB, suggesting that Pmr/TurA and TurB have different oligomerization manner. To investigate hetero-oligomerization mechanism of the three proteins, we performed surface plasmon resonance analysis using TurA and TurB as ligand, and Pmr as analyte. Comparison of affinities and coupling ratio between TurA-Pmr and TurB-Pmr will be discussed.

S28

Sub-session 1.2 - Microbial biodegradation assessment and improvement

P38 MICROBIAL POPULATIONS DURING BIOREMEDIATION OF PAHs IN WOOD SLEEPERS TREATED WITH CREOSOTE OIL B. Slavik1,2*, K. Cajthamlová1, M. Čvančarová1, Z. Křesinová1, T. Cajthaml1,2 1

Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4-142 20, Czech Republic 2 Faculty of Science, Charles University in Prague, Albertov 6, Prague 2 - 128 43, Czech Republic

Abstract Polycyclic aromatic hydrocarbons (PAHs) and their toxic degradation products represent a serious risk for the environment. One way of the risk reduction is application of bioremediation methods. Composting has recently been shown to be efficient for remediation of materials contaminated with PAHs (e.g. sleepers, soil). The process relies on the action of microorganisms able to degrade organic materials. Substrate (agricultural-waste mixture of optimum nutrient ratio or grass from experimental field of Institute of Microbiology) was mixed with woodchips made from sleepers treated with creosote oil and then composted in a special aerated composter. The composts were sampled several times at different thermal phases and after maturation. The scale of PAH degradation was analyzed by GC-MS. The results revealed a significant PAH reduction (up to 97%) of original contamination. The impact of different composting substrates on microbial populations during the degradation process and biodiversity was analyzed with PLFA. In addition, we extracted the metagenomic DNA and analyzed further bacterial and fungal diversity using DGGE and 454 pyrosequencing. DGGE analysis of rDNA was performed using primers gc984f and 1378r (bacteria) and gcITS1f and ITS2r (fungi). Amplicon pyrosequencing was performed using tagged bacterial (eub530f and eub1100r) and fungal (ITS1 and ITS4) primers targeting rDNA genes. Amplicons were pooled and sequenced on Roche GS Junior Titanium Platform. Our results support previous expectations based on PLFA quantification and show dramatic changes during different phases of composting process. The results indicate a high importance of fungal species that show increasing activity in the later phases of the composting process. Acknowledgements This work was funded by grant 525/09/1058 of the Czech Science Foundation and also by Institutional Research Concept no. AV0Z50200510.

P39 A MULTIVARIATE STATISTICAL ANALYSIS OF THE PERFORMANCE OF FLUIDIZED BED BIOREACTORS USED FOR AD SITU REMEDIATION OF WATER POLLUTED WITH PCE Cuauhtémoc U. Moreno-Medina1, Luz M. Bretón-Deval1, Favio Fava2, María T. Ponce-Noyola3, Josefina Barrera Cortés3, N. Rinderknecht-Seijas4, Juvencio Galíndez-Mayer5, Héctor M. Poggi-Varaldo1* 2

1 Environmental Biotechnology and Renewable Energies Group, Dept. Biotechnology and Bioengineering, CINVESTAV-I.P.N. DICAMN Università di Bologna, Italy ; 3Dept. Biotechnology and Bioengineering, CINVESTAV-I.P.N, México; 4ESIQIE-I.P.N., México; 5ENCB del IPN, México DF, México; e-mail : [email protected]

Abstract The PCE biotic treatment is generally carried out in anaerobic conditions. On the other hand, use of zero-valent iron (ZVI) for the remediation of organic aliphatic chlorinated compounds has been successfully demonstrated. The use of hybrid systems, biological-ZVI reactors in the context of bioremediation of waters polluted with PCE seems to be a logical extension. Multivariate statistical analysis is increasingly used for processing results due to its capacity for handling and organizing massive quantity of data. This study uses principal component analysis (PCA) to identify the most relevant variables associated to the performance of fluidized bed bioreactors (FBBRs) operated in three electron acceptors modes for the depuration of a water contaminated with PCE. Four lab scale, mesophilic FBBRs were implemented and fed a water that contained 80 mg PCE/L. In Period 1 two FBBRs were operated in methanogenic denitrifying (MD) regime and two as partially-aerated methanogenic (PAM). One MD and one PAM FBBR were coupled to a side sand-ZVI filter (MD-Z and PAM-Z). Bioreactor consortia at the start of Period 1 were unacclimated to PCE. In Period 2 all FBBRs were switched to methanogenic regime M. PCR-TGGE analysis was carried out as described elsewhere. In general, switching from two electron acceptors mode to M regime significantly improved the dehalogenation efficiency in all bioreactors (p < 0.0001). FBBRs coupled to ZVI generally showed the highest dehalogenation efficiency (21-60 %). Regarding the PCA, the first three PC explained 93% of the variance of experimental data; the analysis succeeded in reducing the number of variables from 41 to 3. The first principal component (PC1) explained ca. 58% of the variance; it was associated with PCE metabolites in bioparticles and biogas trap as well as TKN-biomass and TGGE bands. The PC2 explained ca. 23 % of the variance and was associated with COD and PCE removal efficiencies (COD, PCE), dehalogenation efficiency (dehalog), Cl, and specific oxygen uptake rate (SOUR). Fig. 1a revealed three groups of variables (or ellipses i.e., 1: COD, PCE, dehalog, Cl; 2: CV in biogas trap, PCE and CV in bioparticles and specific methanogenic activity. 3: biomass-TKN, TGGE bands, and PCE metabolites. PC1 represents the overall biotic activity in FBBRs. Regarding biological activities, the SDA was the metabolic activity nearest to PC1, and the highest specific activity in all FBBRs in the two periods. PC2 may represent the FBBRs aerobic activity since the SOUR is the nearest metabolic activity to the PCE, dehalog and Cl. This suggests that the SOUR is a significant dehalogenation biotic activity in selected FBBRs. Bioreactors can be grouped in three clusters that are linked to their PCE removal efficiency and other secondary features (Fig. 2b); Group 1: M-Z and M (PCE 90.5 %, dehalog 15.5 %), Group 2: MD-Z and MD (PCE 92.5 %, dehalog 20 %), and Group 3: PAM bioreactors. This is consistent with and summarizes the performance improvement of methanogenic operation with coupled ZVI filter as reported above. Key words: ad situ remediation, fluidized bed bioreactor, perchloroethylene, principal component analysis

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P40 COMPARISON OF OZONATION AND FUNGAL TREATMENT FOR THE DEPURATION OF AN ANAEROBICALLY PRETREATED MEZCAL VINASSE Vania Robles-González1,2, Nora Ruiz-Ordaz1, Juvencio Galíndez-Mayer1, Noemí Rinderknecht-Seijas3, Héctor M. Poggi-Varaldo4* 4

1 ENCB del IPN, Mexico ; 2Universidad Tecnológica de la Mixteca, México; 3ESIQIE del IPN, Div. Ciencias Básicas, México Environmental Biotechnology and Renewable Energies R&D Group, Depto. de Biotecnología y Bioingeniería, Dept. Biotechnology and Bioengineering, CINVESTAV-I.P.N, México, e-mail : [email protected]

Abstract In Mexico, the manufacturing of mezcal generates huge amounts of mezcal vinasses (MV), that cause a negative environmental impact. Although there is no specific information for mezcal vinasses, similar effluents have been treated by anaerobic digestion (AnD) usually complemented by oxidative chemical post-treatments such as ozonation. In the present work the post-treatment of anaerobically pretreated MVs by batch ozonation and fungal treatment using special pellets of the ligninolytic fungus Trametes versicolor were evaluated. Anaerobically pretreated mezcal vinasses (AP-MV) (Robles-González et al., 2012 b) were subjected to two postreatment processes: ozonation and fungal treatment using pellets of Trametes versicolor in a mixture of sawdust and activated carbon (hybrid or triple pellets). The batch ozonation was performed for 30, 60, 90, and 120 minutes using AP-MV without dilution (COD0=26.7 gO2/L). Batch fungal post-treatment with hybrid pellets was carried out with AP-MV diluted with tap water to a concentration of organic matter of 4.14 gO2/L. Wastewater analyses were performed according to Standard Methods (APHA, 1998); total aromatics compounds (TAC) and total phenolic contents were determined as described in literature. Removal efficiencies of organic matter in the ozonation process were low. The concentration of phenolic compounds decreased below the limit of quantification (20 mg gallic acid/L), after 30 minutes of treatment. The total removal of aromatic compounds was moderate and the values ranged from 15 to 31%. The post-treatment with ozone was very effective in removing color (74 to 80%). On the other hand, fungal post-treatment achieved higher net removal efficiencies of COD than ozonation. However, the efficiencies of color and total aromatic compounds removals were lower. The overall removal efficiency of AnD followed by fungal postreatment was 88.9%, significantly higher than that obtained with the combination AnD plus ozonation (60 min), which was 76.2%. Our results show that fungal post-treatment with ligninolytic fungi was more efficient than post-treatment with ozone in terms of organic matter removal (COD). This could represent an economic advantage in the treatment of effluents with a high content of recalcitrant compounds as in the case of mezcal vinasse. Some authors post-treated vinasses from beet molasses by ozonation, after conventional biological treatment; they observed COD and colour removals of 13% and 76% at 30 min of ozonation, similar to our results. There are no studies on anaerobic treatment of mezcal vinasses followed by post-treatment with ligninolytic fungi. So, our results will be discussed in terms of studies conducted with other recalcitrant effluents. Our COD removal efficiencies in the fungal post-treatment were higher than those reported in literature in assays of post-treatment of anaerobic weak black liquor using T. versicolor who observed moderate removals (32 to 71%). Key words: anaerobic digestion, fungal treatment, mezcal vinasses, ozonation

P41 A DESIGNED AND TAILORED COMPOST FOR DIFFERENT APPLICATIONS: INNOVATIVE PROCEDURE APPLIED TO DIFFERENT COMPOST PROPERTY CHARACTERISATION Alessandra Bonoli1, Alice Dall’Ara2, Silvia Serranti3 1

DICAM, University of Bologna, Italy; 2UTTMATF, ENEA Faenza, Italy; 3Department of Chemical Engineering Materials & Environment, SAPIENZA UNIVERSITA' DI ROMA, Italy

Abstract The first objective in aerobic conversion and composting process is to recycle biowaste, food and agriculture products and animal breeding residue. Compost obtained from different organic waste sources (municipal solid waste, biomass, etc.) is more and more utilized as a product suitable for agricultural purposes reducing at the same time land filling of wastes. It’s possible to obtain a compost that can be used in different applications: as a marketable fertilizer, substrate, soil for environmental restoration etc. Nowadays, for instance, agricultural needs are not connected to waste management. It is an acquired scientific knowledge that the use of very stabilized organic matter protects the biological characteristics of the soil, improves soil fertility, fights back soil erosion and organic matter depletion particularly in warm climate. A particularly stabilized compost can be used also as substrate and growing media with low water requirements. Very often the factor that limits a wider use of compost and organic substrate is the difficulty to manage correctly the maturing process of the raw materials, with a consequent poor agronomic quality of the final product which has to be distributed on the soil. Specific properties can be used also for specific application of stabilized organic material: designed compost for landfill cover, in biofilter preparation for land recovery or in specialized fertilizer for niche agricultural products. In relation with specific application of compost, the utilization of dedicated compost can be considered using additives or enzyme for a tailored and a specific designed material. At the same way engineered system of laying biowaste or manure sanitation through the application of new biological technologies (bio-treatment) can be developed to obtain quality marketable fertilizer, that improves the soil structure, enhances its fertility and rehabilitates for predesertification. The specific properties have to be monitored by a quality control systems. A specific and “ad hoc” applications on compost products characterization can be carried out by dedicated lab test and assessment methods. Main aim of the study was to investigate the possibility offered by new procedures, as Hyperspectral Imaging, to evaluate the compost quality in order to develop control strategies to be implemented at plant scale and to individuate its best utilization field.

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Sub-session 1.2 - Microbial biodegradation assessment and improvement

P42 HORMONAL ACTIVITIES OF NOVEL BROMINATED FLAME RETARDANTS AND THEIR BIODEGRADATION BY WHITE ROT FUNGI M. Ezechiáš1,2, K. Svobodová1, T. Cajthaml1,2 1

Laboratory of Environmental Biotechnology, Institute of Microbiology ASCR,v.v.i., Vídeňská 1083, 142 20 Prague, Czech Republic; 2Institute for Environmental Studies, Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague, Czech Republic

Abstract Over the last years, a number of articles focused on brominated flame retardants (BFR) as new pollutants in the environment. These compounds have been used in high volumes to reduce the flammability of numerous types of polymers and resins commonly found in furniture and electronic components. Some of these flame retardants (octabromodiphenyl ether and pentabromodiphenyl ether) were proved to be toxic and persistent in the environment. Nowadays these substances are also listed as POPs by Stockholm convention. After the phase out of previously used BFR, many new and alternative BFR have been used for commercial applications. However, the knowledge about these new substances and their fate in the environment is limited. In the recent years, several articles have demonstrated that some of these compounds are detected in environmental samples. The compounds have been found to have long transport potencial and to be toxic. This article focuses on biodegradation ability of white rot fungi toward these new pollutants. Trametes Versicolor, Bjerkandera Adusta, Irpex lacteus, Pleurotus ostreatus, Phanerochaete chrysosporium, Dichomitus squalens, Setulipes androsaceus and litter decomposing strain PL13 from CCBAS Institute of Microbiology ASCR have been used in this study. Each fungi grew in malt extract - glucose medium that had been artificially contaminated with s mixture of brominated flame retardants: 1,2-bis(2,4,6tribromophenoxy)ethane (BTBPE), bis(2-ethylhexyl) tetrabromophthalate (BTBP), 2,3,4,5,6-pentabromoethylbenzene (PBEB), 2,3,4,5,6pentabromotoluene (PBT), 2,4,6-tribromophenol (TBP), hexabromobenzene (HBB), 2-allyloxy-1,3,5-tribromobenzene (ATBB), pentabromobenzyl acrylate (PBBA) and decabromobiphenyl (DBB). The fungi were incubated for 7 and 22 days. Afterward, gas chromatography with mass spectrometry was employed to determine concentrations of the tested compounds in the medium. The degradation result from the experiment showed that only three compounds (TBP, ATBB, PBBA) were degraded by the fungi. Flame retardants such as BTBPE, BTBP, HBB or DBB did not exhibit any significant removal. We also used two bioassays with recombinant strain Saccharomyces cerevisiae to investigate hormonal activities of these new BFR. Our data demonstrate the ability of 2,4,6tribromophenol to lower the transcriptional activity of human estrogen and androgen receptor. PBBA and BTBPE also show slightly antihormonal effects. Our findings suggest that persistency of the new BFR being possibly a serious problem. Results also show that TBP could be classified as a new environmental endocrine disruptors. These results emphasize the need for a further research in biodegradability and hormonal activity of brominated flame retardants.

P43 REMOVAL OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) BY SELECTED MICROBIAL STRAINS ISOLATED FROM HIGHLY CONTAMINATED SOIL F. Sannino1, V. Ventorino2, O. Pepe2, A. Piccolo1 1

Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università di Napoli “Federico II”, Via Università 100, 80055 Portici (Na), Italy; 2Dipartimento di Scienza degli Alimenti, Università di Napoli “Federico II”, Via Università 100, 80055 Portici (Na), Italy

Abstract Polycyclic aromatic hydrocarbons (PAHs) constitute a group of priority environmental pollutants in soils and sediments due to their toxic, mutagenic and carcinogenic effects. PAHs may be present at high concentrations at industrial sites associated with petrole, coal tar, gas production and wood preservation industries. The ACNA site, an industrial area of Cengio near Savona, is largely contaminated by different classes of organic compounds and it has been included in the list of national priorities for environmental reclamation. As compared to the physical-chemical treatments, the use of microbial technology to clean up contaminated soils was found to be more efficient, economical and eco-friendly. The aim of this work was to assess the capacity of selected microbial strains, isolated from very same polluted soil of the ACNA site, to degrade and remove the anthropogenic organic compounds present in the aqueous extracts from an ACNA soil. In the extraction experiments, 30 g of soil were kept under reflux in a Soxhlet with 225 mL of an acetone/n-hexane (1:1) mixture for 48 hours. The organic extracts were first dried in a roto-evaporator at 40°C and redissolved in 5 mL of acetone and 145 mL of ultrapure water (final volume of 150 mL). An aliquot of these aqueous extracts was purified by solid phase extraction (SPE) through elution in Bond-Elut C-18 cartridges. Preliminary tests were carried out to optimize the volume of extract to apply and the type of organic solvent to be used for elution. Finally, an amount of 20 mL of extract and 30 mL of organic solvents (10 mL of n-hexane, 10 mL of diethyl ether followed by 10 mL of acetone) were utilized. After extraction with C-18 cartridges, the samples were dried, redissolved with 1 mL of CH2Cl2, containing 100 g/ml octafluoronaphtalene solution in CH2Cl2 as internal standard, and analyzed by GC-MS. The aqueous extracts from the contaminated soil (CSAE) were used at a concentration 40% as growth media of microorganisms, represented by bacteria and mould strains isolated form the same ACNA soil, in order to evaluate by GC-MS analyses the removal of polycyclic aromatic hydrocarbons (PAHs) present in the CSAE. The results obtained both with bacteria and mould selected strains showed a significant removal of the identified pollutants that varied from 28 to 100% depending on the pollutant class. The development and optimization of such extractive, degradative and analytical methodology will allow to assess the effective ability of the isolated microbial strains to be further used in bio and mycoremediation of soils polluted by PAHs and other aromatic compounds.

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P44 INTERACTION BETWEEN DEGRADATIVE PLASMID AND HOST CHROMOSOME DIFFER BETWEEN THREE PSEUDOMONAS HOSTS Y. Takahashi*1, M. Shintani1,2, N. Takase1, Y. Kazou3, F. Kawamura3, H. Hara4, H. Nishida1, H. Yamane1, H. Nojiri1 1

The University of Tokyo, Japan; 2RIKEN, Japan; 3Rikkyo University, Japan; 4Okayama University of Science, Japan

Abstract Carbazole degradative plasmid pCAR1 was originally isolated from Pseudomonas resinovorans, and was proven to be IncP-7 plasmid transferable to other Pseudomonas and Stenotrophomonas bacteria. Its 200,231-bp entire nucleotide sequence has already been determined. To understand the mode of function of pCAR1 as an agent making its host to be carbazole degrader, it is necessary to understand the mechanisms of pCAR1 behaviour and to determine how much its behaviour is changeable in diverse types of host strains. On the other hand, pCAR1 carriage also alters its host strains in their growth, phenotype or cell physiology. While this phenomenon has been empirically known as “plasmid cost” or “metabolic burden,” it has not been well-characterized how much these phenomena are affected by host cell type. Therefore, behavior of pCAR1-harboring strain should be determined by both host’s effects on pCAR1 function and impacts of its carriage on host cell. In this study, such interaction between pCAR1 and chromosome were comprehensively evaluated using three Pseudomonas hosts, P. putida KT2440, P. aeruginosa PAO1, and P. fluorescens Pf0-1. Growth phase-dependent transcriptome analyses of three host chromosomes showed that pCAR1 carriage affected more greatly at the transition and stationary phases in all hosts. Comprehensive phenotype comparisons showed that pCAR1 carriage reduced host fitness, swimming motility, and stress resistance and suggested the reduction of primary metabolic capacities in the TCA cycle and branching metabolisms. Interestingly, the extent of the impacts was different host by host, which was the largest in KT2440 and the lowest in Pf0-1. The impacts were more similar between KT2440 and PAO1 than between other combinations. Comparison of pCAR1 transcriptome in the three hosts showed that the transcriptional pattern from early log to stationary phase of some genes (e.g. transposase gene and transcriptional activator gene) were different, while the genes related to degradation and plasmid maintenance were transcribed similarly in three hosts. The numbers of genes transcribed was the largest in KT2440, which may be the reason of above-mentioned different host cell response. These results reported here suggested the importance of host choice in bioaugmentation using degradative plasmid.

P45 IDENTIFIACATION OF CULTIVABLE AND NON-CULTIVABLE RHIZOSPHERE BACTERIA FROM LONG TERM CONTIMANTED SOIL BY PCBs WITH USING MOLECULAR GENETIC Petr Stursa, Petra Prouzova, Tomas Macek, Martina Mackova Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, ICT Prague, Technicka 3, 166 28 Prague 6, CZ

Abstract A number of persistent substances got to the environment due to human activity during the 20 century. Toxicity for the humans has been detected in many of these substances. Polychlorinated biphenyls (PCBs) belong among such substances. PCBs can be removed from the environment only via physical-chemistry methods, which are very complicated and expensive. Therefore, the attention is focused on use of phytoremediation and bioremediation in the degradation of PCBs. In our experiment plants of tobacco, black nightshade and horseradish were grown in long-term PCB contaminated soil and we focused on description of microbial diversity in the rhizosphere of these plants. Diversity was measured using the analysis of 16S rRNA gene and by MALDI-TOF MS and the results were compared (cultivable microorganisms). In the second part of the experiment, we focused on characterizing non-cultivable micro-organisms using methods of Stable Isotope Probing (SIP) and terminal-restriction fragment length polymorphisms (T-RFLP). To identify non-cultivable strains TRFLP was done with heavy DNA fractions. Genomic libraries were created, containing a part of 16S rRNA to identify the individual peaks of the spectra. These genomic libraries were sequenced and obtained sequences represented different bacterial species contained in heavy DNA. Sequences thus obtained were assigned to individual peaks from T-RFLP profile using In Silico digestion. From the results obtained using SIP it is clear that the vast majority of bacterial species obtained from heavy DNA is completely different from bacteria which we identified by 16S rRNA analysis and MALDI-TOF MS. Dominant representative cultivable species is Rhodococcus and dominant representative non-cultivable species are bacterial genera Hydrogenophaga or Methylophillus. Acknowledgements The work has been supported by projects 525/09/1058, ME 09024, FP7-KBBE-2010-4/265946

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Sub-session 1.2 - Microbial biodegradation assessment and improvement

P46 LEVEL OF ENVIRONMENTAL POLLUTION TRIGGERS COLONIZING Miscanthus sinensis BY ENDOBACTERIA POSSESSING GENES CODING FOR 2,3-DIOXYGENASE S.W. Gawronski1, A.Kacprzyk2 1

Laboratory of Basic Research in Horticulture, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences-SGGW; 2Institute of Biochemistry and Biophysics of Polish Academy of Sciences

Abstract Internal tissues of higher plants are often colonized by endobacteria, which interact with a host plant in different ways and may induce physiological changes in every level of their biological organization. Bacterial endophytes can support plant growth for example by production of phytohormones, N2- fixation. Besides endophytic bacteria are able to degrade aromatic organic compounds and chelating heavy metals taken up by plants from an environment. The main group of bacterial enzymes that catalyze degradation of aromatic compounds are dioxygenases. The aim of this study was to check whether endobacteria colonise plants of Chinese silvergrass (Miscanthus sinensis) and if so, whether there are differences in endobacteria profile in regard to level of environmental pollutions focusing especially on genes involved in degradation of xenobiotics and chelation of heavy metals. Bacteria species identification was carry out using molecular methods with sequence of 16S rDNA gene. Total DNA was isolated from surface-sterilized leaves tissues. In PCR reactions catechol 2,3-dioxygenase and 16S rDNA sequences were amplified. Results showed that endobateria were present in plants growing both at clean, medium and heavy polluted sites. However, only plants collected from heavy and medium polluted sites were colonized by endobacteria possessing catechol 2,3-dioxygenase gene. Most of the identified bacteria belong to genus Bacillus sp.

P47 TRANSGENIC PLANTS IN RHIZOREMEDIATION OF PCB-CONTAMINATED SOIL V. Kurzawova, M. Novakova, M. Chovancova, T. Macek, M. Mackova Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic

Abstract Polychlorinated biphenyls (PCBs) are organic compounds widely spread in the environment. Physico-chemical methods for decontamination of polluted soil are mostly expensive and destroy the landscape. In contrast, biological systems such plants, microorganisms and their consortia are public acceptable, mostly cheaper and don’t destroy the landscape. Bacteria are able to degrade PCBs by anaerobic way (reductive dechlorination) and by aerobic degradation pathway (biphenyl operon). The role of plants in PCB decontamination is important as well. Plants absorb PCBs, transform them to non-phytotoxic form and store them e.g. in their vacuoles or cell walls. The cooperation between plants and microbe can be quite advantageous for PCB rhizoremediation. Plant exudates into the rhizosphere several compounds (e.g. glycosides, amino acids) stimulating the growth and activity of soil bacteria and compounds of their secondary metabolites (e.g. flavonoids, terpenoids, phenolic acids, etc.). Secondary metabolites can serve as a source of carbon and nitrogen. Because of their structural similarity with PCBs they are potential inducers of biphenyl operon encoding for enzymes involved in the bacterial biphenyl degradation pathway. Preparation of transgenic plants with higher production of secondary metabolites with verified ability to induce bacterial degradation of PCBs could be the effective way of polluted soil decontamination. The aim of this study is to prepare transgenic plants Nicotiana tabacum with overexpressed gene for flavonoid-3‘-hydroxylase (AtF3’H), the enzyme converting kaempferol to plant secondary metabolite quercetin. The ability of quercetin to induce the expression of bphA gene, the gene of biphenyl operon, was verified by cultivation of PCB degraders with quercetin as a source of carbon and by detecting the bphA gene. Consequently the AtF3’H gene was cloned to the pQE30 vector to obtain the enzyme with histidine tail (6 x His) in bacteria. His tag will serve for the detection of expression and for the isolation of the obtained enzyme. After the activity test the AtF3’H gene with His tag will be cloned to the plant vector with the root specific promoter that will target the expression of AtF3’H in transgenic plants to roots. This should lead to a higher production of quercetin in plants and consequently to releasing of this compound to the rhizosphere, where this flavonoid can stimulate PCB degradation of present bacteria. Acknowledgements This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project LH 12087) and Czech Science Foundation (project 525/09/1058).

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P48 STIMULATION OF BACTERIAL CHLOROBIPHENYL METABOLISM THROUGH THE ADDITION OF PLANT SECONDARY METABOLITES Lucie Musilova1, Ondrej Uhlik1, Michal Strejcek1, Petra Lovecka1, Vlasta Dudkova1, Cestmir Vlcek2, Jakub Ridl2, Miluse Hroudova2, Tomas Macek1, Martina Mackova1, Katerina Demnerova*1 1

Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic; 2Department of Genomics and Bioinformatics, Institute of Molecular Genetics, Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic

Abstract Interactions between plants and their rhizosphere bacteria are very important for an effective transformation of environmental contaminants which is the essence of rhizoremediation. It has been shown that the presence of a particular plants’ root system highly influences soil microbial community structure. Among others, aromatic pollutant-degrading bacteria are fostered via root exudation, or root turnover. These processes provide plant secondary metabolites, which can promote microbial cometabolism of aromatic pollutants after they were released into soil. The aim of the study was to investigate the influence of selected natural compounds (naringin, limonene, and caffeic acid) and natural materials rich in these compounds (grapefruit peel, lemon peel, and pears, respectively) on the diversity of chlorobiphenyl-metabolizing bacterial populations in long-term PCB-contaminated soil. We have integrated analytical methods of determining PCB degradation with DNA-stable isotope probing (SIP) as a direct method which links the metabolic process with its microbial originators. 13C-DNA, isolated after stable isotope probing, was used to generate 16S rRNA gene amplicons which were further processed by pyrosequencing. Our results show that plant secondary metabolites promote the activity of different chlorobiphenyl-metabolizing microbial populations in soil. Specifically, naringin and limonene in soil foster mainly populations of Hydrogenophaga spp., caffeic acid Burkholderia spp. Similarly, the degradation of individual PCB congeners was influenced by the addition of different plant compounds. Residual content of PCBs was the lowest after treating the soil with naringin. Similar trends were observed after the addition of plant materials, where naringin-rich grapefruit peel addition resulted in the most effective degradation. After the addition of caffeic acid, higher substituted congeners were more degraded compared to all other treatments. Overall, the findings reveal that active utilization of chlorobiphenyls in soil bacteria is promoted by the presence of plant secondary metabolites. The results may therefore be beneficial to bioremediation (especially rhizoremediation) research in a way they permit predicting the behaviour of populations potentially involved in biodegradative processes. Acknowledgements This work was supported by EU project MINOTAURUS within 7th Framework Programme (project no: 265946), the Ministry of Education, Youth and Sports of the Czech Republic (project ME 09024), and Czech Science Foundation (project 525/09/1058).

P49 LOOKING IN ENVIRONEMNT FOR NEW ENZYMES AS A TOOLS FOR BIOCATALYSIS AND BIOREMEDIATION Adam Sobczak1,2, Paweł Krawczyk1, Dorota Tokarska1, Dorota Adamska1, Michał Kamiński1, Marta Błaszkiewicz1, Marcin Ostajewski1, Marcin Gołębiewski1,3, Leszek Lipiński1, Andrzej Dziembowski1 1

Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland; 2Department of Genetics and Biotechnology, University of Warsaw, Poland; 3Department of Biotechnology, Nicolaus Copernicus University, Torun, Poland

Abstract Bacteria during evolution created enormous biodiversity enabling them to spread around the whole planet. Up to date, due to lack of technologies, bacterial biodiversity has not been deeply explored. Lately developed methodology comprising metagenomics and second generation sequencing opened opportunity to analyze biodiversity as well as whole genomes of bacterial communities thriving in different (sometimes extremely contaminated) environments. We have build library of one hundred metagenomic DNA isolated from different environments mainly soil highly contaminated with xenobiotics eg. pesticides. Biodiversity analysis based on 16S rRNA genes showed correlation between bacterial community composition and level as well as character of chemical pollution. Up to date four samples of metagenomic DNA have been analyzed by deep sequencing to determine their potential as a source of new enzymes suitable for biodegradation or biocatalysis. Gene assembly and bioinformatic analysis gave almost 2 mln contigs longer than 150bp, with largest contig 332 kb. Anotation of identified ORF's on KEGG and SEED databases exhibit that 11% of their number might be involved in xenobiotic degradation processes. Despite lower O.T.U number and species richness observed in metagenoms from soils contaminated with chloroorganic pesticides, those samples exhibit 5 times more different genes involved in metabolism of aromatic (xenobiotic) compounds. To explore this plethora of potential enzymes we are developing strategy for highthroughput protein expression and purification. This will allow us perform functional analysis of new enzymes identified in metagenoms, and fish catabolysts of high practical importance for bioremediation and biotransformation.

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Environmental Engineering and Management Journal

March 2012, Vol.11, No. 3, Supplement, S35-S52

http://omicron.ch.tuiasi.ro/EEMJ/

“Gheorghe Asachi” Technical University of Iasi, Romania

Sub-session 1.3. Phytoremediation and mycoremediation Oral presentations THE POPLAR ENDOPHYTE PSEUDOMONAS PUTIDA W619 AS A KEY TO A SUCCESSFUL PHYTOREMEDIATION OF VOLATILE ORGANIC CONTAMINANTS: FROM THE LAB TO THE FIELD Nele Weyens 1, Sofie Thijs 1, Panos Gkorezis 1, Daniel van der Lelie 2, Safiyh Taghavi 2, Jaco Vangronsveld 1 1

Hasselt University, Agoralaan, building D, 3590 Diepenbeek, Belgium; 2Research Triangle Institute (RTI) International 3040 Cornwallis Road - PO Box 12194 Research Triangle Park,NC 27709-2194,USA

Abstract Phytoremediation of volatile organic contaminants is often unsatisfactory because plants and their rhizosphere do not completely degrade these compounds resulting in evapotranspiration through the leaves causing secondary contamination of the ambient air and by consequence undermining the merits of phytoremediation. Under laboratory conditions, endophytic bacteria equipped with the appropriate degradation pathway can be used to improve the in planta degradation of organic contaminants resulting in decreased phytotoxicity and evapotranspiration. In this work, we describe the different experiments that were performed in order to move endophyte-enhanced phytoremediation from labscale experiments towards application on a TCE-contaminated field site. Poplar cuttings were exposed to trichloroethylene (TCE) in 3 different experimental set-ups, more specifically a short-term (days) hydroponics experiment, a midlong-term (weeks) pot experiment, and finally in a long-term (months) field trial. In order to improve the phytoremediation efficiency, poplar cuttings were inoculated with the TCE degrading root endophyte Pseudomonas putida W619. In the short-term hydroponics experiment, P. putida W619 colonized all plant parts except the leaves which resulted in a very significant plant growth promoting effect and a slightly diminished (23%) TCE evapotranspiration. In case the cuttings were grown in pots filled with potting soil, a growth promoting trend and a 41% decreased TCE evapotranspiration were observed after the roots and the stem were colonized by P. putida W619. In the field experiment, 3 months after inoculation, the inoculated root endophyte P. putida W619 could only be re-isolated from the roots. However, also some natural abundant stem endophytes got equipped with the TCE metabolic activity by horizontal gene transfer. Although the inoculation did not significantly affect plant growth, a 90% reduced TCE evapotranspiration was achieved after inoculation. These results demonstrated that the endophyte P. putida W619 could be the key to both, increasing biomass production on marginal, nutrient poor land and also to an optimized phytoremediation of volatile organic contaminants. Furthermore, we can conclude that although we can learn a lot from lab-experiments, it is often very difficult to extrapolate lab-scale observations to real life field applications. However, the combination of the results obtained from these experiments at different levels of complexity provided us with new, interesting knowledge that can be exploited in future field applications.

Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S35-S52

GM PLANTS EXPRESSING BACTERIAL DIOXYGENASES FOR ENHANCED PHYTOREMEDIATION OF ORGANIC POLLUTANTS M. Novakova1, M. Mackova1, Z. Antosova1, J. Viktorova1, L. Trbolova1, M. Sylvestre2, T. Macek1 1

ICT Prague, Faculty of Food and Biochemical Technology, Department of Biochemistry and Microbiology, Technická 5, 16628 Prague, Czech Republic, e-mail: [email protected]; 2INRS-Quebec, Pointe-Claire, H9R 1G6, Quebec, Canada

Abstract Genetically modified (GM) plant can be useful for several reasons. One of them is cleaning up our environment, contaminated soil, water and air in the process called phytoremediation. The aim of this work is to construct and study GM plants with increased capabilities to degrade organic pollutants such as polychlorinated biphenyls (PCBs) and toluene. We have prepared GM plants of Nicotiana tabacum containing genes of bacterial dioxygenases – bphC gene and todC1C2 genes. BphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase which cleaves the aromatic ring of dihydroxybiphenyl and was cloned in fusion with the gene for β-glucuronidase (GUS), luciferase (LUC) or with a histidine tail under the control of CaMV 35S promoter. The todC1C2 genes produce oxygenase ISPTOL (with histidine tail), a component of bacterial toluene-2,3-dioxygenase that can oxidize toluene and other organic pollutants (also biphenyl). Both genes (todC1 and todC2) were cloned under either the constitutive CaMV 35S promoter or inducible RbcS promoter. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. Genetic constructs successfully expressing dioxygenase’s genes were used for preparation of transgenic tobacco plants. The presence of transgenic DNA and its expression into mRNA and protein was already determined in parental and first filial generation of transgenic plants with bphC gene. The ability to remove the toxic substrate 2,3-dihydroxybiphenyl from media was studied with selected transgenic lines. Transgenic line H3 (harboring bphC gene with histidine tail) showed 95 % higher decrease of the substrate content in medium than nontransgenic plants. Further toxic effect of Delor 103 (PCB mix) and selected congeners of PCBs on transgenic tobacco lines was studied, where transgenic plants grew better than nontransgenic on Delor 103 and congener PCB 10. Properties of prepared transgenic plants will be studied more, nevertheless they already sound to be promising in phytoremediation technologies. Acknowledgements Project is supported by grants ME-09024-BIOAROM, EU grant Minotaurus FP7 KBBE-2010-4-265946 and GACR P501/12/P521.

SPATIAL AND TEMPORAL CHARACTERIZATION OF BACTERIAL COMMUNITIES IN A PHYTOREMEDIATION PILOT PLANT AIMED AT DECONTAMINATING POLLUTED SEDIMENTS DREDGED FROM LEGHORN HARBOR AREA Carolina Chiellini1, Renato Iannelli2, Veronica Bianchi2, Giulio Petroni1 1

Department of Biology, Unit of Protistology-Zoology, University of Pisa, Via Volta 4/6, 56126 Pisa, Italy; 2Department of Civil Engineering, University of Pisa, via Gabba 22, 56126 Pisa, Italy

Abstract The Agriport project (Agricultural Reuse of Polluted Dredged Sediments, Eco-innovation EU Project n. ECO/08/239065) aims at developing a new technology for the treatment of polluted sediments dredged from the seabed of a commercial port through phytoremediation processes. Through plant activities and microorganism metabolisms, it is possible to recover dredged saline sediments by decontaminating them and adapting their composition until the obtainment of a techno-soil that is reusable in the terrestrial environment. This is an important advantage from the environmental point of view, and allows to partially solve one of the main problems of most commercial ports, that is the accumulation, storage and disposal of polluted dredged sediments. Aim of the present study is the monitoring of spatial and temporal variation of microbial communities that are present in an experimental phytoremediation plant. The pilot plant is made up of a sealed 80 m3 basin that was filled with a mixture of dredged sediments (70%) and natural soil (30%). It was planted with three plant species, and has been properly cultivated with optimized fertilization and irrigation for two years. Terminal Restriction Fragment Length Polymorphism (TRFLP) was chosen to study the composition of microbial communities in different points of the mixture under treatment. Here we present the results of the first two years of experimentation focusing on the evolution of bacterial community structures. In detail we observed that, starting from a heterogeneous mixture of different microbial communities at time zero, in a short time a microbial community homogeneously distributed in the pilot plant developed. This community is gradually becoming similar to those originally present in the rhizospheres of the three adopted plant species.

S36

Sub-session 1.3 - Phytoremediation and mycoremediation

REMOVAL OF PHENOLIC COMPOUNDS IN CONSTRUCTED WETLANDS MESOCOSMS TREATING SUGAR CANE STILLAGE AT HIGH SURFACE ORGANIC LOAD RATES E.J. Olguín *, R.E. González-Portela, G. Sánchez-Galván, K.L. Tapia-Fierro Biotechnological Management of Resources Network. Institute of Ecology (INECOL), Carretera Antigua a Coatepec No. 351, Xalapa, Veracruz 91070, Mexico, e-mail: [email protected]

Abstract Ethanol production from sugarcane molasses usually generates large amounts of wastewater (stillage) that causes a severe environmental impact. Stillage contains high organic matter, potassium and sulfate concentrations. One of the major problems is that it contains high concentrations of phenolic compounds, especially melanoidins (around 30 mg/L) which induce toxicity in the microbial soil and aquatic biota when disposed without treatment. Our research group has reported the use of sub-surface flow constructed wetlands (SSFCWs) planted with Pontederia sagittata for the treatment of stillage at high surface organic load rates (SOLRs) (50 and 100 gCOD/m2d) without any pre-treatment apart from adjustment at pH 6.0. This system was highly effective at removing COD (85%), BOD5 (82%), TKN (60%), N-NO3 (60%) and S-SO4 (77%). The objective of this work was to evaluate the performance of SSFCWs planted with P. sagittata treating stillage and operating at even higher SOLRs: 94 and 188 gCOD/m2d. Special attention was paid to the removal of phenolic compounds. The SSFCWs were operated at two hydraulic retention times (HRT) (5 and 7 days) using two different filter media, volcanic gravel and ceramic material. Samples were taken at three different sections along the SSFCW length: initial (inlet), middle and final (outlet). The results showed that the removal of the total phenolic compounds (initial concentration was 28 mg/L) was significantly higher (p< 0.05) with volcanic gravel (81%) compared to ceramic material (73%), after 60 days of treatment at a HRT of 7 days. The initial values of COD (4,480 mg/L) and BOD5 (2,500 mg/L) were reduced in 81±2.03% and 79±6.91%, respectively in the SSFCWs packed with ceramic material and volcanic gravel, respectively. Sulfates were removed at an average of 67.5±2.5% for both types of filter media at a HRT of 5 days. Plants showed intoxication symptoms in the initial sections of all SSFCWs (they died after 20 days of treatment), mainly due to the high SOLRs tested. On the other hand, the plants in the middle and final sections maintained a healthy appearance during the whole experimental period even though they did not grow. The residual concentrations of nitrates (5 mg/L) and phosphates (2-6 mg/L) that were found in the effluents after treatment, may serve as additional nutrients in ferti-irrigation of sugar cane plantations. This phytotechnology shows potential in the treatment of stillage or any other agro-industrial wastewater containing high levels of organic matter and phenolic compounds.

ASSESSING THE ABILITY TO TREAT NITROGEN COMPOUNDS IN DOMESTIC WASTEWATER OF A CONSTRUCTED WETLAND WITH DIFFERENT AQUATIC PLANT SPECIES Nguyễn Thị Loan Faculty of Environmental Science, University Of Sciences–Vietnam National University 334 Nguyen Trai Road Thanh Xuan District-Ha Noi-Vietnam

Abstract Wastewater treatment technology by “Constructed Wetlands” with aquatic plants is being widely used in Vietnam. This paper presents research results on the ability to treat the nitrogen compounds (NH3- , NO2- , NO3- and total nitrogen) of domestic wastewater of a constructed wetland system with aquatic plants such as: Pistia, Enydra fluctuans Lour, Phragmites communis, and Cyperaceae; Three experiments with different combinations of aquatic plant species in a three treatment unit-constructed wetland were conducted to find a suitable retention time for each experiment, and to compare the performance of aquatic plants in the proposed systems, based on that the best combined constructed wetland system would be suggested. Results showed that aquatic plants play an important role in wetland systems because they increase the treatment efficiency for nitrogen compounds by 5% in samples without plants and by 50%-98% in samples with plants. Treatment efficiency of all vegetation in the retention time of 12 days reached the highest value, ranging from 50% to over 98%. The treatment efficiency for all nitrogen parameters of Enydra fluctuans Lour and Cyperaceae was better (by approximately 10%) than that of Pistia, and Phragmites communis respectively. The combination of Enydra fluctuans Lour and Cyperaceaehave gave better treatment efficiency and this would be recommended to apply in a constructed wetland system. Key words: aquatic plants, constructed wetland, nitrogen compound

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Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S35-S52

ANAEROBIC DIGESTION OF CORN STOVER AS SUSTAINABLE SOIL MANAGEMENT CONCEPT FOR METAL CONTAMINATED AGRICULTURAL LAND A. Peene, B. Mattheeuws, Jan Smis Organic Waste Systems NV, Dok Noord 5, BE-9000 Gent, e-mail : [email protected]; [email protected], [email protected]

Abstract In some regions agricultural soils are diffusely contaminated with heavy metals. Grain maize cultivated on heavy metal polluted land accumulates metals. Talking in terms of soil remediation, the metal extraction capacity is limited and therefore this concept has to be considered as a sustainable soil management strategy. Organic Waste Systems has developed a concept that combines the “biomass to energy” objective with the gradual rehabilitation of the agricultural land. Grain maize is the input of the process chain. The maize kernel is harvested separately from the rest of the plant. As the degree of absorption of heavy metals in the kernel is rather poor to negligible, it is suitable for cattle feed production. The heavy metals are mainly accumulated in the rest of the plant. The entire maize plant is harvested, but only the corn stover is used as main substrate for a dry digestion process. By means of the DRANCO process corn stover is anaerobically converted into biogas, what subsequently can be converted into heat and electricity. The residual digestate does not return to the fields. This way, part of the contamination will be extracted from the soil. The more heavily contaminated digestate has to get a controlled application. Digestate can also be dewatered and dried, making use of the residual heat of the CHP plant. A classical incineration process including strict exhaust gas treatment can provide the final energy recuperation step from the dried cake, resulting in a very limited amount of ashes (containing the heavy metals) that can be stored or stabilized, e.g. in glazed clinkers. The feasibility of this new concept for sustainable land management is being studied for the Flemish Campine region where agricultural soil is contaminated with Cadmium, Lead and Zinc. By collecting 40 000 tons of corn stover each year up to 35 kg Cadmium, 160 kg Lead and 9 000 kg Zinc can be extracted from the agricultural soil in that region. Additionally 13 000 MWh of renewable electricity can be produced yearly (1.5 MW electrical power). Although there is no full-scale realization yet, the potential for application in Europe is huge. Due to the use of corn maize the accessibility for farmers is high and widely applicable. Moreover, due to the use of the very robust and flexible DRANCO technology maize can be substituted by other crops, e.g. crops with a higher metal accumulation capacity.

MYCOREMEDIATION OF CONTAMINATED SOIL IN FIELD SCALE M. Tuomela*1, K. Jørgensen2, E. Winquist3, K. Björklöf2, E. Schultz2, F. Anasonye1, L. Häkkinen2, M. Räsänen3, J. Sorvari2, E.S. Hartikainen1, K. Steffen1, L. Valentin1 1

Department of Food and Environmental Sciences, P.O. Box 56, 00014 University of Helsinki, Finland, 2Finnish Environment Institute (SYKE), P.O. Box 140, 00251 Helsinki, Finland; 3Department of Biotechnology and Chemical Technology, P.O. Box 16100, 00076 Aalto University, Finland; e-mail: [email protected]

Abstract Ligninolytic fungi are known to be able to degrade organic contaminants with their non-specific enzymes. Thus, attempts have been made to find the best fungi to grow in soil, tolerate toxic xenobiotics, in addition to degrade the contaminants. The most promising fungi belong to litter-decomposing fungi which grow in soil, and in several screenings many promising candidates have aroused. One of the most promising fungal strains found in the Fungal Biotechnology Culture Collection (FBCC) of University of Helsinki is Phanerochaete velutina, FBCC941. This fungus grows into soil when introduced with pine bark, and produced manganese peroxidase (MnP) in solid state cultivations. We performed several field scale (up to 13 t soil) or pilot scale experiments with fungal treatment, in which the inoculation technique and the support material were optimized. The compounds in focus were trinitrotoluene (TNT), polyaromatic hydrocarbons (PAH) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F). Remediation was monitored in order to clarify the activity of the fungus and the role of the native microbial population together with chemical analysis of the soil. Quantitative PCR was used for determination the survival of the fungus in soil together with PAH-degrading bacterial genes in PAH contaminated soil. Decrease in toxicity of the soil was measured with earthworms, cress and clover. In some of the experiments extremely high concentration of PAH (5000 mg/kg) and TNT (>10 g/kg TNT) inhibited the fungal growth, and thus no degradation of the contaminants occurred. Dilution of PAH and TNT contaminated soil with garden compost (ratio 1:1 and 1:20, respectively) reduced the toxicity and enabled the fungal growth to soil, but enhanced also the growth of indigenous microorganisms present either in soil or compost. The good quality of the fungal inocula was found to be essential for the growth in contaminated soil. Severe contamination with molds, such as Trichoderma prevented growth of P. velutina completely, but if contamination was minor, P. velutina was able to outcompete the molds. However, in optimal conditions 93 % of PAHs were degraded by both fungus P. velutina and indigenous bacterial population. P. velutina was also the most efficient degrader of TNT. Degradation of PCDD/F could not be proved due to tremendous variation of concentrations in all soil samples (in average concentration was approximately 10 000 ng/kg WHO-TEQ). More experiments are needed to determine the minimum inoculum-soil ratio and other constraints for the fungal treatment method.

S38

Sub-session 1.3 - Phytoremediation and mycoremediation

INOCULATION OF POPLAR WITH ARBUSCULAR MYCORRHIZAL FUNGI EXERTS A PROTECTIVE ROLE ON HEAVY METAL STRESS BY TRANSCRIPTOME MODULATION A. Cicatelli1, V. Todeschini2, G. Lingua2, P. Torrigiani3, S. Biondi4, S. Castiglione1 1

Dipartimento di Chimica, Università di Salerno, 84084 Fisciano (SA), Italy; 2Dipartimento di Scienze dell’Ambiente e della Vita, Università del Piemonte Orientale, 15121 Alessandria, Italy; 3Dipartimento di Colture Arboree, Universita` di Bologna, 40127 Bologna, Italy; 4Dipartimento di Biologia e.s., Universita` di Bologna, 40126 Bologna, Italy

Abstract Phytoremediation (comprising phytoextraction, phytodegradation, rhizofiltration, phytostabilisation, phytovolatilization) is a technology that uses plants, associated or not with microrganisms, for the treatment of soil and water contaminated by organic or inorganic pollutants. Poplar is a tree species with several features suitable for phytoremediation, including a remarkable ability to tolerate high concentrations of heavy metals (HMs). Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with the roots of the vast majority of land plants; they improve nutrient uptake and can, in some cases, enhance phytoextraction and/or phytostabilisation of HMs while alleviating stress in the host plant. In a field trial on a Zn- and Cu-contaminated site, differences in plant survival and growth were observed among 168 clones originating from natural populations of Populus from northern Italy. One well-performing clone (AL35) had a distinctly higher concentration of both metals in the roots, and was used for investigating physiological and molecular markers associated with HM tolerance, such as metallothioneins (MTs) and polyamines (PAs). In field-grown plants, a higher concentration of the PA putrescine positively correlated with root Cu concentration. In a subsequent greenhouse study, AL35 cuttings pre-inoculated with AMF (Glomus mosseae or G. intraradices) and then transferred to pots containing soil collected from the HM-polluted site displayed growth comparable to that of controls grown on unpolluted soil, in spite of higher Cu and Zn accumulation. Such plants also showed an overall induction of MT and PA biosynthetic gene expression, together with increased free and conjugated PA levels. A genome-wide transcriptomic (cDNAAFLP) analysis allowed the identification of a number of genes, mostly belonging to stress-related functional categories of defence and secondary metabolism, that were differentially regulated in mycorrhizal vs non mycorrhizal plants. The relevance of epigenetic changes in plant adaptation to environmental stresses was evaluated by analysing DNA methylation changes (MSAP method); on polluted soil, the most dramatic effects were observed in mycorrhizal plants. It is concluded that mycorrhizal plants exhibit increased capacity for phytoremediation of HM-contaminated soil, together with improved growth, and that their greater stress tolerance may derive from the protective role of PAs, and the transcriptional regulation of stress-related genes.

Posters P1 EFFECT OF COMBINED POLLUTION OF CHROMIUM AND BENZO (A) PYRENE ON SEED GROWTH OF LOLIUM PERENNE Chigbo Chibuike Onyema, L.C. Batty University of Birmingham, U.K.

Abstract The single and joint effects of chromium (Cr) and benzo (a) pyrene (B (a) P) on the seed germination and the elongation of root and shoot of Lolium perenne were investigated. The results showed that in solution, increasing concentration of Cr could inhibit the germination rate as well as root and shoot elongation of L. perenne. Also, the increasing concentration of B (a) P (1-4mg.L-1) could accelerate the germination rate of L. perenne. The joint toxicity of Cr and B (a) P showed that increasing concentration of Cr and B (a) P could have a significant (p< 0.05) antagonistic effect on the germination rate of L. perenne. In the single factor experiments and joint effect tests of Cr and B (a) P on the seedling growth, it was found that root and shoot elongation was inhibited significantly (p< 0.05) for higher concentration of Cr whereas increasing concentration of B (a) P non- significantly (p> 0.05) accelerated the shoot elongation of L. perenne. However, root elongation was inhibited for lower concentration of B (a) P and was accelerated for higher concentration of B (a) P although results were non- significant. There were significant relationships between the concentration of pollutants and the root and shoot elongation (p< 0.05). Higher concentration of B (a) P with low concentration of Cr had significant (p< 0.05) antagonistic effect on shoot and root elongation of L. perenne in solution tests. Also, low concentration of B (a) P with increasing concentration of Cr had a significant (p< 0.05) synergistic effect on shoot elongation. The toxicity effects of Cr and B (a) P to seed germination or root and shoot elongation are in the following sequence: Root elongation> shoot elongation> germination rate.

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Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S35-S52

P2 USING BIOSURFACTANTS IN PHYTOREMEDIATION OF SOIL POLLUTED WITH PETROLEUM HYDROCARBONS G. Kvesitadze1, E. Karpenko2, G. Khatisashvili1, R. Vildanova2, T. Sadunishvili1, N. Gagelidze1, G. Adamia1, L. Amiranashvili1, M. Pruidze1, N. Kuprava1 1 Durmishidze Institute of Biochemistry and Biotechnology, Agrarian University of Georgia. David Agmasheneblis Kheivani, 10th km, 0159 Tbilisi, Georgia; 2Lviv Department of Physical-Organic Chemistry Institute, National Academy of Sciences of Ukraine, Naukova str. 3a, Lviv 79053, Ukraine, e-mail: [email protected]

Abstract Phytoremediation ecotechnologies based on joint application of ecological potential of plants and microorganisms are actively used for remediation of chemically contaminated environment. However, the efficiency of phytoremediation is sufficiently decreased due to the low mobility of pollutant molecules. The similar cases appear while dealing with long-term contamination of soil with crude oil, when light hydrocarbons of oil have volatilized and the remaining components with long chains form resin mass are hardly removable. The problem could be solved via improvement of phytoremedition technologies by application of surface-active substances, capable to emulsifying resin mass, and improving its penetration to plants. The biosurfactants produced by specially selected microorganisms fit this purpose. The advantages of biogenic surfactants if compared with their synthetic analogues are their higher surface activity, efficiency at extreme temperatures and pH, biodegradability, and positive influence on plants growth. In the presented work the results of using microbial surfactants rhamnolipids, rhamnolipid biocomplex PS and trehalose lipids) in model phytoremediation experiment targeted to clean the soil artificially contaminated with crude oil (the level of initial contamination by total petroleum hydrocarbons (TPH) equals 27 500 ppm) have been shown. Preliminary selected according to oil degradation ability bacterial consortium (consisting of oil-destructor strains of Pseudomonas and Bacillus) and alfalfa (Medicago sativa) were applied in the experiment. Mass of the contaminated soil samples was equal to 7.5 kg. Suspension of bacteria (1.2 l) and solutions of biosurfactants (100 mg/500 ml) were introduced to each soil sample at the beginning of the experiment. On 14th day after inoculation plants were sowed in soil samples. The experiment was continued during 3 months at greenhouse conditions. The chromatographic analysis of contaminated soil samples after remediation process have shown that as a result of abiotic and biotic processes (volatilization of hydrocarbons and their assimilation by aboriginal microflora) TPH content in contaminated soil was decreased by 45% during the three months. The use of plants without bacterial consortium and biosurfactants resulted in soil decontamination by 63%. Plants and bacteria jointly were capable to assimilate 82% of oil hydrocarbons. The application of biosurfactants resulted in further intensification of bioremediation process: in cases of trehalose lipids and rhamnolipids the maximum cleaning effects by 96 and 98%, correspondingly are achieved. The results clearly indicate the efficiency of using biosurfactant preparations for the enhancement of phytoremediation process.

P3 MOLECULAR BIODIVERSITY OF ARBUSCULAR MYCORRHIZAL FUNGI (AMF) IN TRACE METALS CONTAMINATED SOILS AND THEIR ROLE IN SOIL PHYTOREMEDIATION S.E. Hassan, M. St-Arnaud, M. Hijri Université de Montréal, Département de sciences biologiques, Institut de recherche en biologie végétale (IRBV), 4101 rue Sherbrooke Est, Montréal, QC, H1X 2B2, Canada

Abstract We assessed the indigenous arbuscular mycorrhizal fungi (AMF) community structure from the roots and associated soil of Plantago major (plantain) growing on sites polluted with trace metals (TM), and on unpolluted sites. Uncontaminated and TM contaminated sites containing As, Cd, Cu, Pb, Sn, and Zn, were selected based on a survey of the TM concentration in soils of community gardens in the City of Montreal (Canada). Total genomic DNA was extracted directly from these samples. PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE), complemented by cloning and sequencing, as well as direct sequencing techniques, were used together to investigate AMF community structure. We found a decreased diversity of native AMF (assessed by the number of AMF ribotypes) in soils and roots harvested from TM polluted soils compared to unpolluted soils. We also found that community structure was modified by TM contamination. Various species of Glomus and Scutellospora were the most abundant ribotypes detected in unpolluted soils; ribotypes of G. etunicatum, G. irregulare/ G. intraradices and G. viscosum were found in both polluted and unpolluted soils, while ribotypes of G. mosseae and Glomus spp. (B9 and B13) were dominant in TM polluted soils. The predominance of G. mosseae in metal polluted sites suggests the tolerance of this species to TM stress, as well as its potential use for phytoremediation. A greenhouse trial was conducted to investigate the role Glomus irregulare and G. mosseae on cadmium (Cd) uptake by sunflower plants grown in soil complemented with three Cd concentrations (0.75, 10, and 30 mg kg-1). Ten weeks after sowing, root colonization, plant biomass, and Cd, Zn, and Cu concentrations in plant tissues were determined. We found that root mycorrhizal colonization rates were not significantly affected by Cd treatments. At low soil Cd concentration (0.75 mg kg-1), G. irregulare-inoculated plants had significantly higher shoot Cd and Zn concentrations than plants inoculated with G. mosseae and non-inoculated plants. At 10 mg kg-1 of Cd concentration in soil, no significant difference in shoot TM concentrations was found between plants inoculated with G. irregulare and non-inoculated plants. While, At 30 mg kg-1 of Cd concentration in soil, G. mosseae-inoculated plants had significantly lower shoot Cd and Zn concentrations and biological exchange factor (BCF) values than plants inoculated with G. irregulare and non-inoculated plants. The results indicated that these AMF strains mediate different tolerance strategies to alleviate TM toxicity in their host plants.

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Sub-session 1.3 - Phytoremediation and mycoremediation

P4 BIODEGRADATION OF 17α-ETHINYLESTRADIOL BY EDIBLE WHITE ROT FUNGUS–A MECHANISTICAL STUDY Zdena Kresinova1,2, Martin Ezechias1,2, Tomas Cajthaml1,2 1

Laboratory of Environmental Biotechnology, Institute of Microbiology ASCR, v.v.i., Division Ecology, Vídeňská 1083, 142 20, Prague 4, Czech Republic, e-mail: [email protected]; 2Institute for Environmental Studies, Science Faculty, Charles University of Prague, Benátská 2, 128 01, Prague 2, Czech Republic

Abstract The presence of trace amounts of endocrine disrupting chemicals (EDCs) in the environment is of great scientist interest around the world. Long-term exposition to EDCs can cause adverse effects on water organisms and humans, because of their negative influence of hormonal systems. Bioremediation represents in many cases effective, non-expensive and environment-friendly solution. However, the knowledge of degradation mechanisms including the transformation products is of high importance. In this study, degradation of synthetic estrogen 17ethinylestradiol (EE2) was performed using widespread edible ligninolytic fungal strain Pleurotus ostreatus. In order to elucidate EE2 degradation pathways, a broad set of in vivo and in vitro experiments has been performed. The study involved extracellular and intracellular degradation experiments with the fungal culture, purified ligninolytic enzymes and isolated microsomal fractions. EE2 derived metabolites were identified using gas chromatography coupled with mass spectrometry technique. Generally, the EE2 transformation led to more polar compounds and involved mainly hydroxylation and methoxylation mechanism, but in some cases also dehydrogenation and ring fission of B- and C-rings. Changes of estrogenic activities during the biodegradation were monitored by a recombinant yeast assay and the results did not always correlate with the EE2 concentration decrease. The biodegradation results and the metabolites structures proved a possible involvement of both extracellular and intracellular enzymes in fungal EE2 removal mechanisms. Acknowledgements This work was funded by grants No. TA01020804 of the Czech Technology Agency.

P5 UTILIZATION OF ARBUSCULAR MYCORRHIZAL FUNGI FOR THE PROTECTION OF TOMATO PLANTS (SOLANUM LYCOPERSICUM) OF Cr(VI) TOXIC CONCENTRATIONS Yazmín Carreón-Abud, Miguel Martínez-Trujillo Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo Morelia, Michoacán, México, e-mail: [email protected]

Abstract Chromium (Cr) is a highly toxic non-essential metal for microorganisms and plants. In the cytoplasm, Cr toxicity is mainly related to reduction processes of Cr (VI) to Cr (III) through intermediate unstable states which form free radicals (Cervantes et al., 2001: FEMS Microbiol. Rev. 25: 335). Many agricultural systems are contaminated with heavy metals due to irrigation with industrial or residual waters. In Mexico, the tomato (Solanum lycopersicum) is considered the most important horticultural species for its production value. Under natural conditions, over 80% of plants are colonized by arbuscular mycorrhizal fungi (AMF) (Smith and Read, 2007: Mycorrhizal Symbiosis). The extensive extraradical hyphal network of these fungi allows plants to increase nutrient uptake and translocation, and may increase tolerance to stress conditions, including the presence of heavy metals (Gaur and Adholeya, 2004: Current Sci. 86:528). Due to the problem of contamination by heavy metals (including Cr) and the need to counter it, in this work it was determined that the association of several species of AMF to tomato plants, has several benefits. Tomato seeds were germinated and transplanted at 21 days, to pots and grown under greenhouse conditions, were watered with nutrient solution and exposed to different concentrations of Cr (VI) (0, 1000, 3000 and 6000 ppm). In each treatment, half of the plants (30) were inoculated with a consortium of the following species: Glomus invermaium Hall, Glomus mosseae (Nicol & Ger) Gerd & Trappe, Glomus aggregatum and Gigaspora Scheck & Smith sp. aff. margarita. The plant survival was significantly improved in the treatments with AMF (70%) compared to the control without fungi (22%), in the treatment of 6000 ppm of Cr (VI). The dry weight of aerial parts increased 25% by the presence of AMF in the treatment of 3000 and 6000 ppm, while the root dry weight increased 300% and 30% by the presence of fungi in Cr (VI) at 1000 and 3000 ppm, respectively. We consider that the protection of tomato plants by association with AMF can be used as a strategy to improve the crop production in areas contaminated by Cr (VI) and possibly other metals.

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P6 USING ARBUSCULAR MYCORRHIZAL FUNGI TO ENHANCE PLANT GROWTH IN MAIZE (ZEA MAYS) IN SOILS WITH HIGH CONCENTRATIONS OF ALUMINUM María del Rocío Madrigal-Pedraza1, Mayra E. Gavito2, Miguel Martínez-Trujillo1, Yazmín Carreón-Abud1 1

Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo; 2CIECO, Universidad Nacional Autónoma de México, Morelia, Michoacán, México, e-mail: [email protected]

Abstract Approximately 30% of the world’s total land area consists of acid soils, and as much as 50% of the world’s potentially arable lands are acidic. It is known that generally plants grown in acid soils due to Al solubility at low pH, have reduced root systems and exhibit a variety of nutrient-deficiency symptoms, with a consequent decrease in yield. Among the mechanisms employed by crop plants that enable them to tolerate toxic levels of Al are those that exclude Al from the root apex and those that allow the plant to tolerate Al accumulation in the root and shoot. Maize is the main crop cultivated in Mexico, including the tropical areas, where the toxicity by aluminum reduces its yield. Although Al-tolerant genotypes from maize have been reported, this alternative does not work for all regions Under natural conditions, 80% of plants are colonized as symbiotic associations with arbuscular mycorrhizal fungi (AMF), which are found in most plant systems and climates. In addition to improving the nutrition of plants, it has been observed that there are variable effects of AMF in the interaction of plants with metals. A range of factors including the inherent properties of the fungus, the ability to capture the heavy metal by plants are characteristics that may influence the uptake of metal in the soil by mycorrhizal plants. This paper evaluates the effect of three types of AMF inocula to confer tolerance to maize plants grown in soil supplemented with aluminum. We used three different mycorrhizal inocula: 1) Native population (obtained of soils with high concentrations of soluble aluminum, 2) Acaulospora delicata, 3) Gigaspora gigantea. The aluminum concentrations used were 0, 50 and 100 ppm. The seeds of the variety NB9, were germinated and grown in special pots of PVC under greenhouse conditions. It was observed that only the native inoculum provided a significant increase in dry weight of root and stem, increasing the plant biomass by 20% compared to the control without AMF. Although the native micelia growth was only 50-90% compared to the A. delicata and G. gigantea, the effect on the plant growth was higher. The results show that the AMF can serve to increase the tolerance of maize plants to aluminum, when adequate inocula are used, particularly by fungal populations derived from acid soils.

P7 BIOREACTOR OPTIMIZATIONFOR THE TREATMENT OF INDUSTRIAL WASTEWATERS BY MEANS OF A FUNGAL STRAIN Federica Spina, Antonella Anastasi, Alice Romagnolo, Valeria Tigini, Valeria Prigione, Giovanna Cristina Varese Université de Caen Basse-Normandie - UR ABTE - F-14032 Caen, France Napoca, Romania

Abstract The wastewaters outgoing from the industries can cause serious environmental damages on the whole ecosystem. Both public opinion and institutions are becoming more and more aware about the risks and of course, they claim for deep controls on the effluents discharged. It stands to reason that appropriate wastewater treatments are mandatory. It should be also considered that the composition of these effluents changes continuously; so that a single approach, able to degrade always and each compound, seems to be unrealistic. From an applicative point of view, it should be hypothesized an approach combing different techniques, which could exploit their potential towards different molecules. At the moment, several processes are under investigation, but they often have some drawbacks in terms of economical and environmental sustainability. In the present study, several fungal strains have been tested towards real industrial wastewaters. The efficacy of the treatment was monitored following the decolourisation percentage and the modification of other parameters as the chemical and biological oxygen demand. Since some abiotic parameters could deeply limit even the survival of living organism, in some cases their control is required. In this study, working with very alcaline effluents (pH up to 12), the pH control was often a necessary step. Moreover, since these wastewaters lack of organic compounds useful for the fungal growth, the addition, even in low amount, of nutrient could allow the development of a more active biomass, improving the final efficacy of the treatment. From an applicative point of view, the potential of the fungus should be evaluated monitoring its capability to compete with the autochthonous bacterial microflora of the wastewaters. According to this, all the experiments were carried out with non sterile effluents and in not axenic working conditions. Moreover, as above mentioned, any new technique should be able to work with the already existing ones, hopefully compensating the shortcomings shown by other processes. With this aim, in the present study, the fungal treatment was compared with the activated sludge one, already in use in the wastewater treatment plant of interest, in order to define if the two techniques could work in a synergic way. Finally, anytime a process shows promising potential, it should be always considered whether it could be scaled-up to higher volumes than laboratories ones. Different reactor set-up’ and operative strategies have been tested in order to find out the best solution which coupled interesting bioremediation efficiency with higher working volumes.

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P8 NEW INSIGHT INTO FUNGAL DEGRADATION OF POLYCHLORINATED BIPHENYLS Tomáš Cajthaml*1, Monika Čvančarová1,2, Zdena Křesinová1,2, Alena Filipová1, Tatiana Stella1,3, Stefano Covino1 1 Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic; 2Institute of Environmental Studies, Faculty of Science, Charles University, Benátská 2, CZ-128 01 Prague 2, Czech Republic; 3University of Tuscia, Via San Camillo De Lellis s.n.c., 01100 Viterbo, Italy

Abstract Polychlorinated biphenyls (PCBs) due to their persistency and toxicity represent a serious environmental problem. A high number of studies have been published about bacterial transformation of PCBs; however, PCB transport into the bacterial cells represents a limiting factor for their effective degradation. Moreover, probably the most important biphenyl-upper-pathway leads to production of chlorobenzoic acids (CBA) that represents dead-end toxic metabolites inhibiting further PCB transformation. On the other hand, information about fungal PCB degradation and the respective mechanisms is limited. The aim of this study was to explore degrading capabilities of ligninolytic fungal representatives (LF) towards polychlorinated biphenyls under various physiological conditions. The degradation was tested in axenic cultures of the fungi in various liquid media that were artificially spiked with a technical mixture of Delor 103; however, also in a historically contaminated soil. An attention was also paid to elucidation of the degradation mechanisms performing degradation experiments with various isolated enzymes or sub-cellular fractions and to degradation of fungal PCB transformation products that were detected i.e. CBA. The degradation results were evaluated from many perspectives including analytical quantification, qualitative analysis of the transformation products, ecotoxicological monitoring and microbial population analyses. Generally, the results showed that the LF are very efficient in PCB removal when e.g. Pleurotus ostreatus 3004 degraded more than 90% of PCBs in low nitrogen-mineral liquid medium in 42 days and Irpex lacteus removed more than 75% of PCBs. The soil degradation experiment revealed that out of the most toxic PCB congeners, Pleurotus ostreatus and Irpex lacteus were able to remove 51% and 34% of PCBs, respectively. The CBA degradation experiments revealed that these typical bacterial PCB dead-end products are transformed by the fungi even more rapidly than the original compounds. On the other hand, the toxicity monitoring showed that after degradation of PCBs under model conditions, the acute toxicity increased in several cases. An attention was also paid to detection of PCB metabolites produced during the degradation using mainly mass spectrometry techniques, when hydroxyl and methoxyl PCB derivatives and further ring fission products were detected resulting in production of CBAs and related compounds, that were further transformed. The results suggest that both intracellular (P-450 monooxigenase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase) and extracellular (ligninolytic) enzymatic systems are involved in the biotransformation of PCB by LF. Acknowledgements This work was funded by grant 525/09/1058 of the Czech Science Foundation.

P9 MYCOREMEDIATION OF PCBS DEAD-END METABOLITES: IN VIVO AND IN VITRO DEGRADATION OF CHLOROBENZOIC ACIDS BY THE WHITE ROT FUNGUS Lentinus tigrinus T. Stella1, S. Covino2, Z. Křesinová2, A. D’Annibale1, M. Petruccioli1, T. Cajthaml2 1

Department for Innovation in Biological, Agro-Food, and Forestry systems (DIBAF), University of Tuscia, Viterbo, Italy 2 Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

Abstract Chlorobenzoic acids (CBA) are ubiquitous organic contaminants with different degree of chlorination. Besides their use as herbicides, these compounds are often found in the environment in association with polychlorinated biphenyls (PCBs). It is noteworthy, in fact, that the aerobic bacterial co-metabolism of PCBs often leads to the accumulation of CBAs as dead-end products. Several CBA isomers possess endocrine-disrupting activity and exhibit toxicity towards aquatic organisms (i.e. ciliates, Daphnia sp., algae and fishes) and genotoxicity towards higher plants. Considering the remarkable bioremediation potential of fungi towards a wide array of aromatic pollutants, the aim of the present work was to assess the CBA-degrading capability of Lentinus tigrinus CBS 577.79 and to clarify the CBA-biotransformation mechanisms. In vivo experiments were performed using axenic cultures, under both stationary and shaken conditions, in either complex or mineral media. Seven day-old cultures were spiked with a mixture of 12 CBA (mono-, di- and tri-CBA) (CBAM) to reach a final concentration of 120 mg/L. Chlorobenzoates did not exert inhibitory effects on fungal growth and on the production of the ligninolytic enzymes, such as laccase and manganese peroxidase (MnP). In particular, spiking of the CBAM stimulated laccase activity with respect to the relative control (nonspiked cultures). The large majority of CBAs was efficiently removed by L. tigrinus cultures with the sole exceptions of 2,6-DCBA, 2,3,6and 2,4,6-TCBA, the partial depletion of which was only observed in MEG-submerged cultures. The structural identification of CBA degradation products (i.e., chlorinated benzoaldehydes and benzyl alcohols, chlorotoluenes and chlorophenols) suggested that the transformation of these compounds was the result of a combined action of both extracellular and intracellular enzyme systems. In vitro experiments with purified laccase and MnP, in the presence and in the absence of redox mediators, showed that these enzymes were not involved in the initial breakdown steps of chlorobenzoates. Conversely, in vitro tests carried out with the extracted microsomal fraction confirmed the involvement of cytochrome P450 enzymes in CBA degradation process. Moreover, the Vibrio fischeri assay showed that the fungus was able to partially remove the toxicity associated with the CBAM.

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P10 SELECTION OF MICROFUNGI WITH HIGH LIPOLYTIC ACTIVITY AND THEIR LIPASE CHARACTERIZATION S. Chinaglia1, L.R. Chiarelli2, G. Valentini2, A.M. Picco1 1

Department of Earth Science and Environment, Laboratory of Mycology, University of Pavia, Via San Epifanio 14, 27100 Pavia 2 Department of Biochemistry “A. Castellani”, University of Pavia, Via Taramelli 3/b, 27100 Pavia

Abstract Lipases are serine hydrolases that catalyze both the hydrolysis and the synthesis of esters from glycerol and long chain fatty acids. They are the third largest enzyme group based on their market value, finding extensive applications in chemical, pharmaceutical, food and leather industries. Additionally, a promising field for the application of lipases and lipolytic microorganisms is in the environmental management, in particular in the biodegradation of plastic and hydrocarbon and in treatment of waste with high fat content. Lipids are major organic matters in municipal and some industrial wastewater and solid wastes and can cause severe environmental pollution. Several research reports and patents describe the use of active lipid degrading microorganisms and/or enzyme pools developed in the laboratory for the biological treatment of effluents or waste with high fat and oil concentrations. In view of the variety in applications, there has been a renewed interest in the development of sources of lipase. Numerous microbial species produce lipases with different enzymological properties but fungi are known to be more potent lipase producers. The aim of this study was to screen and select microfungi with high lipolytic activity from a strains pool isolated from different sources. Furthermore lipase characterization of two mesophilic strains isolated from wastes selected as good lipase producers was achieved. A semiquantitative evaluation of the lipolytic ability of strains was carried out on solid media including, as carbon source, synthetic and natural fatty substrates. The ability to hydrolyze these compounds was estimated with the measurement of the precipitation/clearing zone around the colony. Among the screened strains, two of the most active towards all the substrates tested, Penicillium solitum and Cladosporium cladosporioides, were chosen for biochemical characterization of lipolytic enzymes. A preliminary ecophysiological characterization allowed to indicate their thermal and trophic preferences and their growth rate. Moreover, their extracellular lipases were purified to homogeneity and biochemically characterized. The lipase activity was determined spectrophotometrically using p-nitrophenyl esters as substrates. Substrate specificity and the effects of pH and temperature on lipase activity were studied. Both lipases showed maximal activity at alkaline pH. However they differed in substrate specificity and in thermal stability. Lipase from P. solitum was more active towards long-chain substrates; whereas lipase from C. cladosporioides versus short-chain substrates. Finally, the enzyme from C. cladosporioides was considerably more thermostable than the P. solitum one (T50, nearly 19°C higher). These results are interesting to develop a preliminary consortium for waste treatment.

P11 DEGRADATION OF ENDOCRINE DISRUPTING CHEMICALS AND REMOVAL OF ESTROGENIC ACTIVITY BY LENTINUS TIGRINUS AND ITS EXTRACELLULAR ENZYMES S. Covino1, T. Stella2, Z. Křesinová1, A. D’Annibale2, M. Petruccioli2, T. Cajthaml1 1

Academy of Sciences of Czech Republic, Prague, Czech Republic; 2University of Tuscia, Viterbo, Italy

Abstract Endocrine disrupting compounds (EDCs) are environmental chemicals capable of interfering with natural hormones in human and wildlife endocrine systems. The heterogeneous family of EDCs comprises a wide range of substances (i.e. persistent organic pollutants (POPs), surfactants, plasticizers, estrogens and personal care products ingredients) which may cause adverse effects. Concerning the biodegradation of EDCs, it is of note that ligninolytic fungi and their extracellular enzymes exhibit a remarkable potential for the removal of these compounds. Therefore, the main objective of the present study was to investigate the degradation capacity of the basidiomycete Lentinus tigrinus towards four representative EDCs, i.e., 17α-ethinylestradiol (EE2), bisphenol A (BPA), nonylphenol (NP) and triclosan (TRC). Whole-cell bioconversion experiments were conducted under both stationary and shaking conditions using two standard media, namely malt extract-glucose (MEG) and low-nitrogen Kirk’s medium (LNKM). Fungal cultures were then spiked with either EE2, BPA, NP or TRC. Moreover, in order to evaluate the involvement of L. tigrinus lignin-modifying enzymes in the degradation process, in vitro treatments of the four EDCs were performed with purified laccase and MnP isoenzymes, under both mediated and non-mediated conditions. The outcome of in vivo and in vitro incubations was assessed also by determining the residual estrogenic activity of ethyl acetate-extracted reaction mixtures. Regardless of the treatment typology, EE2, BPA and NP were effectively degraded by L. tigrinus cultures, and the processes were accompanied by the complete removal of the estrogenic activities associated to those culture extracts. Contrarily, TRC was more recalcitrant and it was not significantly degraded in shaken cultures conducted on LNKM. Accordingly, a high residual estrogenic activity was found in ethyl acetate extracts of the latter samples. As for the enzymatic treatments, laccase was more efficient than MnP in the oxidation of EE2 and BPA under non-mediated conditions, while MnP oxidized NP and TRC at a faster rate than the former. In mediated reactions, best degradation performances towards EE2, BPA and TRC were observed with the laccase/mediator system while NP was more susceptible to MnP oxidation. The estrogenic activities of EE2 and BPA were significantly removed by all in vitro treatments and their removal extents were not significantly affected by the type of treatment. A high impact of the treatment typology, conversely, was observed for NP and TRC, the residual estrogenic activities of which were best removed by the laccase and laccase/mediator systems. Acknowledgements This work was funded by grant No. TA01020804 of the Czech Technology Agency.

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P12 ECO-EFFICIENCY ANALYSIS OF A FUNGAL BIOREMEDIATION METHOD Markus Räsänen*1, Erika Winquist1, Marja Tuomela2, Matti Leisola1, Jaana Sorvari3 1

Department of Biotechnology and Chemical Technology, P.O. Box 16100, 00076 Aalto University, Finland; 2Department of Food and Environmental Sciences, P.O. Box 56, 00014 University of Helsinki, Finland; 3Finnish Environment Institute (SYKE), P.O. Box 140, 00251 Helsinki, Finland; e-mail: [email protected]

Abstract A novel fungal bioremediation method was compared to more conventional soil clean-up technologies using an eco-efficiency analysis tool developed in the Finnish Environment Institute. The study focused on a treatment of PAH-contaminated soil using six alternative soil remediation methods: hazardous waste landfilling, thermal treatment, bitumen stabilization, aerated fungal treatment, fungal treatment without aeration and aerated garden waste compost treatment. For the eco-efficiency analysis, data was compiled on the costs, environmental effects and achievable risk reduction of these alternative remediation techniques. The fungal treatment method was a main focus and data for its eco-efficiency appraisal was generated in a separate field study using 2 tons of PAH-contaminated soil. The soil was first diluted 1:1 (mass ratio) with garden waste compost, which resulted in the concentration of approximately 1400 mg PAH/kg soil (sum of 16 EPA PAH). During a 5-month-treatment the PAHs degraded similarly in both the fungal treatment pile (inoculated with white-rot fungus Phanerochaete velutina FBCC941, obtained from the Fungal Biotechnology Culture Collection of University of Helsinki) and the control pile (without fungal inoculum). The concentrations of the PAHs after 5 months were approximately 100 mg/kg (fungal treatment) and 80 mg/kg (control). The degradation of the PAHs took virtually place in both soil piles during the first 3 months of the experiment. In the eco-efficiency analysis, thermal treatment turned out as the most eco-efficient method, which lowered the risks effectively and economically, without significant environmental effects. The second most eco-efficient method was bitumen stabilization, but it is quite questionable method regarding sustainability because stabilization doesn’t remove the pollutants from the soil, and it always requires a disposal. Aerated garden waste compost treatment and fungal treatment without aeration with low inoculation ratio did also well in the eco-efficiency comparison. It can be concluded that fungal treatment could have potential in remediation of PAH-contaminated soils, as well as other contaminated soils, when properly optimized. Without aeration and with optimized compost to soil ratio garden waste compost treatment could offer a highly eco-efficient method for remediation of PAH-contaminated soil.

P13 EVALUATION O MYCROCISTIN BIODEGRADATION BY WILD YEASTS FOR APPLICATION IN WATER TREATMENT Gisele Maria de Andrade de Nobrega*1, T.A. Miguel2, D.D. Lopes3, C.L. Yokoyama4, F.C. Pagnocca5, E.K. Kuroda6, K. Tsuji7, A.R. Coelho8, E.H. Hashimoto8, S.Garcia9, L.D. Paccola-Meirelles1, O. Kawamura10, K-I. Harada11, E.Y. Hirooka9 1

Department of General Biology, Center for Biological Sciences, State University of Londrina, Londrina-State of Paraná, Brazil; 2Department of Food Science and Technology, Center of Agricultural Sciences, State University of Londrina; 3Department of Genetics, Federal University of Rio Grande do Sul; 4 Department of. Food Science and Technology, Center of Agricultural Sciences, State University of Londrina; 5State University of São Paulo, Institute of Biosciences of Rio Claro; 6Department of Construction, Center of Technology and Urbanism, State University of Londrina; 7Kanagawa Prefectural Institute of Public Health, Chigasaki, Kagawa, Japan; 8Federal Technological University of Paraná, Campus Francisco Beltrão; 9Department of Food Science and Technology, Center of Agricultural Sciences, State University of Londrina, University Campus. P.O.Box 6001. 86051-990, Londrina-State of Paraná, Brazil; 10Department of. Biochemistry and Food Science, Faculty of Agriculture, Kanagawa University, Japan; 11Faculty of Pharmacy, Meijo University, Nagoya, Aichi, Japan; e-mail:[email protected]

Abstract Yeasts are one the most important safe microrganisms in fermentative process nowadays, and play an important role, with emphasis on non- pathogenic. Many studies show the potential of this microrganisms in biologic control. Mycrocistins (MC) are produced by toxic cyanobacteria such Microcystis aeruginosa, which occur in eutrophic water environmental and cause undesirable economy loss, as well as human and animal health hazards. Conventional water treatment has been ineffective in MC removal and the application of chemical agents has caused negative environmental impact. Therefore using yeasts for biocontrol would be a promising strategy to avoid MC hazard in water supply. In this work, 31 yeasts strains isolated from anthill and sugar and bioethanol plants, were tested for MC degradation. The yeasts were analyzed for MC degradation potential with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Yeast cells suspension was added to 8 ml of solution containing 1 mg/l of MC (lyophilized crude extract prepared with cell mass of Mycrocystis spp. Strain TAC 95). After 96 h of incubation at 30oC by shaking at 100 rpm MC levels were evaluated by ELISA. Negative control was crude cell extract of strain TAC 95 without yeast cells. Positive control was carried out by adding S. microcystinivorans strain B9 to the crude cell extract suspension prepared with TAC 95. B9 strain is characterized as having a potent MC degrading activity, showing 97% to 99% of MC degradation. Yeasts MC degradation rates varied from 0% to 70%, and the highest degradation was observed when using strain VI08R, which is a Saccharomyces cerevisiae, hence, a GRAS degree microorganism. S. boulardii commercial strain, another GRAS degree yeast, and also presented high MC degradation rate (53%). The data indicated that tested yeasts isolates can be a promising strategy for MC degrading and their application can be more explored with promising results to water treatment. Acknowledgements Financial Support: NANOBIO/CAPES Foundation, Ministry of Education/BRAZIL. Araucária Foundation and Paraná Fund (Paraná State Grant).

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P14 ENZYME ACTIVITIES AND RESPIRATION AS BIOINDICATORS OF THE BIOLOGICAL QUALITY OF Pb-Cd-CONTAMINATED SOIL UNDER AIDED PHYTOSTABILIZATION Jacek Krzyżak1, Grażyna Płaza1, Rosa Margesin2, Daniel Wasilkowski3, Agnieszka Mrozik3 1

Institute for Ecology of Industrial Areas, Environmental Biotechnology Department 6 Kossutha St., 40-844 Katowice Poland; 2Institute of Microbiology, University of Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria; 3University of Silesia, Department of Biochemistry, 40-032 Katowice, 28 Jagiellońska St., Poland

Abstract The ecological evaluation of recultivation and remediation management systems requires special attention to be given to the use of soil health bioindicators, based on soil biological properties. Specifically, microbial properties related to the size, structure, activity and biodiversity of the microbial populations are the most promising biological indicators. Microorganisms respond quickly to the different stressors, and they rapidly adopt to environmental conditions. This adaptation potentially allows microbial analyses to be discriminating in soil health assessment, and changes in microbial populations and activities may therefore function as an excellent indicator of change in soil health. Thus, microbiological measurements could be used as soil biomarkers for biomonitoring, soil remediation efficiency, and as tools for evaluation of soil quality criteria or Ecological Risk Assessment. Microbial indicators have been broader applied to describe of microbial communities in polluted sites and to evaluate the remediation processes, i.e., their capacity to restore soil health. The aim of the study was to evaluate the short-term effect of aided phytostabilization on the microbial indicators such as respiration and key enzyme activities (dehydrogenase, acidic and alkaline phosphatases, protease, ß-glucosidase) in a pilot-scale (plot) experiment. Pb-Cd highly contaminated soil was amended with lignite and lime to reduce metals bioavailability and vegetated with Festuca arundinacea grass species. After amendments application the exchangeable metal concentrations significantly decreased. Cd and Zn bioavailable forms were reduced by about 70 % and 60 %, respectively. The reduction of As bioavailability was 69.3 %. Amendments increased the pH and organic matter content in the soil. Moreover, surface of amended soil was densely covered by tall fescue. The presented aided phytostabilization option significantly increased the enzymes activities. The activities of dehydrogenase, acidic and alkaline phosphatases were high (averages 17.10 µg TPF/g s.m. 24h., 128 µg PNP/g s.m. h. and 145 µg PNP/g s.m. h, respectively) in the treated soils, and significantly increased close to control soil values. Also, soil respiration, substrate-induced soil respiration and ß-glucosidase activity were significantly higher in the treated soil compared to the control soil, while the opposite was observed for protease activity. There were significant differences between enzymes activities and respiration in treated and untreated soils. The in situ aided phytostabilization approach of Pb-Cd contaminated soil proposed in this study increased enzymes activities and respiration, and reduced microbial stress in remediated soil, showing that aided phytostabilization can be a suistainable option for heavy metals contaminated soil management.

P15 LASER STIMULATION OF NATIVE TRICHOPHYTON MENTAGROPHYTES VAR. GRANULOSUM AS A NEW METHOD FOR THE ENHANCEMENT BIODEGRADATION OF PETROCHEMICAL HYDROCARBONS

Jan W. Dobrowolski1, A. Budak2, D. Trojanowska2, M. Rymarczyk3, J. Macuda3 1

AGH, Krakow, Poland; 2CM UJ; 3AGH

Abstract Referring to laser photostimulation of different species of plants for better adaptation to the contamination of soil with petrochemical pollutants; our team initiated study on application of laser biotechnology for the enhancement biodegradation of hydrocarbon. We found the algorithm for efficient photostimulation of the growth of fungus Trichophyton mentagrophytes var. granulosum, activity of N-acetylobeta glucozoamidase and significantly increased the biodegradation rate of different hydrocarbons, including PAH. Dermatophites were isolated from the samples of ground from the area of the oldest in the world region of oil exploitation in south east Poland, using standard procedure of homogenization of the samples of ground in physiological solution of NaCl; followed by cultivation of the fungi on solid Sabouraud mileau (bioMerieux). Incubation was taking place for 3 weeks at 27°C. Four species of Trichophyton were isolated. T. mentagrophytes var. granulosum was cultivated on mileau supplemented with agar. Inoculum of density 9 x108 spors of the fungi in 1 ml was treated with coherent light of 670 nm from medical laser of 20mW, or by light 473 nm of laser diode of 20 mW. The time of exposure was 3 repetitions of 1 sec or 30 sec. The activity of 19 enzymes of control and photostimulated fungi were analysed after 14 days of incubation by APIZYM (bioMerieux) kit. In laser treated fungi the activity of important in bioenergy of cells enzyme Nacetylo-beta-glucozamidaze was enhanced (in comparision with control material). After 50 days of cultivation of the fungi in liquid mileau contaminated with crude oil (in Erlenmeyer bulbs), the concentration of oil hydrocarbons in control and experimental groups was analised by Fourier’ s method of infrared spectroscopy. Biodegradation rate of all the tested groups of hydrocarbons (especially polycyclic aromatic hydrocarbons) was much higher in the samples containing laser stimulated Trichophyton mentagrophytes varietas granulosum, than in the control groups. Experimental study focused on further enhancement of biodegradation of polycyclic hydrocarbons in oil contaminated soil – using different algorithms of laser stimulation of whole consortium of microbs from the samples of soil contaminated for a very long time–is in progress.

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P16 PHENOLIC METABOLITES, OXIDATIVE ENZYMES AND MICROBIAL ACTIVITY IN THE RIZOSPHERE OF Cyperus laxus IN PHYTOREMEDIATION OIL SPILL IMPACTED-SITES PROCESS Noemí Araceli Rivera Casado1, María del Carmen Montes Horcasitas1, Refugio Rodríguez Vázquez1, Fernando José Esparza García1, Armando Ariza Castolo1, Josefina Pérez Vargas2, Octavio Gómez Guzmán1, Graciano Calva Calva1* 1

Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. IPN No. 2508, Col. San Pedro Zacatenco 07360 México D.F.; 2 TESE, México, e-mail : [email protected], [email protected]

Abstract Although it has been proposed that phytoremediation processes may be mediated by chemical and enzymatic interactions between contaminant compounds and plant metabolites there are few reports into systems that produce such compounds. With the objective to investigate such interactions, in this work the phenolic profile in a Cyperus laxus phytoremediation system was studied. The presence of complex compounds, in the rhizospheric soil and the plant tissue, derived from chemical interaction between polyaromatic hydrocarbons (PAH) and plant phenolics was investigated. Cyperus laxus is a pioneer plant species in this oil spill impacted-sites of the tropical wetlands of Tabasco, México, and was able to remove c.a. 270 g of total hydrocarbons (340)/kg soil when greenhouse-cultivated in soils from the impacted sites. The PAH and phenolic HPLC-UV profile showed that in both soil and plants (root, bulb and leaf), most PAH were not chemically free but conjugated, typically with flavonoids and phenylpropanoids putatively produced by Cyperus. In both bulb and leaves anthracene, acenaphthene, phenanthrene and fluoranthene were the most common PAH, and protocatechuic acid, coniferyl alcohol, p-hydroxybenzoic acid, and compounds with structures like chrysine, quercetin and luteolin were the most common phenolics. Typically, conjugation of PAH in both soil and plants was mainly with quercetin or hydroxy-3-methoxybenzoic acid. The presence of conjugated PAH with plant phenolic compounds, suggests chemical interactions, some of the probably mediated by enzymatic activity. Preliminary results from assays for peroxidases, catechol oxidases, dioxygenases and polyphenol oxidases from Cyperus plants over some PAH and phenolics seems to confirm such hypothesis. For example, oxidative enzymatic reactions of anthracene with plant extracts yielded structures-like o-quinone, o-phenols and 9,10-anthroquinone, the typical oxidative anthracene metabolites described for fungal enzyme. Further analysis of the enzymatic products by MS-TOF showed also the presence of acids, aldehydes, ketones and alcohols as principal products. These results suggest that as part of the phytoremediation mechanism, Cyperus laxus produces phenolic compounds able to interact with PAH in both the rhizospheric soil and the plant tissue. Thus, the oxidative enzymes and phenolics from Cyperus, which could be secreted to the rhizosphere or not, could act as chemical mediators to produce the conjugated compounds detected in this work. These compounds finally should be directly metabolized by the plant itself and by the rhizospheric microorganisms.

P17 PHYTO-MYCOREMEDIATION: MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF ARBUSCULAR MYCORRHIZAL FUNGI FROM A HEAVY METAL POLLUTED ASH DUMP DOWNTOWN VENICE Alessandra Turrini1, Stefano Bedini1, Emanuele Argese2, Manuela Giovannetti1 1

Department of Crop Plant Biology, University of Pisa; 2Department of Molecular Sciences and Nanosystems, Ca’ Foscari University, Venezia

Abstract Sacca San Biagio is an island in the central lagoon of Venice (Italy) that hosted a municipal solid waste incinerator operating from 1973 to 1984 and producing ashes that were distributed in a thick layer all over the island. The island, that presents high levels of heavy metals (mainly Cu, Pb and Zn), in the last 25 years has been spontaneously colonized by plants and soil biota and represents a unique site for the study of microorganisms thriving in heavy metal polluted environments. Among soil microorganisms, arbuscular mycorrhizal (AM) fungi (AMF) are important plant symbionts living in association with the roots of most land plants (80%) and occurring also in heavy metals contaminated soils. In such soils, AMF are critical in the establishment and fitness of plants, affecting the physico-chemical characteristics of the soil and enhancing metal immobilization. For these reasons phyto-mycoremediation utilizing AMF as plant inoculants is regarded as a promising strategy to heavy metal pollution remediation. We investigated the mycorrhizal status of the different plant species colonizing Sacca San Biagio, 67% of which resulted mycorrhizal. Though, no AM fungal spores could be isolated from the rhizosphere. Since spores were not available we used PCR-based techniques to identify AMF within plant roots. The analyses revealed the presence of nine AM fungal sequence types, two of which new to science. In order to isolate AM fungal symbionts able to live in such harsh environment, trap cultures with plants and ashes originating from Sacca San Biagio were set up. After five months’ cultivation five spore morphotypes were retrieved, morphologically identified and molecularly characterized. DNA was extracted from single spores and amplified using different primer pairs overlapping regions of the ribosomal DNA repeat units: the small subunit (SSU), the internal transcribed spacer (ITS) region and the large subunit (LSU) rDNA. The selection of ruderal plant species and the isolation of AM fungal strains from Sacca San Biagio will allow the detection of suitable stress-adapted plant-AM fungal associations to be used as biotechnological tools for land restoration programs of heavy metal contaminated areas.

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P18 HALOPHYTES PRESENT NEW OPPORTUNITIES IN HEAVY METAL PHYTOREMEDIATION E. Manousaki, N. Kalogerakis* Department of Environmental Engineering, Technical University of Crete, Chania, Greece

Abstract Halophytes are of significant interest since these plants are naturally present in environments with an excess of toxic ions and research findings suggest that these plants also tolerate other environmental stresses, especially heavy metals as their tolerance to salt and to heavy metals may, at least partly, rely on common physiological mechanisms. Therefore, halophytic plants have been suggested to be naturally better adapted to cope with heavy metals compared to glycophytic plants commonly chosen for heavy metal phytoremediation research. Under these considerations, halophytes are potentially ideal plants for phytoextraction or phytostabilization applications of heavy metal polluted saline and non saline soils. Furthermore, a novel process for the phytoremediation of heavy metal contaminated soils termed phytoexcretion has been recently introduced based on findings that some salt-excreting halophytes use their excretion mechanism in order to remove the excess of toxic metal ions from their sensitive tissues and on the idea of using plants as biological pumps for heavy metals. At the Laboratory of Biochemical Engineering & Environmental Biotechnology several Mediterranean halophytic and salt tolerant plants (Atriplex halimus, Halimione portulacoides, Limoniastrum monopetalum, Limoniastrum cornariarum, Tamarix parviflora, Tamarix smyrnensis, Nerium oleander) are under investigation for the remediation of Pb and Cd contaminated saline and non saline soils. The results confirm that halophytes are able to tolerate high concentrations of metals in soil and in some cases also accumulate the metals providing promising opportunities for phytoextraction and phytostabilization applications. Moreover, salt-excreting halophytes such as H. portulacoides, L. monopetalum, T. parviflora and T. smyrnensis were found to excrete the metals from the leaf tissues on their leaf surfaces in order to adapt to the metal burden presenting new opportunities for phytoexcretion purposes.

P19 CONNECTIONS BETWEEN RHIZOBACTERIAL COMMUNITIES OF REED SEDIMENTS AND LAND-USE IN ZHANGYE CITY AREA (GANSU PROVINCE, CHINA) L. Borruso 1*, J. Köbbing2, L.L. Yu2, K. Ott2, N. Thevs2, H. Ping3, S. Zerbe1, L. Brusetti1 1 Faculty of Science and Technology, Free University of Bolzano/Bozen, piazza Università 5, 39100 Bolzano, Italy; 2Institute of Botany and Landscape Ecology, Greifswald University, Grimmer Straße 88, 17487 Greifswald, Germany; 3Chinese Research Academy of Environmental Science, 8 Dayangfang Bei Yuan Road., Chaoyang District, 100012 Beijing, China; e-mail: [email protected]

Abstract Zhangye is a Chinese city (Gansu province) of 1,200,000 inhabitants, located within a big oasis surrounded by Gobi desert and high arid mountains. As many other Chinese cities, Zhangye is characterized by a rapid urban growth, and strong industrial progress including mining, buildings, energy production, metallurgy, machinery assembly, transportation, tourism and agriculture. In recent years the water demand has been dramatically restricted due to the excessive water use in socio-economic systems. Moreover, the widespread environmental pollution has caused a significant decrease in water quality. The city area is characterized by the widespread presence of Phragmites australis (Cav.) Trin (common reed), growing within the urban area, the industrial zone and the surrounding agricultural area. Furthermore an extended reed stand national park is located downstream Zhangye, in the northern territory. Reed stands are recognized to be resistant to the most normally-occurring disturbances, playing an important role for water saving and purification. P. australis root-associated bacteria, however, can broaden the positive effects of the phytoremediation, mineralizing organic molecules or sequestering heavy metals. Since bacterial communities are easily affected by even small changes of the microenvironment, we aimed to investigate the bacterial communities associated to P. australis rhyzosphere in diverse territorial spots in Zhangye, correlating these data with its geographical, urban and social features. Fifty-four samples of P. australis root-associated sediments were carefully chosen into flowing water of wastewaters treatment channels, as well as of industrial, urban and agricultural drainage channels. Microbiological analyses have been assessed by Denaturing Gel Gradient Electrophoresis (DGGE) and Automated Ribosomal Intergenic Spacer Analysis (ARISA). Chemical analyses of the concentration of heavy metals and nutrients have been done. Highly polluted sediments where found among the industrial area, where abnormal concentrations of zinc, arsenic, cadmium and lead, in particular, have been monitored. Detrended Correspondence Analysis of the fingerprinting bacterial community patterns showed high similarity of the replicated samples within the same sample site. The rhyzobacterial communities clustered accordingly with the local land use: samples from the highly polluted industrial area were well separated from the samples of the urban area characterized by an organic pollution. The samples from the agricultural suburbs, from the extensive reed park nearby the city and from the adjacent river clustered accordingly with the relative distance from the urban and industrial areas.

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Sub-session 1.3 - Phytoremediation and mycoremediation

P20 COMBINED BIOLOGICAL AND PHYSICOCHEMICAL PROCESSES FOR TREATMENT OF BAKER’S YEAST WASTEWATER Sahand Iman Shayan, Maryam Hosseini, Sabra Rostami, Sirous Ebrahimi Biotechnology Research Center, Sahand University of Technology, Tabriz, 51335-1996 Iran

Abstract Molasses, a by-product from sugar industry, is used as basic substrate and nutrient source in a large number of fermentation industries, such as ethanol, citric acid and yeast production. The wastewater from these fermentation processes presents high organic load and dark brown colour. Treatment of highly polluted industrial wastewaters among molasses fermentation wastes is still challenging issue. Baker’s yeast wastewater is a high strength molasses based wastewater consists of heavy organic and inorganic materials like melanoidine that is the dark brown colour with high COD (11000-80000 mg/L). Baker’s yeast wastewater cannot be completely degraded using biological anaerobic or/and aerobic treatment processes. After conventional biological treatment, most of the organic load is removed but nevertheless the dark brown colour still persists and it can even increase due to repolymerization of coloured compounds. Therefore, a combination of biological and advanced oxidation processes have been suggested. In this study, three different sequences of ozone, anaerobic and aerobic treatment systems were examined for treatment of baker’s yeast wastewater: Ozone- Anaerobic- Aerobic, Anaerobic- Aerobic- Ozone and Anaerobic- Ozone- Aerobic. The accumulative COD and colour removal efficiency achieved after each stage of combinations were as follows: Ozon-Anerobic-aerobic: COD (41, 86, 91%); Colour (95, 72, 89%) Anaerobic- Aerobic- Ozone: COD (67, 86, 99%); Colour (35, 56, ~100%) Anaerobic- Ozone- Aerobic: COD (69, 81, 91%); Colour (36, 93, 95%) The most effective combination was the sequence of "Anaerobic-Aerobic–Ozone". In the Ozon-Anerobic-Aerobic sequence after anaerobic treatment the colour increased. In this contribution, the feasibility and engineering aspect of the biological and ozonation processes will be discussed and a comparison between different processes for decolourizing and COD removal of baker’s yeast wastewater in further details will be presented. Key words: aerobic, baker’s yeast wastewater, decolourization, ozone

P21 HIGH CONCENTRATION OF COPPER AND ZINC INFLUENCES MICROBIAL BIODIVERSITY OF ZEA MAYS L. RHIZOSPHERE AND SELECTS MULTIMETAL RESISTANT BACTERIA STRAINS S. Matrella, G. Vigliotta, A. Cicatelli, S. Castiglione Department of Chimical and Biology, University of Salerno, Italy

Abstract Heavy metal contamination of soil is a widespread global problem due to persistence in the environment. Moreover, heavy metals are a significant toxic factor to biota, they can accumulate in biological systems and be introduced into food chain via different mechanisms. Bioremediation is a promising alternative to physic-chemical methods of remediation, involving both plants and their associated rhizosphere microorganisms in the degradation and/or removal of pollutants. Soil microorganisms play important roles in the recycling of plant nutrients, maintenance of soil structure, detoxification of noxious chemicals, and in the control of plant pests and growth. Thus, rhizosphere bacteria can promote phytoremediation capability through different mechanisms as: release of chelants, acidification, redox changes and reduction of phytotoxicity of the pollutants. In addition, plants can promote bacteria growth secreting specific secondary metabolites able to reduce toxic effect of contaminants. The goals of this work were to evaluate the bacterial diversity in the rhizosphere of Z. mays plants grown on Cu and Zn contaminated soil and to identify microorganisms potentially useful in phytoremediation processes. Bacterial biodiversity was assessed both in situ, by direct analysis of rhizosphere microorganisms present in contaminated and pristine soil, and by analyzing the evolution of the isolated community on rich medium in the presence of increasing concentrations of different metals. Sequences analysis of 16S rDNA from selected isolated stains revealed seven different phylogenetic groups: Flavobacterium, Bacillus, Chrysiobacterium, Pseudomonas, Agrobacterium, Stenotrophomonas, Lysinibacillus. Metals reduced biodiversity, shifting the community towards the Gram positive Bacillus and Lysinibacillus genera. In addition, two bacteria strains (C7 and C31) were isolated as highly tolerant to Zn and Cu (MIC value of 11.0 and 3.0 mM, respectively for both strains), and resulted also multimetal resistant (MIC values for Ni, Ag , Cd, Pb, Cr, of 1.0, 1.0, 0.1, 5.3, and >23.0 mM, respectively for both strains ). Polyphasic characterization of C7 and C31 identified them as two strains of Stenotrophomonas maltophilia, a species isolated in environments highly contaminated with heavy metals, also known for its ability to produce antifungal compounds that inhibit a large number of plant pathogens. In conclusion our study shows that the application of Cu and Zn strongly modify the microbial bio-diversity of the Z. mays rhizosphere and select some of the resistant bacteria strains potentially useful for improving the capacity of this crop to remediate heavy metal contaminated soil.

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P22 ANTI CANCER DRUG FROM ALGAE AND WASTEWATER TREATMENT: A DOUBLE WINNER Faraz M. Harsini1, Milad Dehghani1, Mohammadreza Mehrnia1, Mohammad H. Sarrafzadeh1, S. Shahab Edin Farazandeh mehr1, A. Sadeghian Haghighi2 1

School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran; 2Department of Computer Engineering, Islamic Azad University of Tehran, Tehran, Iran

Abstract Microalgae are one of the most promising sources for sustainable wastewater treatment in the last decades. Algae used in wastewater treatment facilities to reduce the need for greater amounts of toxic chemicals than are already used. On the other hand, optimization of microalgae production is receiving a growing attention due to their potential to produce drugs, vitamins and energy. It is predicted that cancer will cause 12 million deaths worldwide in 2030, over 30% of the world’s population are anaemic due to iron deficiency and 250,000 children only in South East Asia become blind because of Vitamin A deficiency. These statistics demonstrate the importance of producing more drugs and nutrients with less cost. Microalgae can be a remarkable solution for all these problems. The purpose of the present study is twofold. First, the ability of algae to remove pollutants can be gainfully used in bioremediation of many toxic chemicals. Biodegradative capabilities of microalgae for treatment of phenols, fenamiphos, other organophosphorus pesticides, and toxic chemicals ranging from simple monocyclic to more complex polycyclic pollutants are the primary focus of this study. It also involves the ability of algae for nitrogen and phosphorous removal, carbon dioxide capture and conversion of diluted nutrients from animal wastes. Second, these advantages of algae may increase production of algal biomass. This concept can be an essential step in drug development using algae for more production of beta-carotene, Vitamin B-12, Vitamin E, antibiotics, probiotics, iron and other nutrients and vitamins. Furthermore, anti cancer drugs have been created from blue green algae. This study has been carried out to provide a process, obtaining multi-beneficial effects of microalgae.

P23 nirK-TYPE DENITRIFIER COMMUNITY COMPOSITION AND DENITRIFICATION ACTIVITY IN A BUFFER STRIP B. Boz1, M. Bottegal1, Md.M. Rahman2, F. Fontana1, M. Basaglia1, B. Gumiero3, S. Casella1 1 Department Agronomy Food Natural resources Animals and Environment (DAFNAE), Università di Padova, Agripolis, Viale dell’Università 16, 35020 Legnaro (Pd), Italy; 2Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh; 3Department of Experimental and Evolutionary Biology UNIBO Via Selmi 3 40126 Bologna

Abstract Excess of chemicals can be often overcome by microorganisms, even if the time required could be too long. In order to speed these processes specific microbial populations should be supported. Leaving aside the possibility to augment the size and nature of the microbial community, the most suitable approach is to manipulate the environment in a way that will promote specific microbial populations and activities. In the case of nitrogen, especially nitrate deriving from agricultural managements, its concentration is generally found to increase in water flow. High nitrogen contents may be reduced by forcing the water flow through a buffer strip specifically designed and possibly afforested with suitable plant species. The management of this riparian buffer may definitely affect the soil microbial activities, including denitrification, as well as the composition of the community. The purpose of this study was to verify if the particular hydraulic management, the suspension of farming practices and the development of the woody and herbaceous vegetation actually produced a change in terms of denitrifying microbial community composition as compared to that of a neighbouring agricultural area. In order to monitoring whether the required microbiota was present and functioning in the experimental buffer area, denitrification was constantly followed and the data obtained were compared to those deriving from a direct investigation on the microbial composition of the soil population. This latter analysis was performed both as a general bacterial community analysis and as a targeted determination of specific bacterial groups involved in denitrification. In particular, this investigation was focused on nirK gene, encoding for nitrite reductase, an enzyme essential for the conversion of nitrite to nitric oxide, and considered the key step in the denitrification process.

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Sub-session 1.3 - Phytoremediation and mycoremediation

P24 PREPARATION OF TRANSGENIC PLANTS WITH ENHANCED HEAVY METAL ACCUMULATION J. Fiser1, E. Neumannova1, J. Viktorova1, M. Novakova1, M. Vrbova2, P. Kotrba1, M. Mackova1, T. Macek1 1

Institute of Chemical Technology in Prague, Faculty of Food and Biochemical Technology, Department of Biochemistry and Microbiology, Technicka 5, 16628 Prague, Czech Republic, e-mail: [email protected]; 2Agritec s.r.o., Zemedelska 16, 787 01, Sumperk, CZ

Abstract Phytoremediation has among bioremediation techniques the highest potential for decontamination of heavy metals. This method uses plants for accumulation, transformation or degradation of organic and inorganic pollutants. Plants exploitable to decontaminate inorganic pollutants have to take up, transfer and accumulate high concentrations of metals from the contaminated soil into harvestable shoots. To make the process effective, these plants should form also a large amount of biomass. To find suitable plant species fitting this requirement can be solved by preparation of transgenic plants overexpressing proper binding domains. The aim of this work is to prepare genetically modified plants of Linum usitatissimum with increased ability to bind heavy metals and plants of Nicotiana tabacum containing genetic elements allowing remediation of environment contaminated by both inorganic and organic pollutants together. CUP1 gene encodes a yeast protein metallothionein known for high affinity to heavy metals. In order to increase metal accumulation ability, the gene CUP1 was fused with gene for an additional metal binding domain. In this case a polyhistidine tail was the peptide of choice yielding the construct HisCUP. With the aim to prepare transgenic plants containing HisCUP gene two plant vectors were prepared: (1) using plasmid pNOV2819 (Syngenta) containing a gene for phosphomannose isomerase enabling selection of transgenic plants on medium with mannose, in this vector HisCUP gene was cloned under the control of RUBISCO promoter; (2) using plasmid pGreen0019 enabling selection of transgenic plants on medium with antibiotic kanamycin. In this case HisCUP gene was cloned under the control of constitutive CaMV 35S promoter. First the transient expression in tobacco plants was accomplished and the presence of mRNA of HisCUP gene in plant tissue was confirmed. Further plants of Linum usitatissimum AGT-952 were transformed by prepared vectors with HisCUP gene using agrobacterial infection. The presence of HisCUP gene was confirmed by PCR in eight plant regenerants grown in medium with kanamycin. With the aim to prepare plants able to remove simultaneously organic and inorganic pollutants, HisCUP gene was inserted into the genome of the transgenic tobacco plants containing bphC gene. BphC gene encodes bacterial enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase involved in bacterial degradation pathway of PCBs (polychlorinated biphenyls). Transgenic plants expressing bphC gene together with HisCUP gene thus could solve the problem of mixed soil contamination. Acknowledgements This project was supported by 7th FP of EU, project no: 265946 (MINOTAURUS), grants of the Czech Science Foundation (projects 525/09/1058, P501/12/P521) and MSMT AMVIS LH12087.

P25 PLANT–MICROBE INTERACTIONS IN PCB CONTAMINATED SOIL Petra Prouzova, Eva Hoskovcova, Eva Bedrlikova, Petr Stursa, Katerina Demnerova, Martina Mackova Department of Biochemistry and Microbiology, ICT Prague, Technicka 3, Prague 6, Czech Republic, 166 28

Abstract Plant-microbial interactions in the rhizosphere offer very useful means for clean-up of environments contaminated with recalcitrant organic compounds such as PCB. The microorganisms raise the availability of the compounds and the plants help in the extraction and removal of such compounds and supply nutrients for microorganisms. Secondary metabolites released by plants could play a significant role in cometabolic degradation processes. This work is focused on studying the influence of caffeic acid and naringine, as compounds belong to secondary metabolites, on microbial diversity and degradation in the PCB contaminated soil planted by tobacco (Nicotiana tabacum) and horseradish (Armoracia rusticana). Cultivated microorganisms were isolated from rhizosphere after 4 months cultivation. The potential PCB degraders, microorganism which were able to grow on mineral medium with biphenyl as sole carbone source, were identified by MS MALDI-TOF. Genus Arthrobacter was mostly found and then Pseudomonas and Burkholderia in soil samples of both plants. All these bacteria belong to well-known PCB degraders. The total DNA was isolated from all soil samples and noncultivated bacteria were identified after 16S rRNA analysis. The wide range of PCB and chlorobenzoates degrading bacteria were found (Rhizobium, Sphingomonas, Achromobacter, Burkholderia, Arthrobacter, Microbacterium, Alcaligenaceae). For a better understanding what kind of bacteria were really actively involved in PCB degradation the SIP (Stable Isotope Probing) method was employed. The change of PCB content in different soil samples was followed. The significant decrease of PCB content in horseradish samples was measured after four months cultivation. Acknowledgements The work was supported by GA ČR 525/09/1058 and ME 09024.

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Session 1/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S35-S52

P26 ENHANCEMENT OF PHYTOREMEDIATION ABILITY OF Medicago sativa BY THE MYCORRHIZAL FUNGUS Glomus intraradices Stefano Bedini1, Emanuele Argese2, Manuela Giovannetti1 1

Department of Crop Plant Biology, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy; 2Department of Molecular Sciences and Nanosystems, Ca’ Foscari University of Venice, Calle Larga Santa Marta, Dorsoduro 2137, 30123 Venezia, Italy

Abstract Phytoextraction and phytoimmobilization are promising technologies for the reclamation of heavy metals polluted areas based on the ability of plants to extract metals from the soil and accumulate/ immobilize them in their tissues. Arbuscular mycorrhizal (AM) fungi (AMF) are key organisms of the soil/plant system able to increase plant nutrition and soil physico-chemical characteristics and are considered a promising tool for improving the efficiency of phytoremediation. AMF, that form symbioses with most of terrestrial plant species, occur also in heavy metal rich soils, where they may alleviate plant metal stress as a result of a better nutritional status of mycorrhizal plants. Moreover, they may directly interact with soil metals by means of dense extraradical hyphal networks producing, glomalin, a metal binding protein. Here we tested the effect of a strain of the AM fungus Glomus intraradices, isolated from serpentine soils, on the growth and metal accumulation of the common forage legume lucerne (Medicago sativa L.) grown in a multi-metal (Cd, Cu, Pb) polluted substrate. Plant biomass, mycorrhizal colonization and metal concentration in the plants organs were measured after 4 months’ growth. Substrate multi-metal pollution strongly affected plant growth and mycorrhizal colonization. Plants biomass as well as the distribution of the metals in plant organs were significantly different between mycorrhizal and non-mycorrhizal plants. Mychorrizal plants showed larger growth and higher content of heavy metals both in shoots and roots, compared with non-mycorrhizal plants.

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Environmental Engineering and Management Journal

March 2012, Vol.11, No. 3, Supplement, S53-S74

http://omicron.ch.tuiasi.ro/EEMJ/

“Gheorghe Asachi” Technical University of Iasi, Romania

Session 2 ORGANIC WASTE MULTIPURPOSE BIOREFINERIES FOR THE CONVERSION OF HIGH ENVIRONMENTALLY IMPACTING MATRICES AND EFFLUENTS INTO BIO-BASED CHEMICALS, MATERIALS AND FUELS Keynote lecture ZEROWASTEWATER: SHORT-CYCLING OF WASTEWATER RESOURCES FOR SUSTAINABLE FACTORIES AND CITIES OF THE FUTURE Willy Verstraete LabMET, Ghent University

Abstract We currently deal with water in a non-sustainable way. We load it up with heat, chemical energy, nutrients and discharge it to a treatment plant.Up to know, we rarely invest to recover effectively the energy, the nutrients, the water. This is mainly due to the low exergy of wastewater. The key strategy is to avoid dilution but rather to up-concentrate the discharged water as much as possible upfront. In the latter case, a wastewater treatment plant can be upgraded to become a “bio-refinery”. Clearly, a variety of biotech processes qualify to be integrated in such new zero waste water approach. Moreover, these processes are already at such stage that they can compete with conventional “dissipative” technology in terms of costs and reliability.

Sub-session 2.1. Food processing and agro-industrial waste biorefinery Oral presentations EXPLOITATION OF STARCH INDUSTRY BY-PRODUCT TO PRODUCE BIOACTIVE PEPTIDES FROM RICE PROTEIN HYDROLYSATES Lucilla Dei Più 1, Annalisa Tassoni 2, Diana Isabella Serrazanetti 1, Maura Ferri 2, Elena Babini 2, Andrea Gianotti 1 1

Department of Food Science, University of Bologna; 2Department. of Experimental Evolutionary Biology, University of Bologna

Abstract A significant growth of interest in new plant food ingredients was observed in recent years in food science. The improvement of their functional properties may increase their application in processed foods. Protein hydrolysis characterising both traditional and the industrial food, is recognised as responsible for many beneficial effects such as elimination of allergenicity, production of flavours or flavours precursors and improvement of sensorial or nutraceutical quality. Small peptides and free amino acids have the advantage of being absorbed in the intestine without any digestion in the stomach. This explains their use in many formulas such as diets for nursing infants or sick adults and as stimulants for people liable for allergy development. The most bioactive peptides are generally characterised by molecular weights lower than 10kDa. The production of peptides through hydrolytic reactions seems to be the most promising technique to form proteinaceous antioxidants since peptides have higher antioxidant activity than raw proteins. Recently it was reported that bioactive peptides, derived from enzymatic hydrolysis of various plant proteins, possess antioxidant activity. In our study, a rice starch industry byproduct (provided by a local company “Amideria Il Cervo”) was hydrolysed both with commercial proteases (alcalase, neutrase, flavourzyme, lypaine) and microbial enzymes from whole cells of Bacillus spp. SDS polyacrylamide gel electrophoresis showed the presence of a 15kDa peptide in the non-hydrolysed substrate. The hydrolysates obtained by alcalase (30’ at 55°C and pH8) were characterized by two high molecular weight peptides of about 50 kDa, while those treated with neutrase (15, 30, 60 and 90’ of hydrolysis at 40°C and pH 6.5) showed peptides with molecular weight lower than 10 kDa. Whole cell proteolytic activity was very low. Antioxidant activity was determined by ABTS method. According to our results the antioxidant activity of the small peptides (< 10 kDa) was higher in the hydrolysates obtained by whole microbial cells than in those obtained by commercial enzymes. Arginine was the most abundant amino acid in microbial hydrolysates, followed by alanine, tryptophan, valine, leucine and lysine.

Session 2/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S53-S74

SYSTEMS FOR BIOHYDROGEN AND BIOELECTRICITY GENERATION: A CRUCIAL COMPONENT OF BIOREFINERIES P. Robledo-Narváez1,2, E. Rios-Leal3, N. Rinderknecht-Seijas4, A. Ortega-Clemente5, M.T. Ponce-Noyola1, H.M. Poggi-Varaldo*1 1

Centro de Investigación y de Estudios Avanzados del I.P.N., Environmental Biotechnology and Renewable Energies R&D Group, Dept. of Biotechnology and Bioengineering; 2ITSTB, Dept. of Research, Tierra Blanca, Veracruz, México; 3Central Analítica,, Dept. of Biotechnology and Bioengineering, CINVESTAV; 4ESIQIE-IPN, Division Basic Sciences, México D.F., México; 5ITBR, Ver., México; e-mail: [email protected]

Abstract Biohydrogen is a sustainable option for energy as it can be produced from organic waste through fermentation processes involving dark fermentation and photofermentation. Very often biohydrogen is included as a part of biorefinery approaches that reclaim organic wastes. The latter are an abundant source of renewable and low cost substrate that can be efficiently fermented by microorganisms. The experience shows that series systems show a better efficiency that only one stage on substrate conversion to hydrogen. The aim of this paper is to review several topics related to fermentative biohydrogen and its combination with selected bioenergy processes: (i) biohydrogen by fermentative process, (ii) bioenergy from selected processes that can be combined with dark fermentation, such as microbial fuel cells and microbial electrolysis cells, (iii) biocatalysts relevant to fermentative biohydrogen and other bioenergy processes, (iv) increased bioenergy production from wastes by series and/or sequential processes based on dark fermentation and (v) trends on improvement of biohydrogen yields. Dark fermentation (DF) and photofermentation (PF) are the emblem fermentative processes that yield H2 from organic matter. In DF about one third (at most) of the substrate is converted to hydrogen; desirable and economic substrates are organic wastes such as sewage, domestic and municipal, agro-industrial waste etc. The use of these substrates as well as fermentative mixed cultures in DF is feasible and desirable since it has environmental and economic advantages compared to the use of pure strains and commercial, simple substrates such as glucose, etc. DF can also be conducted in solid substrate fermentation mode when the substrate is an organic solid waste. Besides its good process performance, there are other benefits of SSF such as avoiding leachate generation and treatment, application of high loading rates leading to compact size reactors and relatively small process footprint etc. Yet, DF might show low yields of biohydrogen due to process deviations such as lactic fermentation, methanogenic, acetogenic, and sulphate-reducing consumption of hydrogen, among other causes. Overcoming and/or preventing these deviations need more research. As DF produces fermented by-products (fatty acids and solvents), there is an opportunity for further combining with other processes that yield more bioenergy. Photoheterotrophic fermentation is one of these processes. Special types of bacteria (photosynthetic heterotrophs such as non sulphur purple bacteria) can thrive on the simple organic substances produced in DF (low molecular weight organic acids, for instance) and light, to give more hydrogen. PF can be combined with DF in series, in sequence, or co-cultivation. It is estimated that DF combined with DF can increase the H2 yield by 50% or more, depending on the substrate, lighting regime, and microbes. DF provides a basic process for the development of large-scale to the production of H2 and a significant process in biorefinery approaches. In both direct and inverse cascading schemes of biorefineries, DF biohydrogen can increase the clean energy harvesting from organic wastes. Increasing H2 production yields is a significant task in present research agenda. This can be accomplished by increasing the biohydrogen or bioenergy harvesting with combined processes, by genetic modification of the biocatalysts involved and metabolic engineering, by good process engineering practice and scale up, among other measures. DF combined with PF and MFC and MEC appear as an alternative capable of attaining this goal. So far, two stage systems seem to be good options which allow treatment in a first step by dark fermentation of higher concentrations of feedstock without sterilization. Lab scale tests have shown a significant gain of biohydrogen or bioenergy production for several substrates. However, nutrient concentrations (i.e., N) and dark color in the first stage effluents can be problematic for PH. In practice, effluent conditioning may be required (N removal, dilution, color removal, pH adjustment) before feeding to PH in order overcome inhibitory effects. In addition, interesting experiments with sequential and co-cultivation of DF and PF have shown good results. H2 yields in DF can be impaired by several factors such as lactic fermentation, methanogenesis, autotrophic acetogenesis etc. Metabolic engineering and genetic applications to biohydrogen processes can significantly contribute to the improvement of microbial metabolic capabilities and boosting biological H2 production and overcoming deviations. Although there has been significant progress, more studies are necessary. The design of full scale reactors and ancillary equipment for DF, PH, and other bioenergies is lagging behind. So far no cost effective approach has been developed. Scale-up and demonstration projects of DF combined with other biohydrogen and bioenergy processes are a must if we want an early integration of these processes to biorefinery setups for increasing the sustainability of biorefineries. Key words: biohydrogen, dark fermentation, organic wastes, photofermentation, review

S54

Sub-session 2.1 - Food processing and agro-industrial waste biorefinery

ENGINEERING AMINO ACID PRODUCING Corynebacterium glutamicum FOR ACCESS TO ALTERNATIVE CARBON SOURCES C. Matano, J.W. Youn, J. Lindner, T. Meiswinkel, V.F. Wendisch Genetics of prokaryotes (bio vi), Universitaet Bielefeld, Bielefeld, Germany

Abstract The Gram-positive soil bacterium Corynebacterium glutamicum is an important industrial producer of various amino acids, with an annual production of about 2.5 million tons. These established large-scale biotechnological processes currently rely on glucose, fructose and sucrose as carbon sources. An emerging issue is the intensive utilization of media based on starch and molasses, the main industrial sources of glucose and sucrose. A desirable advance, in the perspective of an environmental sustainability, is to broaden the spectrum of substrates usable by C. glutamicum, developing producing strains able to utilize or co-utilize wastes from agro-industry and food-industry as carbon sources. The development of strains able to utilize hitherto inaccessible carbon sources has been made possible through the characterization of already existing pathways in C. glutamicum, as well as the engineering of heterologous pathways in order to improve carbon substrate utilization, with a particular attention to those available from the agro- and fisheries industries. The efficient utilization of wastes from marine food-processing industries into useful products has become an environmental priority. Initially, the attention was focused on a more efficient use of agro-industrial wastes, such as arabinose and xylose, deriving from lignocellulosic hydrolysates. But access to new carbon sources has also been engineered, in particular, for utilization of glycerol and amino sugars. Glycerol arises in large quantities in the biodiesel production process as major by-product of plant seed oil transesterification with methanol. Amino sugars have an undisputed potential because they could serve both as carbon and as nitrogen source for fermentation processes. Progress and future challenges of approaches to alternative carbon sources for C. glutamicum will be discussed.

IMPROVED ORANGE PEEL WASTE PRETREATMENTS FOR BIOETHANOL PRODUCTION G. Santi, S. Crognale, M. Moresi, M. Petruccioli, A. D’Annibale DIBAF, University of Tuscia, via S.C. de Lellis s.n.c., Viterbo, Italy

Abstract Citrus fruits world production has been estimated to be over 88 million tons per year, and oranges alone account for about 55% of such amount. Orange peel waste (OPW) is the solid residue of orange juice production. It consists of peels, membranes, cores, juice sacs and seeds and represents over 50% of the processed fruits. Its commercial uses are limited and its disposal is of great concern from the environmental point of view. In this study, second generation bioethanol production from OPW was investigated. The feedstock was chemically characterized and exhibited low phenols and lignin contents (1.19±0.01 and 1.9±0.08% w/w, respectively) and high cellulose and free glucose and fructose concentrations (26.11±2.12, 6.67±0.69 and 6.81±0.36% w/w, respectively): such properties made OPW a good putative candidate for bioethanol production. The raw material was suspended in an aqueous solution of sulfuric acid (0.5% v/v) at a solid concentration of 160 g L-1 and pretreated in a novel lab-scale direct steam injection apparatus (DSIA), where different reaction temperatures (130 to 200 °C) and times (90 to 500 s) were tested. The resulting slurries were analyzed for the presence of degradation products, and further depolymerized in shaken flasks using a commercial cellulase preparation (Cellic C-Tec2, Novozymes). The clarified liquor was then fermented in shaken cultures, under repeated-batch conditions, using an industrial Saccharomyces cerevisiae strain. It was found that pretreatments at 200 °C for 90 s and 180 °C for 150 s led to the highest glucose yield after enzymatic hydrolysis (52.30±0.34% w/w) and to the highest ethanol yield (50.35±0.89% w/w) and productivity (2.04±0.03 g L-1 h-1) after fermentation. Since the latter pretreatment (180 °C for 150 s) led to a lower concentration of inhibitors (i.e. formic acid, phenols and 5-hydroxymethylfurfural), such condition was chosen for a scale-up experiment, in which solid concentration in the DSIA was tripled and the entire process feasibility was assessed at the bench-reactor scale. The scaling up was successful since both glucose yield (63.92±3.22% w/w) and ethanol productivity (5.61±0.08 g L-1 h-1) were higher than in shaken flask experiments. On the basis of a mass balance including all the hexoses released after pretreatment and enzymatic hydrolysis the overall process yield at the bench-reactor scale would amount to about 140.13 L bioethanol per metric ton dry OPW.

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Session 2/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S53-S74

THE USE OF A VEGETABLE WASTE-DERIVED FERMENTATION PRODUCT FOR H2 PRODUCTION BY ANOXYGENIC PHOTOTROPHIC BACTERIA Alessandra Adessi1, Giovanni Colica1, Roberto De Philippis1,2 1

2

Department of Agricultural Biotechnology, University of Florence, Italy Institute of Chemistry of Organometallic Compounds (ICCOM), CNR, Florence, Italy

Abstract Research on photobiological H2 production processes is nowadays pointing towards the utilization of low cost substrates as sources of reduced carbon for H2 generation. In this connection, photofermentation often stands as a downstream process, subsequent to other fermentation processes where carbohydrates are converted to organic acids. The aim of this study was the evaluation of the feasibility of a two stage process costituited by an acidogenic phase, where the dark fermentation of vegetable residues (raw residues coming from the central vegetable market of Florence) is carried out by the autochthonous chemoheterotrophic microflora residing on the vegetables, followed by a hydrogen photoevolution phase, where the liquid fraction of the fermented residues is utilized by purple non sulfur bacteria for H2 production. The dark acidogenic phase produced a fermentation broth mainly containing lactic and acetic acids and ammonia. The fermentation broth was diluted with distilled water until ammonia concentration reached a value of 35 mg L-1; after the dilution, the concentration of the soluble products was: lactic acid 2.6 g L-1 and acetic acid 0,7 g L-1. Three Rhodopseudomonas palustris strains were used: strain AV33, isolated from a trophic lake (Averno Lake, Italy) and selected for its interesting capability of using lactate-rich substrates; strain 42OL, used in an up-scale of the process from reactors of 250ml to 3 L to 11 L of volume; a mutant strain (CGA676, insensitive to ammonium) used in order to test the possibility to avoid medium dilution. Under lab conditions, strain AV33 produced H2 at an average production rate of 9.7 ± 1.3 ml (H2) L-1 h-1 (in 0.25 L bioreactors). Strain 42OL produced hydrogen at a rate of about 16mL ml (H2) L-1 h-1 (in 0.25 L bioreactors), at a rate of 2–3mL ml (H2) L-1 h-1 (in 2.5 L bioreactors) and at an average rate of 11 ml (in H2) L-1 h-1 and a maximum rate of about 17 ml (H2) L-1 h-1 (11 L bioreactor). Using strain CGA676 (mutant insensitive to ammonium), the production of hydrogen occurred with no need of diluting the fermentation product, reaching an average rate of 3.9 ± 0.8 ml (H2) L-1 h-1. In conclusion, the two step process seems to be promising for coupling H2 production with the treatment of vegetable waste- derived fermentation products. Acknowledgments This study was partially supported by MIPAAF; (project IMERA), MATTM; (project PIRODE) and by MIUR and CNR (project EFOR).

UPGRADE OF WASTE GLYCEROL TO SHORT-CHAIN POLYHYDROXYALKANOATES CO-POLYMERS M.M. Da Fonseca1, J.M. Cavalheito 1, R. Raposo 1, M.C.D. De Almeida1, M.T.Cesãrio1, E.Pollet 2, H. Diogo 3, C.C. De Carvalho1 1

Ibb, Instituto superior técnico, Lisboa, Portugal; 2Lipht, Uds, Strasbourg, France; 3Cqe, Instituto supe, Lisboa, Portugal

Abstract Polyhydroxyalkanoates (PHAs) are polyesters synthesized under unbalanced growth by many bacteria as intracellular storage compounds. Depending on the ratio of the incorporated monomeric units, polymers with very different physical-chemical properties can be obtained, suitable for applications in fields such as agriculture, medicine and pharmacy. Recently, studies on PHA incorporation in composite materials and on blending with other bioplastics have raised attention. Since the 80s, ca. two dozen companies have invested efforts in developing industrial processes that could launch PHAs in the market at competitive prices. Nowadays, a few more than a dozen produce at industrial scale and, apparently, most of them use noble C sources as feeding streams. Evaluation studies for high scale production have concluded that 48% of the total production costs are ascribed to the raw materials, the C source accounting for 70–80% of the total cost. Aiming at reducing these costs, the development of high productivity cultivation processes using as major C source waste glycerol (GRP), a by-product of the biodiesel industry, is being tackled in our group using a Cupriavidus necator strain. The results presented here focus on the fed-batch production of the copolymer P(3HB-co-4HB) in 2 liter STRs. Incorporation of 4HB monomers was promoted by butyrolactone. Propionic acid, a stimulator of 4HB accumulation, increased two-fold the 4HB molar ratio, but also acted as 3HV precursor, yielding P(3HB-4HB-3HV). Cells were able to change the composition in fatty acids of the cellular membrane as response to the precursors used for co-polymer production. Dissolved oxygen (DOC) was a key parameter for the specific PHA accumulation and the volumetric productivity. By manipulating DOC and cultivation time, P(3HB-4HB) polymers with 4HB molar % between 11 and 22%, thus presenting a wide spectrum of thermal and mechanical properties, were obtained. High polydispersity indexes and multiple melting points indicated that the produced PHAs are polymeric blends. In short, (i) PHA production from GRP shows a large integration potential with biodiesel plants and (ii) prospective PHA applications in various areas are envisaged, namely in the medical field.

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Sub-session 2.1 - Food processing and agro-industrial waste biorefinery

Posters P50 COMMERCIALIZATION OF NEW VALUE CHAINS IN THE BIOECONOMY Manfred Kircher, Achim Marx CLIB2021

Abstract Already today the bio-economy contributes significantly to the gross domestic product and it will gain even more relevance in future. Examples across the broad field of biotechnology (fuel, fine- and bulk chemistry) and the value chain (biomass, intermediates, products) of the bioeconomy will be presented. Case studies for the rapid commercialization of breakthrough technologies will demonstrate the disruptive power of biotechnology esp. in the field of second and third generation bioprocesses. Emerging industries need basic scientific input from academia, innovative process- and product development in young enterprises (SME) and established companies (technology push) and a market willing to absorb new products (market pull). How these elements of innovation interact will be illustrated for the example of bio-fuels. Finally, the role of open innovation, clusters and global partnering in pushing the bio-economy will be discussed.

P51 PROCESS INTENSIFICATION IN BIOTECHNOLOGY: TOWARDS AN INTEGRATED BIOREFINERY Ludo Diels, Wouter Van Hecke, Heleen De Wever, Bert Lemmens, Luc Van Ginneken, Karolien Vanbroekhoven Flemish Institute for Technological Research (VITO), Mol, Belgium

Abstract The strong growth of the world population and accompanying social development leads to pressure on the climate, scarcity of resources, environmental problems and reduction in biodiversity. One of the possible human mitigation responses is the development of a bio-based economy inspired by the use of renewable resources (biomass) and the full fractionation and transformation of it into food/feed – materials/chemicals – energy in the so-called cascade-approach. Biorefineries, already existing for some decades now, were mostly focused on the food production chain. Nowadays, however, we are moving towards an integrated biorefinery in which either all compounds present in the biomass will be valorised as much as possible, or new biomass streams (including biowaste) will be tackled. New challenges exist in the disclosure of lignocellulosic biomass (second generation) in order to valorise not only the food components but also the straw, fibres, leafs, etc. Disclosure of these rather persistent molecules in an environmentally-friendly and sustainable way is still an important challenge. On the other hand, the extraction of several high-added value compounds (e.g., polyphenols) can be a success by using rather mild disclosure methods in order to keep its glycosilated forms intact and active. Although great expectations exist and a lot of development is going on at lab and pilot scale, still many problems need to be solved. It is known that between 50 and 70% of all the costs of a biorefined product are linked to it’s downstream processing. The integration of fermentation or bioconversion processes with subsequent separation or purification processes will become the challenge of the coming years in order to allow a real breakthrough of the integrated biorefinery of the future. The integrated recovery of the produced molecules or the (in-situ) removal of inhibiting chemicals from the process will keep them under certain threshold limits leading to more efficient conversion processes or reduction in feedback inhibitions. The economical value of this integrated approach lies in the increase in conversion efficiency (less substrate that is not used) at higher rates and less energy consumption in the further downstream processing. Removal of organic acids by electrodialysis and related concepts will allow to produce acids without concomittant production of huge amounts of waste products (such as gypsum). Recovery of volatile alcohols by integrated pervaporation systems will reduce toxicity in the fermentation broth and lead to a first concentrated product flow. Other technologies such as membrane distillation, membrane extraction, adsorption or crystallization have also large potential in these integrated systems. An important discussion in these integrated systems concerns the need for a first (membrane) separation of the cells from the broth allowing a cleaner process to proceed for the further separation of the produced molecules. In fact, such membrane-based fermentors can probably become standard technology in the future. Not only fermentation (whole-cell based) processes can be integrated, but also enzyme reactors are under development. The immobilization of enzymes on beads or membranes can save on enzyme cost and improve the quality of the produced new molecules. Challenges and problems of these integrated conversion-separation processes will be discussed in detail leading to new insights in future biorefineries including (semi-) continuous processes compared to batch processes.

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Session 2/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S53-S74

P52 NEW ADVANCES IN THE INTEGRATED MANAGEMENT OF FOOD PROCESSING BY-PRODUCTS IN INDIA AND EUROPE: SUSTAINABLE EXPLOITATION OF FRUIT AND CEREAL PROCESSING BY-PRODUCTS WITH THE PRODUCTION OF NEW FOODS AND FEEDS (NAMASTE EU) Fabio Fava1, Keith Waldron2, Carlos Bald3, András Sebők4, Jan Broeze5, Victor M. Garijo6, Hans-Georg Brendle7 1

University of Bologna, Italy; 2Institute of Food Research, UK; 3AZTI Tecnalia, Spain; 4Campden BRI, Hungary; 5Agrotechnology & Food Innovations, NL; 6 Grupo Leche Pascual, Spain; 7J. Rettenmaier & Söhne Gmbh, Germany

Abstract Huge amounts of by-products are generated by plant processing industry; in particular, fruit and cereal are among the most relevant surplus of Indian and European food industry. They are usually disposed of in landfills, since their chemical and microbiological instability, which leads to oxidation and spoilage, represents the major impediment to their exploitation (reuse or recycling). Nonetheless, such matrices are indeed unexploited sources of a number of valuable compounds potentially profitable in the formulation of novel feeds and foods. The remarkable opportunity of designing novel strategies to obtain added value products from the biotechnological treatment of these byproducts, hence satisfying the urgent need to valorize them, is the main target of recently EC-funded FP7 project NAMASTE-EU (Joint EC & DBT-India- call: KBBE-2009-2-7-02: Valorisation of by-products in food processing). NAMASTE aims at developing new laboratory-scale protocols and processes for the sustainable exploitation of citrus, pomegranate, mango, as well as wheat and rice bran surpluses via their (bio)conversion into either beverages, snacks, probiotics, prebiotics (dietary fibers), edible fractions enriched in active molecules (vitamins, oligosaccharides, minerals, aminoacids, antioxidant) used for the preparation of such products. Among the main results achieved so far in the project, there are, among others: - development of EU-India shared procedures for the selection, physical/chemical and biological treatment of citrus and wheat processing by-products aimed at obtaining bioactive molecules; - development and assessment of new (bio)technologies for their valorization as ingredients in the formulation of novel food products and feeds; - development of EU-India shared procedures for the assessment of the quality, chemical and microbial safety of new food products and feed, as well of the environmental and economical sustainability of the processes used; The following project outcomes are expected. - Obtainment of transnational plans for sustainable valorisation of fruit and cereal industry by-products. - Generation of a common India/EU’s knowledge-base and combined outlook in the field of environmentally sustainable food processing strategies. - Explore the setting up of new joint ventures, such as trade deal between Industries and EU and Indian consortia, which might promote the accession to new and emergent food markets.

P53 PRODUCTION OF POLYHHYDROXYALKANOATES (PHAs) FROM USED FRYING OILS AND POLYMER RECOVERY USING DIFFERENT STRATEGIES M. Scandola1*, L. Martino1, A. Scoma2, M. Cruz3, F. Freitas3, A.R. Gouveia3, M.A.M. Reis3** 1

G.Ciamician’ Dept.-University of Bologna, via Selmi 2, 40126, Bologna, Italy; 2DICAM-University of Bologna, via Terracini 28, 40131, Bologna, Italy; 3REQUIMTE-Chemistry Department, FCT/Universidade Nova de Lisboa, Portugal, Campus da Caparica, 2829-516; e-mail: *[email protected]; **[email protected]

Abstract Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by many prokaryotic microorganisms, as intracellular reserve materials. Depending on their composition, PHAs display strongly different mechanical properties ranging from hard to soft materials, thus representing an alternative to synthetically produced polymers. In the present study PHA is produced by Cupriavidus necator DSM 428 using waste frying oil from the catering industry, in fed-batch bioreactors cultivations. A polyhydroxybutyrate homopolymer (PHB) was obtained by using the waste oil as the sole substrate, while the use valeric acid as a co-substrate resulted in the production of a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(HB-co-HV) copolymer, with 23% HV content. At the end of the cultivation, the two biomasses, with biopolymer contents of 81.3% (w/w) of PHB and 42.3% (w/w) of P(HB-co-HV), were subjected to different extraction procedures, namely: solvent extraction with chloroform, chemical digestion using either sodium hypochlorite or sulphuric acid, and enzymatic treatment with Alcalase CLEA® or lysozyme. In some cases, heat-shock pretreatments were carried out. The recovered polymer was characterized by thermal analysis and by X-ray diffraction. Finally, the efficiency of the different recovery methods was evaluated.

S58

Sub-session 2.1 - Food processing and agro-industrial waste biorefinery

P55 INFLUENCE OF CARBON SOURCES ON THE PRODUCTION AND CHARACTERIZATION OF THE EXOPOLYSACCHARIDE (EPS) BY Bacillus sphaericus 7055 STRAIN Murat Yilmaz1, Gokcen Yuvali Celik2, Belma Aslim3, Dilsad Onbasili2* 1 Nigde University, Faculty of Science and Arts, Department of Biology, Campus, 51200, Nigde-Turkey; 2Erciyes University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology 38039, Kayseri-Turkey; 3Gazi University, Molecular Biology Research Center, 06830, Ankara-Turkey

Abstract In this study, EPS produced by five Bacillus spp. strains was determined. The only one strain (B. sphaericus 7055) was selected due to its high EPS production and it was investigated by growing this strain in LB broth medium containing various carbon sources. The highest EPS production of this strain was found in medium containing fructose. However, the effect of different concentrations of fructose and molasses on EPS production by the strain was studied. The maximum EPS yield of the strain 7055 was recorded with 2.5% (w/v) fructose, also the highest EPS production was found in 2.5% (w/v) molasses. The strain 7055 was found to contain (98.6%) galactose and (1.4%) glucuronic acid in control medium wheareas the composition of the strain 7055 [2.5% (w/v) fructose] was found to be (99.9%) neutral sugar and (0.1%) Glucuronic acid while the strain 7055 [2.5% (w/v) molasses] was found to contain (65.9%), neutral sugars and (34.1%) glucuronic acid. Key words: Bacillus, different carbon sources, exopolysaccharide (EPS), sugar beet molasses

P56 METABOLIC ENGINEERING IN Enterococcus faecalis FOR ITS USE IN BIO-ETHANOL PRODUCTION FROM WHEY N.F. Rana, S. Gente, J.M. Laplace, Y. Auffray USC INRA 2017 Laboratoire de Microbiologie de l'Environnement, Université de Caen Basse-Normandie Espalande de la Paix 14032 Caen cedex, France

Abstract Lactose is an interesting carbon source for the production of several bio-products by fermentation, primarily because it is the major component of cheese whey, the main by-product of dairy activities. However, the yeast Saccharomyces cerevisiae, the microorganism more widely used in industrial fermentation processes, does not have a lactose metabolization system. Thus, strain development programs through metabolic engineering are required for the implementation of lactose-to-ethanol processes with increased productivity. In this study the possibility of using whey permeate as a raw material for the production of ethanol using Enterococcus faecalis was examined. Enterococcus faecalis is a lactic acid bacterium that metabolizes many carbon sources and tolerates harsh environmental conditions. This study presents the potential of genetically modified E. faecalis JH2-2 strain for the production of bioethanol from glucose and lactose. E. faecalis has two lactate dehydrogenase genes (ldh) involved in lactate production. The deletion of these ldh genes led to increased production of formate, acetate and ethanol. The wild type JH2-2 converts only 4,8 % of the sugar to ethanol, but after the deletion of ldh genes this level is increased up to 15,2 %. To enhance ethanol production ability of E. faecalis mutant ∆ldh1/∆ldh2, a pyruvate decarboxylase gene from Clostridium acetobutylicum was inserted in its genome under the control of the strong ldh1 promoter. The recombinant PDC was found to have specific activity of 17 U/mg and ethanol yield was increased to 19 %. Moreover the induced PDC expression was also observed in the ldh deficient mutant ∆ldh1/∆ldh2 with an expression vector pMSP3535 inducible with nisin. Using this strategy, the specific activity of the PDC was found to be 47 U/mg with an ethanol yield of 40 %. Transcriptional analysis of E. faecalis JH2-2 and its mutants by a qRT-PCR approach reveals that the deficiency of the lactate dehydrogenase resulted in the upregulation of the genes involved in the production of formate and acetate. Whereas the genes encoding enzymes implicated in acetoin production are down-regulated. Out of four adh genes in E. feacalis only adhE and adhZn are expressed. This study presents a successful cloning of the pdc gene of C. acetobutylicum in E. faecalis and it also reveals the efficiency of molecular modifications for the use of E. faecalis for bio-ethanol production. Further optimization and modifications can make a promising combination of genetically modified E. faecalis for the production of ethanol from waste materials such as whey.

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Session 2/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S53-S74

P57 A NEW APPROACH ON THE BIOHYDROGEN PRODUCTION PROCESS VIA METHANE GENERATED FROM EFFLUENTS J.O.B. Carioca1,2, O. Araújo 3, C. Morais 4, S. Macambira 5, S. Galdino 5, A. Siqueira 6, E. Lima 6, J. Furlan 7 1

Universidade Federal do Ceará, Depto. de Tecnologia de Alimentos, Campus do Pici, Bloco 858, Fortaleza, Brazil; 2Centro de Energias Alternativas e Meio ambiente CENEA, Fortaleza, Brazil, e-mail:[email protected]; 3Escola de Química da UFRJ; 4Universidade Federal de Pernambuco; 5Instituto Federal de Educação e Tecnologia; 6RENORBIO; 7Universidade de Fortaleza

Abstract This paper analysis a new biohydrogen technological route generated from cashew residues in the Brazilian Northeast Agribusiness via methane production. It is knew that agribusiness produces a large quantity of residues, mainly those generated in the food sector, where there is a plenty availability of sugar contained effluents, appropriated to produce methane or even biohydrogen. That is the case of the pseudo fruit of cashew, the peduncle, which is considered an exotic fruit due to its tannin tasty. There is in the Brazilian Northeast, about of 800.000 hectares of cashew plantations where the pseudo fruit of cashew is largely wasted. After the seventies, world agricultural and land use are suffering a challenging pressure to produce biofuels to replace fossil fuels. Inside this context, Methane and biohydrogen has gained a considerable attention, due to its sustainable conditions. Recently, the world automobile industry pointed out their challenging project to replace the actual inefficient intern combustion motors by batteries or hydrogen fuel cell driven vehicles, towards a world green mobility, free of CO2 – emissions for small cars or even the thermoelectric power generation. So, biohydrogen produced from renewable feedstocks, mainly wastes, will assume a strategic position in the next decades. A broad analysis of the state of art of different biofuels technologies where made in previous authors publications. In this paper, it will be focused one analysis on the use of a new technological process route to produce biohydrogen using methane produced from effluents containing sugar, avoiding environmental problems. The idea is to combine the use of two well known technologies, the methane produced through the efficient UASB reactors associated with a catalytic methane cracking process to generate hydrogen and reduced carbon, which has large applicability in the metalwork industry, instead of the use of the classical light and dark fermentations. The previous analysis on methane produced technologies indicates that UASB reactors presents high yield and efficiency, and it is a well established technology, as well as the catalytic cracking of methane which has been considered a major technological route to produce biofuels in the recent analysis made by the German chemical industry. This seems to be a strategic and promising technological route to be focused aiming at to convert wasted bioresources into biofuels to attend future environmental sustainable requirements, as well as, the needs of the transport sector, power generation and the chemical industry. The technological route developed could be used to process other effluents or even other biomass type like microalgae, which has a great potential to be produced in the Brazilian Northeast.

P58 BIO-HYDROGEN PRODUCTION BY Escherichia coli WDHL AND BACILLUS SP. USING WHEAT STRAW HYDROLYSATE AS SUBSTRATE Zazil Donaxí Alvarado Cuevas1, Arturo Sánchez2, Leandro G Ordoñez1, José Tomás Ornelas Salas3, Antonio De León Rodríguez1 1 Camino a la Presa San José 2055, Lomas 4ª Sección, C.P. 78216, San Luis Potosí, SLP, México, Phone: +52(444)8342000, e-mail: [email protected]; 2Unidad de Ingeniería Avanzada, CINVESTAV Guadalajara, Av. Del Bosque 1145, Col. El Bajío 45019, Zapopan, Jal. México; 3Facultad de Ciencias, Universidad Autónoma de Guadalajara. Av. Patria 1201, Lomas del Valle, 3a Sección, C.P. 45129, Zapopan, Jalisco, México

Abstract Biological production through dark fermentation is one approach to generate renewable hydrogen since it utilizes a wide range of carbon sources, such as industrial and agricultural wastes. The wheat straw is source of carbohydrate by enzymatic, thermal and chemical hydrolysis. The goal of this work was to produce bio-hydrogen by Bacillus sp. isolated from an anaerobic sludge and Escherichia coli WDHL using wheat straw hydrolysate (WSH) as substrate. The hydrolysis was performed using sulfuric acid 0.72% (v/v) during 1 h at 121°C. Carbohydrate content was determined by the DNS method for reducer sugars (RS) and using an HPLC. Fermentations were performed in 110 mL serological bottles using HP medium plus WSH at 20 g/l RS. Gas production was measured using a liquidreplacement device and bio-hydrogen was measured by gas chromatography. The results showed that both microorganisms consumed the fermentable carbohydrates, however 50% of RS remained at the end of the cultures. The hydrogen production occurred in the first 8 h of culture and E. coli WHDL produced 3.2-fold bio-hydrogen higher than Bacillus sp. The wheat straw hydrolysate is an attractive rawmaterial source of carbohydrate fermentable for the bio-hydrogen production. Acknowledgements Partial financial support of SENER-CONACyT Grant 150001.

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Sub-session 2.1 - Food processing and agro-industrial waste biorefinery

P59 CHEESE WHEY WASTE AS RAW MATERIAL FOR THE BIO-HYDROGEN PRODUCTION BY ESCHERICHIA COLI WDHL: IMPORTANCE OF AMINO ACIDS AVAILABILITY Zazil Donaxí Alvarado Cuevas1, Leandro G. Ordónez1, José Tomás Ornelas Salas2, Antonio De León Rodríguez1 1

División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica. Camino a la Presa San José 2055, Lomas 4ª Sección, C.P. 78216, San Luis Potosí, SLP, México. Phone: +52(444)8342000, e-mail: [email protected] 2 Facultad de Ciencias, Universidad Autónoma de Guadalajara. Av. Patria 1201, Lomas del Valle, 3a Sección, C.P. 45129, Zapopan, Jalisco, México. Phone: +52(33)36488463, e-mail: [email protected]

Abstract Cheese whey (CW) is the by-product from cheese production and represents an 85-90% of the total volume of processed milk. Only a minor proportion is used in the food industry and for animal feeding. The rest is a matter of concern because of the risk of being a pollutant if it is not disposed in a proper way. However, CW could be an inexpensive potential raw material for bio-hydrogen production by fermentative processes, since its high content of lactose and lactalbumin. The goal of this work was to produce bio-hydrogen from CW by Escherichia coli WDHL, which was genetically improved for this proposal. Since, E. coli does not assimilate complex proteins, the effect of an enzymatic proteolysis and addition of external amino acids were also evaluated. The experiments were performed in 110 mL serological bottles and 1-L bioreactors using HP medium with 20 g/l CW. The effect of 1000 U/l pancreatin and addition of tryptone 2.75 g/l as external amino acids source were evaluated. Gas production was measured using a liquid-replacement device and the bio-hydrogen was measured by gas chromatography. The results showed that both strategies improved the bio-hydrogen production. The use of pancreatin yielded 75% bio-hydrogen higher than the cultures control. Whereas, the bio-hydrogen production increased up 350% using tryptone. The results indicate that cheese whey is an attractive raw material for the bio-hydrogen production, but an external amino acids source is needed. Acknowledgements Partial financial support of SENER-CONACyT Grant 150001.

P60 BIOCHEMICAL METHANE POTENTIAL (BMP) TEST OF RESIDUAL BIOMASS FROM AGRO-FOOD INDUSTRY Mariangela Soldano, Nicola Labartino, Claudio Fabbri, Sergio Piccinini CRPA-Research Centre on Animal Production, Reggio Emilia, Italy; CRPA Lab- Environment and Energy Section, Reggio Emilia, Italy

Abstract Anaerobic digestion is a bio-energy chain that allows exploiting with high efficiency crop and animal residues, wet and dried biomass, producing a biofuel (biogas/biomethane), ideal for conversion into electricity and/or heat or used for transport. The CRPA Lab, Environment and Energy Section of Tecnopolo of Reggio Emilia, has a systems to determination of the Biochemical Methane Potential (BMP) of different biomass and the real efficiency/reliability of a biogas plant. This parameter expresses the amount of biogas/methane potential obtained from the degradation of biomass, and expressed normal cubic meters of biogas or methane per kg of volatile solids. The paper reports the results obtained from the physico-chemical characterization and BMP tests of various residues and by-products generated by the agro-food industry. These biomass are of considerable interest to the high supply of organic matter and the absence of unwanted fractions; so they are suitable for sending in anaerobic digestion and a possible solution for their recovery. They can also represent an interesting alternative to energy crops whose use in anaerobic digestion is currently a issue of great discussion. The BMP laboratory plant (in batch) consists of reactors that are glass bottle with a 1.500 ml working volume. They are maintained at 36°C ± 2°C by a thermical room. Substrate is mixed with an inoculum that is a digester sample in a biogas plant and a salt solution. The measure of the amount of biogas produced is done by two methods: manometric and mass system. In the first case the measurement is done directly in the digester by measuring the increase in pressure in the headspace due to the generation of CO2 and CH4. In the second case, the measurement is performed when analyzing the biogas quality with a thermal dispersion mass sensor. The system allows evaluating the kinetics of reaction and substrate utilization rate by microorganisms. The article reports the BMP test of 5 biomass of interest from agro-food industry. It was analyzed the curve of cumulative methane production and the curve of the rate of volatile solids degradation of a BMP test of rapeseed cake (residues after extraction of the oil, comes from different).

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P61 BIOETHANOL PRODUCTION FROM DAIRY EFFLUENTS: IMPROVEMENT OF THE PROCESS EFFICIENCY Francesca Zoppellari, Laura Bardi Agricultural Research Council - Research Centre for the Soil-Plant System - Turin Research Group, Environment Park - Regione Piemonte, Settore Fitosanitario, Via Livorno, 60 (A2 LAB) 10144 TURIN, Italy

Abstract Whey and scotta are effluents from dairies, coming respectively from cheese and ricotta processing. Whey contains minerals, lipids, lactose (5-6%) and proteins and its BOD is about 30-60 g/l; scotta stills contains mainly lactose and at present it is not reused in any way, thus representing a waste whose disposal is a significant cost for dairies. Alternative destinations of these effluents as culture broth for biotechnological transformations can be a way to reach both goals of improving the added value of the agro-industrial processes and of reducing their environmental impact. In this work we investigated the way to produce bioethanol from lactose of whey and scotta and to optimize the fermentation yields using the yeast Kluyveromyces marxianus var. marxianus. Batch fermentations were carried out in fermenter at a laboratory scale, with row whey and scotta as culture media in anaerobic conditions. Different temperatures were tested, in order to check if the thermotolerance of the chosen yeast could be useful to improve the ethanol yield. The residues of lactose and galactose were analyzed at the end of each fermentation. The best ethanol yield was reached from scotta at 37°C, but with a high lactose residue and a final low ethanol concentration, indicating a stuck fermentation. The maximum ethanol concentration, even if with a lower yield than scotta, was reached with fermentations of whey at 37°C, in which the sugars were almost completely exhausted. The increase of temperature to 40°C did not improve the ethanol production, neither with whey nor with scotta. Both matrices can be considered suitable for ethanol production. Even if the final ethanol concentration reached with scotta can still be increased, by reducing the sugars residues, the good yields obtained indicate that this effluent, that at present is only considered a waste, can become a source. The fermentation can be efficient also at low temperatures, that allow to maintain low the costs of the processes.

P62 BATCH TESTS OF BIOLOGICAL HYDROGEN PRODUCTION FROM FOOD INDUSTRY WASTES BY FOUR THERMOTOGA THERMOPHILIC STRAINS IN 0.12-L MICROCOSMS AND IN A 19-L FERMENTOR A. Alberini*1, S. J.Mendes1, G. Bucchi 1, C. Manfreda2, M. Cappelletti 2, D. Pinelli1, S.Fedi2, F. Fava3, D. Frascari1 1 Department of Chemical, Mining and Environmental Engineering, University of Bologna, Via Terracini 28, Bologna, Italy; 2Department of Biology, University of Bologna, Via Irnerio 42, Bologna, Italy; 3Department of Civil, Environmental and Material Engineering, University of Bologna, Via Terracini 28, Bologna, Italy

Abstract This work is aimed at evaluating the feasibility of a cost-effective process of biological H2 production from food industry wastes under thermophilic conditions. The H2-producing performances of 4 Thermotoga strains (T. neapolitana, T. petrophila, T. naphtophila, T. maritima) were compared at 77 °C by means of tests conducted in 120-mL batch bioreactors containing a nutrient-rich growth medium additioned with glucose, molasses or cheese whey as carbon source. For all the substrates tested, T. neapolitana resulted the bestperforming strain under suspended-cell conditions, with a 0.9-1.9 mmol Lmedium-1 h-1 H2 production rate at an 8-10 g L-1 initial substrate concentration, and a 1.6-2.6 mmolH2 mmolmonosaccharide consumed-1 yield. To compare the 4 strains also under attached-growth conditions, preliminary tests were conducted with glucose-growing T. neapolitana, with the goal to select the best biomass carrier among 4 porous materials utilized in biofiltration applications. The best results were obtained with the carrier characterized by the highest interfacial area, equal to 2.1 m2 g-1. Also under attached-growth conditions, T. neapolitana resulted the best strain for all the 3 substrates tested, with a 1.21.8 mmol Lmedium-1 h-1 H2 specific production rate. Further tests, aimed at simplifying the growth medium composition, led to encouraging results. For example, in the case of molasses a minimum medium composed only by NH4Cl, K2HPO4, NaCl, buffer and cysteine resulted – in comparison with the ATCC 1977 complete medium – in a 73% reduction of medium cost and in a 12% increase of the H2/substrate yield. Finally, the process scale-up to a 19-L reactor is in progress. The preliminary results indicate that, in the scale-up of batch H2 production from glucose by T. neapolitana, the values of the three monitored yields (H2/substrate, organic acids/substrate, biomass/substrate) did not show significant variations, whereas the H2 volumetric production and the initial H2 production rate resulted to be significantly affected by the concentration of the initial inoculum. Acknowledgements Project financing by the Italian Ministry of Agriculture, Food and Forestry (MIPAAF) under grant “Combined production of hydrogen and methane from agricultural and zootechnical wastes through biological processes (BIO-HYDRO)” is acknowledged.

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Sub-session 2.1 - Food processing and agro-industrial waste biorefinery

P63 COMPARISON OF BIOHYDROGEN PRODUCTION IN FLUIDIZED BED BIOREACTOR AT ROOM TEMPERATURE AND 35°C K.M. Muñoz-Páez 1, J. García-Mena 2, M.T. Ponce-Noyola 3, A. Ramos-Valdivia 3, I.V. Robles González 4, N. Ruiz-Ordáz 5, L. Villa-Tanaca 5, N. Rinderknecht-Seijas 6, R. De Philippis 7, H.M. Poggi-Varaldo *1 1

Environmental Biotechnology and Renewable Energies R&D Group, Dept. Biotechnology and Bioengineering. CINVESTAV-IPN. P.O. Box 14-740, México D.F.; 2Dept. Genetic and Molecular Biology, ibidem; 3Dept. Biotechnology and Bioengineering, ibidem; 4NOVA Universitas. Oax., México; 5 ENCB-IPN. México, D.F.; 6ESIQIE- IPN, México D.F., Mexico; 7Department of Agricultural Biotechnology. University of Florence. Firenze, Italy; *email: [email protected]

Abstract In the last 10 years, interest on biohydrogen has increased exponentially. Recent studies have found that favorable immobilized-cell anaerobic hydrogen production systems include anaerobic fluidized bed reactors. Even though AFBR exhibits positive features for the production of biogases such as H2 it has been used primarily for the treatment of wastewater. To our knowledge, there are no studies for H2 production by AFBR at low temperatures. Therefore, this work was aimed to evaluate the H2 production in an anaerobic fluidized bed reactor using two incubation temperatures: room temperature and 35 °C. The laboratory-scale AFBRs consisted of glass columns loaded with 1 L of granular activated carbon (1-2 mm diameter) colonized by an anaerobic consortium (with heat-shock pretreatment, 90ºC) . Sucrose (8 g/L) was used as substrate without addition of buffer solution. The hydraulic residence time was 1 day; the incubation temperature was 35ºC (AFBR-M) and room temperature (AFBR-R). The average H2 concentration in the biogas in AFBR-R was 1.8 times superior than AFBR-M (54% and 31% respectively. pH ranged from 4.85 to 4.0 despite no buffer solution was added. It was observed CH4 in the biogas of AFBR-R until the pH dropped down to 4.5. The H2 productivity in AFBR-R was 2.3 times superior that in AFBR-M (1332.9 and 577.2 mLH2/Lbed day, respectively. Acetic acid (HAc), Butyric acid (HBu), Propionic acid (HPr), Ethanol (EtOH) and Butanol were the soluble microbial products (SMP) detected in this fermentation. The volatile fatty acid (VFA) contributed to most of the SMP at both temperatures with ratios VFA/SPAFBR-A of 26.3 and VFA/SPAFBR-M 4.2 (VFA/SP: volatile fatty acid/solvents products). Our results agree with those found by Gadhamshetty et al. (2009) in batch reactors operated at 22°C and 37°C in a CSTR reactor, they observed that the H2 production was 1.3 times superior in a reactor operated at lower temperatures (22ºC). This result could be attributed to the gradual changes in pH induced by slower kinetics at lower temperature. In conclusion the H2 production in an AFBR at room temperature showed encouraging results for H2 production in submerged fermentation of soluble substrates.

P64 SYNTHESIS OF BIODIESEL FROM HYDROLYSATES OF Arundo donax Domenico Pirozzi, Abu Yousuf, Gaetano Zuccaro, Rosaria Aruta, Filomena Sannino Dipartimento di Ingegneria Chimica, Facoltà di Ingegneria, Università di Napoli Federico II, P.le Tecchio, 80, 80125 Napoli, Italy; Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Facoltà di Scienze Biotecnologiche, Università di Napoli Federico II, Via Università 100, 80055 Portici (Napoli), Italy

Abstract The market penetration of the Ist generation biodiesel is limited by the relatively high cost of the feedstock (i.e. vegetable oils and animal fats), the insufficient availability of fertile land, as well as the social and environmental problems caused in different developing countries. New promising perspectives are offered by the hydrolysis of lignocellulosic materials to obtain fermentable sugars, as a large range of waste materials can be recycled, such as non-food parts of crops, forest products, and industry wastes. Though so far the hydrolysates of lignocellulose have been mainly used for the production of bioethanol, they can be alternatively employed for culturing oleaginous microorganisms, producing more than 20% of their weight in the form of lipids. These lipids are mainly triglycerides, potentially exploitable as feedstock for the synthesis of biodiesel. In this study, we demonstrate that the oleaginous yeast Lipomyces starkeyi can be grown in the presence of lignocellulose hydrolysates obtained from different samples of Arundo donax. L. starkeyi were cultured in the presence of the raw hydrolysate of A. donax (ADH) with no external organic supplement. A preliminary dilution was required to obtain a satisfactory cell growth, due to the presence of some products of the lignocellulose hydrolysis that inhibit the cellular growth (furfural, phenolic compounds, etc.). The yeast growth was accompanied by the complete consumption of the reducing sugars and a significant reduction of TOC. In order to remove the inhibitors, the ADH was treated following three different protocols: (a) overliming treatment with concentrated Ca(OH)2, (b) neutralization with NaOH and adsorption on active carbons, (c) overliming followed by neutralization and adsorption. These treatments made possible the growth of L. Starkey in the presence of undiluted raw ADH. The experimental data demonstrate that the best results are obtained by the method (b).

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P65 EVALUATION OF CAROB PULP AS FERMENTATION SUBSTRATE FOR BIOHYDROGEN AND ORGANIC ACIDS PRODUCTION: SUGARS RICHNESS VS TOXICITY POTENTIAL M. Lima1, J. Ortigueira2, L. Alves2, S.M. Paixão2, P. Moura*1,2 1

ISCSEM–Instituto Superior de Ciências da Saúde Egas Moniz, Quinta da Granja, Monte de Caparica, 2829–511, Caparica, Portugal; 2LNEG-National Laboratory of Energy and Geology, Bioenergy Unit, Estrada do Paço do Lumiar 22-1649-038, Lisboa, Portugal; *e-mail: [email protected]

Abstract Carob pulp (fruit without seed) represents about 90% of the total dry weight of the fruit containing a very high percentage of readily soluble sugars (up to 54% w/w). This high sugar content renders this raw material very suitable as fermentation substrate, e.g. in the production of biofuels. In this study, we tested the potential of carob pulp for the production of biohydrogen and organic acids by Clostridium butyricum and two microbial consortia. Since the use of carob pulp may be limited by its content in compounds with antimicrobial properties, a toxicological and chemical characterization was carried out. Carob pulp was submitted to an aqueous extraction in order to obtain a sugar-rich extract and spent solids. These solids were subsequently washed to recover residual sugars and phenolic compounds. Both carob extract and spent solids were used as carbon sources in fermentation experiments. The specific rate of hydrogen and butyrate production in the fermentations of carob pulp extract by C. butyricum attained 0.24 and 0.1 mmol.h-1.g-1, respectively, which corresponds to higher values than the ones obtained with glucose. The fermentation by the microbial consortium (LE58) produced mainly acetic acid and generated 0.13 mmolh-1g-1 hydrogen. By the contrary, the inclusion of the spent solids in the fermentation media inhibited microbial growth. In order to better understand this antimicrobial potential and its possible correlation with phenols concentration, the spent solids were submitted to different extraction conditions. The resulting liquids were characterized and its toxicity was evaluated. The highest concentration of gallic acid equivalents (GAE) was obtained by using distilled water at 100 ºC, after 30 minutes of extraction. The concentration of total phenols, and sugars and pinitol reached 16.6 ± 0.4 mg GAE and 339 ± 31 mg sugars / g d.w. of spent carob pulp, respectively. All waters obtained from the extraction processes inhibited the growth of Pseudomonas putida. Shorter extraction times result in higher sugars:total phenols ratios and lower toxic levels. On the contrary, the water with the lowest sugar concentration, obtained under the severest extraction condition tested, produced the highest toxicity level of 16.54 toxic units. The results showed that the upgrading of carob pulp for the biological production of hydrogen and organic acids is possible. Any fermentation process optimization aimed at upgrading spent carob pulp by the use of residual sugars must consider short extraction times and explore the different solubilisation profiles of carob’s sugars and phenolic compounds.

P66 ROBUST YEASTS FOR THE CONVERSION OF LIGNOCELLULOSIC HYDROLYSATES INTO ETHANOL Lorenzo Favaro, Alberto Trento, Marina Basaglia, Sergio Casella Department of Agronomy Food Natural resources Animals and Environment (DAFNAE), Università di Padova, Agripolis, Viale dell’Università 16, 35020 Legnaro (PD), Italy

Abstract In recent years increasing attention has been devoted to the production of bioethanol from lignocellulose. The process requires a pretreatment of biomass, which may result in the formation of inhibitory compounds. Ethanol production from biomass hydrolysates is the subject of a number of works, but only limited research has been conducted on looking for yeasts able to both tolerate inhibitors and ferment sugars with high yield. This study aimed at the selection of robust yeasts suitable for the industrial scale bioethanol production. One hundred yeast strains, mainly newly isolates belonging to Saccharomyces cerevisiae, were screened for their fermentative abilities at different temperatures in minimal media supplemented with high glucose and/or xylose concentrations. Moreover, the strains were evaluated for inhibitor tolerance in rich and defined broth having increasing concentrations of weak acids (acetic, formic, lactic acid) and furans (furfural and 5-hydroxymethyl-2furaldehyde). The effects of pH value and high sugars levels on yeast inhibitor tolerance were also considered. Ten S. cerevisiae strains showed outstanding ethanol yield from glucose at both 25 and 40°C. Moreover, their fermentative abilities were not affected by the presence in the medium of high xylose concentrations. Among them, few isolates were able to tolerate well different levels of inhibitors. Their tolerance was influenced by the pH in the medium and high sugar levels seemed to enhance their ability to withstand weak acids. The newly isolated S. cerevisiae F17 and S. cerevisiae MEL2 were further studied for their fermentative abilities in a defined medium supplemented with 100 g/L glucose, 50 g/L xylose and increasing concentrations of inhibitors. Both yeasts exhibited interesting ethanol yield within 48h, producing about 48 g/L ethanol. The selected strains have been adopted for the production of ethanol from real lignocellulosic hydrolysates and the most tolerant yeasts for an evolutionary engineering approach to further improve their robustness. Their genetic modification for the integration of different genes (glycoside hydrolases and pentose metabolising enzymes) will also be addressed.

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Sub-session 2.1 - Food processing and agro-industrial waste biorefinery

P67 BIOENERGY FROM LIGNOCELLULOSIC WASTES: PREATREATMENT, ENZYMATIC HYDROLYSIS AND ETHANOL PRODUCTIVITY B. Ribeiro, S. Alves, L. Baeta-Hall, L. Duarte, F. Carvalheiro, José C. Duarte* LNEG-Laboratório Nacional de Energia e Geologia, I.P, Bioenergy Unit: Estrada do Paço do Lumiar, 22, 1649-038 Lisboa, Portugal

Abstract Biovalorization of wastes from wheat straw, and other plant materials which constitute a low-cost lignocellulosic biomass, into valuable compounds was studied. For bioethanol conversion there are mainly three processes involved: (i) pretreatment for hemicellulose removal from lignocellulosic biomass, (ii) hydrolysis of cellulose to produce reducing sugars, and (iii) fermentation of the sugars to ethanol using an ethanologenic microorganism. Waste biomass pre-treatments were applied: autohydrolysis and acid hydrolysis were compared. Enzymatic hydrolysis was studied using a commercial cellulase mixture (Celluclast 1.5 L and Novozym 188, Novozymes A/S, Denmark) at different conditions. The influence of different parameters such as temperature (35-60ºC), incubation time (1-7 days) and enzyme loading (10-15 FPU/g and 0.2-0.4 mL/g polysaccharides) during the hydrolysis process was evaluated. The optimisation criterion was the fermentable sugar yields, which were analysed by HPLC. The best results for the wheat straw hydrolysis were obtained for the enzymatic loading: Celluclast 10 FPU/g polysaccharides + Novozym 188, 0.2 ml/g polysaccharides at 55ºC for 48 h. These optimal saccharification conditions for wheat straw pretreated (WSP) allowed a final process yield of ~60% in hydrolisate sugar content. Other waste materials are also compared. The potential for ethanol production by fermenting wheat straw hydrolisate (WSH) with two yeast strains of S. cerevisiae (strains F and K from our collection) and a bacterial strain of Z. mobilis (strain CP4 a gift from LO Ingram) was evaluated. Batch fermentation tests of the WSH showed an ethanol yield of 74%, 79% and 58%, respectively. Supplementing the WSH with peptone and/or yeast extract had no effect on yield for yeast fermentation. However, the ethanol production by Z. mobilis was highly increased when yeast extract was added to the hydrolisate, corresponding to an ethanol yield of 98%. Moreover, further pretreatment optimization of lignocellulosic materials to remove lignin can significantly enhance the hydrolysis of cellulose improving the bioethanol yield and in addition the recovered lignin can also be converted into valuable compounds. These results show that an integrated exploitation of these lignocellulosic wastes from agricultural production is economically possible and highly advantageous for energy and chemicals production.

P68 COMPUTER-BASED ARTIFICIAL INTELLIGENCE STRATEGIES IN MODELING AND OPTIMIZATION OF MICROBIAL BIOPROCESSES OPTIMIZATION CASE OF BIOGAS GENERATION FROM WASTES E.B. Gueguim Kana Discipline of Microbiology, University of KwaZulu-Natal, Pietermaritzburg Campus, e-mail: [email protected]

Abstract Microbial fermentations produce a wide range of valuable foods, fuels and chemicals, most of which are complex to be produced through conventional chemical processes. The industrial viability of such processes requires an optimal combination of physico-chemical process parameters. Traditional modeling and optimization strategies suffer from large computational burden and are unable to consider the interaction effects of the various process parameters. Emerging Artificial Intelligence (AI) tools such as Artificial Neural Network (ANN), Genetic Algorithm (GA), Fuzzy Logic (FL), Ant Algorithm (AA) and Particle Swarm Optimization (PSO) are being considered in the design of optimal production media and process operating conditions. In this work, the techniques and procedures of ANN, GA and PSO are detailed. Their application is illustrated by previous findings in: (1) an improvement of 8.64% biogas production form mixed co-substrates of sawdust and others; (2) the minimization of acidification time in yogurt production by Lactobacillus bulgaricus and Streptococcus thermophilus using a non-linear temperature profile (3) a comparative evaluation of AI strategies with the Response Surface Methodology (RSM) on modeling and optimization of citric acid production by Aspergillus niger on seven process parameters, in which the AI optimized process showed an experimental production 6.68 g/L against the RSM predicted 3.35 g/L; (4) an improvement of fermentative biohydrogen production from mixed agrowastes. ANN coupling GA is a more efficient strategy to navigate the optimization search space for fermentation development, and its prospect is further discussed.

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BIOETHANOL PRODUCTION FROM MIXED SUGARS USING Scheffersomyces stipitis Isabella De Bari1, Paola De Canio 2, Daniela Cuna 1, Federico Liuzzi 1, Patrizia Romano 2, Angela Capece 2 1 ENEA Italian National Agency for New Technologies, Energy and Sustanaible Economic Development, C.R. Trisaia S.S. 106 Jonica 75026 Rotondella (MT), Italy, e-mail: [email protected], Phone:(+39) 0835974313; 2Basilicata University, Department Bi.Di.BAF–Campus Macchia Romana–Via Ateneo Lucano 10, 85100 Potenza, Italy, e-mail: [email protected], Phone (+39)0971205576

Abstract Bioethanol can be produced from different biomasses, including lignocellulosic feedstocks, which can contain comparable amounts of 5carbon and 6-carbon sugars. In order to obtain an effective conversion of these biomass carbohydrates, suitable microorganisms are required for the fermentation step. In this paper the ability of Scheffersomyces stipitis to ferment mixed syrups has been investigated in pure culture and in co-cultures with other yeasts species. Both the performance of free and entrapped cells were explored. The performance of the Schef. stipitis strain NRRLY-11544, was initially tested by using synthetic broths containing different sugars concentrations, with a xylose fraction of 50%. According to the diauxic behavior of Schef. stipitis, the sugars consumption occurred sequentially, achieving an average process yield of 75%. To overcome this drawback, the cells immobilization was tested. Silica-hydrogel films and alginate beads were used as immobilization carriers. The sugars uptake in the entrapped cells occurred simultaneously. In all tests, xylose consumption was almost completed when the process time was prolonged. As common trend the ethanol level decreased at increasing the xylose fraction in all the systems investigated. This phenomenon appeared less evident at higher sugars level, probably due to a favored fermentative metabolism according to the Crabtree effect. However, the fermentation with both free cells and immobilized cells yielded comparable results in terms of xylose consumption and ethanol yields, but the immobilization offered the advantage of enabling the fermentation of subsequent batches of sugars with similar performances over several weeks. The use of Schef. stipitis and S. cerevisiae in free co-cultures ensured faster processes. However, the rapid production of ethanol by S. cerevisiae, inhibiting Schef. stipitis, caused a stuck of the process, resulting in a lower ethanol yield than pure culture fermentation. Finally, co-cultures of inhibitors-adapted Schef. stipitis and S. cerevisiae, co-immobilized in alginate gel beads, have been used in order to explore their co-fermentation ability of an un-detoxified enzymatic hydrolyzates, obtained from a steam pretreated corn stover. The results indicated that, in the enzymatic hydrolyzates, adaptation increases the xylose consumption from Schef. stipitis by 62% and improves the ethanol yield by 17%, respect to wild type yeast. Overall the use of immobilized and adapted co-cultures, could be a feasible strategy for the bioethanol production from lignocellulosics. Nevertheless the ethanol inhibition on Schef. stipitis metabolism is still a problem. Further investigations on fermentation strategy by cocultures are in progress in order to develop a process providing the online sequestration of ethanol.

P70 BIOMETHANE PRODUCTION FROM TOBACCO PLANTS WITH MODIFIED CELL WALL Marianna Villano1, Fedra Francocci2, Felice Cervone2, Giulia De Lorenzo2, Mauro Majone1 1

Department of Chemistry, Sapienza University of Rome; 2Department of Biology and Biotechnology “C. Darwin”, Sapienza University of Rome

Abstract Plant biomasses represent an abundant source of lignocellulosic material for renewable biofuel production. The conversion of plant cell wall polysaccharides into simple fermentable sugars (saccharification) is typically regarded as a major bottleneck for the bioconversion process. The recalcitrance to saccharification is largely due to the complex structure of the cell wall that is composed of a cellulosehemicellulose network embedded in a matrix of pectin that limits the exposure of cellulose to hydrolysis. It has been previously demonstrated that the modification of homogalacturonan, the main component of pectin, by the expression of an attenuated variant of the polygalacturonase II of the fungus Aspergillus niger (hereafter named PG plants), improves enzymatic saccharification with respect to the unmodified plants. In this work, two transgenic tobacco PG plants (lines PG16 and PG7, expressing respectively high and low level of the transgene), were used to evaluate the effect of cell wall modifications on anaerobic fermentation of plant tissues into methane gas. To this aim, samples of both transgenic and wild type plants were incubated (at 35°C) in closed reactors in the presence of an anaerobic sludge as inoculum. Control tests lacking plant biomass or containing a corresponding amount of glucose (instead of plant biomass) were also setup. Each condition was tested in triplicate to evaluate its reproducibility. The reactors were analyzed at regular intervals for organic acids (the main intermediates resulting from the fermentation of saccharification products) and methane gas, the end-product of anaerobic degradation of plant biomasses. The production rate of organic acids (namely acetate, propionate, and butyrate) with PG16 modified tobacco was substantially higher than with the wild-type, confirming that the expression of AnPGII can enhance the rate of saccharification. Interestingly, the final conversion yield of tobacco plants (both modified and wild-type) into methane was over 90% (on a COD basis) and only slightly lower than that observed with pure glucose. In conclusion, these results suggest that residuals from tobacco plants have a high potential for conversion into biofuels.

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P71 Si-MICROBIAL SYMBIOTIC EFFECT ON THE ROOT FORMATION OF THE CULTIVATED PLANTS Vladimir Matichenkov Effo, Ibbp ras, Pushchino, Russia

Abstract Modern agriculture requires the reduction of the traditional fertilizer application and increasing the quality of products. The production of the agricultural plants depends on the root formation. Our and literature data showed that root formation can be regulate by application of the special microorganisms (micorhiza) or by active forms of Si. Aim of this study was to determine the major factors for root formation of the cultivated plants. The investigations were conducted with sand, sandy-clay and clay soils and with using corn and barley as tested plants. The active Si (as diatomite from Central Russian region at the rate 1 t/ha) and concentrated monosilicic acid (100 mg/l of Si), mycorhiza and standard NPK fertilizers were used in the experiment. After 2 months the biomasses of the cultivated plants were measured (root, stems and leaves). The CO2 emission from cultivated soil and content of azotobacters were tested as well. The obtained data showed that the application of the conventional fertilizers reduced the microbial activity of the soil and amount of the azotobacters, while the biomass of the stems and leaves were increased significantly. The application of the active Si (both solid and liquid forms and mycorhiza increased the soil microbial activity and azotobacted content. The biomass of the whole plant (roots, stem and leaves) were increased as well under optimization of Si plant nutrition and microbial substances application. The maximum effect was obtained for combination of the active Si and mycorhiza. The root mass was increased on 2.3 times and azotobacter amount on 3.4 times, compare with control. The obtained data confirm the theory that the conventional fertilization has negative influence on the soil-microbial system. The additional amount of the fertilizers crushed the balance between plant, soil microbes and soil biochemical equilibrium. In the opposite the application of active forms of Si and mycorhiza reinforces the soil-plant-microbial system. In the result the productivity of the cultivated plants is increased as well. These data give possibility for elaboration the new system for sustainable agriculture, which will not conflict with natural balance in the soil matrix.

P72 OPTIMIZATION OF PRODUCTION CONDITIONS FOR Trichoderma sp. P25 AS A BIOCONTROL AGENT BY USING SOLID STATE FERMENTATION S. Sözer, S. Sargın, R. Eltem, F. Vardar Sukan Ege University Faculty of Engineering, Department of Bioengineering 35100 Bornova, İzmir, Turkey

Abstract Biological control is an environment friendly approach which promotes the use of specific microorganisms to protect plants against plant pathogens and pests instead of chemical treatments. The biocontrol agent (BCAs) market has been growing continuously over the last few decades due to the adverse environmental impacts of chemical pesticides. Trichoderma type fungi, can be used to control a wide spectrum of soil-borne plant pathogens. Therefore, Trichoderma based BCAs are in common use since they have both plant growth stimulation characteristics and high activity on soil bioremediation. For the production of BCAs conidial mass of Trichoderma is the most proficient propagule since they withstand downstream processing especially in the drying stage. Although, there is also abundant literature on the use of conventional synthetic media like glucose, cellulose, soluble starch, and molasses to produce Trichoderma spp. it is difficult to produce conidia in submerged culture. Moreover, the cost and availability of raw materials are quite important considerations for the commercial productions of BCAs. Therefore solid state fermentation is an attractive alternative since it offers many advantages through the efficient use of agro industrial wastes. Conidia are produced more abundantly than chlamydospores in SSF. In this study Trichoderma sp. P25, which was formerly evaluated with respect of its mycoparazitic activity, lytic enzyme activities, sporulation rate and was demonstrated to have a potential as a biocontrol agent properties, was used for the production of micropropagules in SSF. The effects of various organic nitrogen sources, initial pH were investigated in wheat bran based medium. The effect of incubation temperature, initial moisture content and cultivation time on micropropagule production was further optimized by Response Surface Methodology. The results indicate that SSF is a feasible alternative production method for the production of BCAs.

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A HIGH-ADDED VALUE PRODUCT FROM TOMATO POMACE CONVERSION: -L-ARABINOFURANOSIDASE FROM Pleurotus ostreatus FOR LIGNOCELLULOSE CONVERSION Antonella Amore*1, Angela Amoresano1,2, Leila Birolo1,2, Vincenza Faraco1,2 1 2

Department of Chemical Sciences, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo, via Cinthia, 4 80126 Naples, Italy; School of Biotechnological Sciences, University of Naples “Federico II”; *Presenting author: e-mail: [email protected]; **Corresponding author: email: [email protected]

Abstract 2nd generation bioethanol produced from lignocellulose is one of the best alternatives to fossil fuels to achieve the targets established by Directive 2009/28/CE. Its advantages include low net greenhouse gas emissions, abundance and geographically more even distribution of lignocellulose raw materials, minimization of the potential food-versus-fuel conflict. (Hemi)cellulases are needed for lignocellulose hydrolysis, leading to the production of fermentable sugars. The major cost driving factor in lignocellulose-to-ethanol process is the high cost of (hemi)cellulolytic enzymes, so that reduction of their production costs and improvement of their performances are required to improve process competitiveness. Solid state fermentation (SSF) is an economic and environmentally friendly alternative to conventional process for industrially relevant enzymes production. On the other hand, white-rot fungi (e.g. Pleurotus ostreatus) represent the most appropriate microorganisms for producing enzymes through SSF because of the similarity between their natural environment and the conditions in which SSF processes are carried out. In this work, an -L-arabinofuranosidase produced by the fungus Pleurotus ostreatus (PoAbf) during SSF on tomato pomace was identified, and the corresponding gene and cDNA were cloned and sequenced. The amino acid sequence similarity to the other -Larabinofuranosidases indicated that the enzyme can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L-1) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da. Mass spectrometry analyses led to the localization of a single O-glycosylation site. The enzyme is highly specific for -L-arabinofuranosyl linkages, it followed Michaelis–Menten kinetics with a KM of 0.64 mM and a kcat of 3010 min-1 when assayed with p-nitrophenyl -L-arabinofuranoside, and exhibited an optimum pH of 5.0 and an optimal temperature of 40 °C. PoAbf is also able to hydrolyze natural substrates, besides some tested oligosaccharides. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The durable activity showed by rPoAbf in comparison to the other -L-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties. Design of PoAbf mutants was carried out in order to deepen both the role of its catalytic residues and the effect of glycosylation on enzyme stability.

P74 VANILLIN PRODUCTION FROM WHEAT BRAN WITH Pseudomonas fluorescens BF13-1P Elena Dal Bello1, Stefano Rebecchi1, Andrea Negroni1, Giulio Zanaroli1, Diana Di Gioia2, Maurizio Ruzzi3, Fabio Fava1 1

Dept. of Civil, Environmental and Materials Engineering, University of Bologna, Bologna, Italy; 2Dept. of Agroenvironmental Sciences and Technologies, University of Bologna, Bologna, Italy; 3Dept. for Innovation in Biological Agro-food and Forest systems, Tuscia University, Viterbo, Italy

Abstract Relevant amounts of wheat bran are produced by the Mediterranean Countries, where they are mainly employed in the formulation of animal feeds but also extensively discharged as a waste. Wheat bran contains relevant amounts (5-6 % w/w) of ferulic acid, i.e., a precursor for microbial vanillin production, which can be partially released from the cell wall polysaccharides via selected enzymatic treatments. We recently optimized a batch process for the bioconversion of food-grade ferulic acid into vanillin with resting cells of Pseudomonas fluorescens BF13-1p. In this work we investigated the possibility of i) increasing vanillin concentration in a fed-batch process, ii) using the biomass for consecutive bioconversion steps and iii) using ferulic acid from wheat bran hydrolysates as the bioconversion substrate. P. fluorescens BF13-1p cells were grown in a 2-L stirred tank reactor on LB medium at 30°C, pH 6.8 and induced for 1 h with 2.5 mM ferulic acid after 4.30 h of growth. Batch bioconversion experiments were performed in shaken flasks (150 rpm, 30°C) with 6 g (wet weight)/L cells in saline phosphate buffer pH 7.0 or in wheat bran hydrolysate. Fed-batch bioconversions with repeated spikes of foodgrade ferulic acid 2.5 mM and 5 mM allowed to obtain vanillin concentrations (3.5 mM and 6.4 mM, respectively) 20% lower than those obtained using the same total amount of ferulic acid in the batch process. Conversely, biomass could be efficiently reused in a second bioconversion process, allowing to obtain vanillin concentration, molar yields and bioconversion selectivity comparable those obtained after the first bioconversion (4.3 mM, 82.8% and 87.5%, respectively), while only 1.1 mM vanillin and 22% molar yield were obtained after the third use of biomass, due to the rapid accumulation of the by-products vanillic acid and vanillyl alcohol. Finally, bioconversions performed using crude bran hydrolyzates as substrates showed unsatisfactory vanillin molar yields (13.6%) and bioconversion selectivity (15%) due to vanillin oxidation to vanillic acid. The process efficiency slightly improved when the hydrolyzate pH was maintained at 7.0, whereas vanillin production comparable to those obtained with food-grade ferulic was obtained when ferulic acid occurring in bran hydrolyzates was purified from sugars on ISOLUTE ENV+ columns. Acknowledgements This work was supported by the EC within the FP7 under Grant Agreement No. 245267 (NAMASTE project).

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Sub-session 2.1 - Food processing and agro-industrial waste biorefinery

P75 INCREASING THE FEED VALUE OF OLIVE OIL CAKE BY SOLID STATE CULTIVATION OF THE WHITE-ROT FUNGUS Fomes fomentarius Mohamed Neifar, Amany Ayari, Abdellatif Boudabous, Ameur Cherif, Atef Jaouani Laboratoire Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Campus Universitaire, 2092, Tunis, Tunisia

Abstract Olive oil cake (OOC) generated by the oil industries, well implanted in Tunisia, represents a major disposal and potentially severe pollution problem. Treatment of OOC with white-rot fungi can enhance the nutritive value of this agro-industrial waste. In the present study, solid state fermentation was used to cultivate Fomes fomentarius on OOC. The effect of several parameters such us humidity, time of incubation and physical chemical pretreatment was evaluated. The efficiency of fungal treatment was so estimated by analysing the chemical composition and in vitro digestibility of the resultant substrate. The results show a significant (p < 0.05) increase in crude protein contents from 6.48% for the control to 22.32% for treated OOC. There were also consistent significant decreases (p < 0.05) in the values obtained for NDF, ADF and ADL (respectively by 23.43, 13.77 and 10.86%). Therefore, significant differences were observed in the hemicellulose and cellulose contents. This result suggests that fungal treatment of OOC resulted in improved crude protein and digestibility, hence its potential in ruminant nutrition. Key words: in vitro digestibility, Olive oil cake, solid state fermentation, white-rot fungi

P76 BIOTRANSFORMATION OF AGRICULTURAL WASTES WITH INNOVATIVE ENZYMES A. Morana1, S.M. Paixão2, S.A. Ladeira3, L. Alves2, E. Ionata1, F. La Cara1 1

Istituto di Biochimica delle Proteine–CNR, Napoli, Italy; 2LNEG–Instituto Nacional de Energia e Geologia IP, U. Bioenergia, Lisboa, Portugal; 3UENF-Universidade Estadual do Norte Fluminense Darcy Ribeiro, LTA-CCTA, Campos dos Goytacazes-RJ-Brasil

Abstract Among potential alternative bioenergy renewable resources, lignocellulosics have been identified as the prime source of biofuels and other value-added products, due to their high polysaccharides content. The partial or total degradation of the polysaccharide fractions is an ambitious target that can be efficiently reached with eco-friendly enzyme-based process. The description of new hydrolytic enzymes is an important step in the development of techniques which use lignocellulosic materials as a starting point for fuel production and/or value added products (ex: xylo-oligosaccharides). So, the main goal of this work is the study of new hydrolytic enzymes from non-conventional microorganisms for the production of fermentable sugars from some agricultural wastes. In this context, a screening of some microorganisms, including thermophilic and halophilic bacteria, was carried out from which the strains Anoxybacillus sp. strain 3M and Geobacillus thermodenitrificans were selected, due to their interesting xylanolytic activities. Herein, we show the characterization of the xylanolitic activities of both strains and compare the potential of their xylanases to transform two types of raw materials: brewer’s spent grain (BSG) and grape cane. These residues were subjected to several pre-treatments to allow a better release of the sugars by the action of the enzymes. In particular the materials were treated with ammonium (10% v/v) and potassium hydroxide (10% v/v) in the following operational conditions: 70º C for 22h and 121° C for 30 min, for ammonia and potassium hydroxide soaking, respectively. The results obtained showed that for both strains the presence of xylanolytic enzymes was revealed in the culture supernatant supplemented with xylan, with the highest amount produced in the late stationary phase (1.34 U/mL for Anoxybacillus sp. strain 3M and (0.95 U/mL for G. thermodenitrificans). The hydrolysis tests of pretreated BSG and/or grape cane with the novel enzymes from both strains are in progress for the polysaccharide fraction degradation to xylo-oligosaccharides or xylose. In the hydrolysis studies with G. thermodenitrificans xylanases the highest xylan degradation yield was obtained in grape cane, after 48 h incubation. In this assay, 25% xylan was hydrolyzed to xylose, revealing the presence of both xylanase and beta-xylosidase activities in the supernatant.

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SCREENING OF NOVEL YEAST INULINASES AND FURTHER APPLICATION TO BIOPROCESSES S.M. Paixão*, P.D. Teixeira, T. Silva, A.V. Teixeira, L. Alves* LNEG–Instituto Nacional de Energia e Geologia, IP, Portugal

Abstract Inulin is a carbohydrate composed of linear chains of -2,1- linked D-fructofuranose ue through a sucrose-type linkage at themolecules terminated by a glucose resid reducing end. The tubers of several plants, such as Jerusalem artichoke, chicory, dahlia and yacon, contain a large amount of inulin. This polymer can be actively hydrolyzed by microbial inulinases to produce glucose, fructose and value-added products as inulo-oligosaccharides. The glucose and fructose produced by inulin hydrolysis can then be used in bioprocesses. Great attention is focused on renewable sources in fuel ethanol production and Jerusalem artichoke is one of the most interesting materials among unconventional raw materials, due to the high production of fermentable sugars. Tubers contain about 25% dry matter, consisting mostly from carbohydrates, being inulin the most important one, reaching 50-60% of dry matter. This work is focused on Jerusalem artichoke inulin applications either for bioethanol production or fossil fuels biodessulfurization (BDS) process. For bioethanol production, is advantageous the use of a microorganism that produces the enzymes required for a direct conversion of biomass to ethanol (consolidated bioprocessing - CPB). CBP is gaining increasing recognition as a potential breakthrough for low-cost biomass processing. In this context, the inulinase biosynthesis potential of some yeasts strains isolated from different sources was evaluated in a medium containing Jerusalem artichoke juice (25% v/v), as well as their ability to simultaneously produce ethanol from the fermentable sugars released from Jerusalem artichoke’s inulin hydrolysis. Three novel strains presenting extracellular inulinase activity (1678 U/L) and able of a CBP from inulin to ethanol were isolated. The best microorganism was the yeast strain Talf 1, producing 60g/L of bioethanol from Jerusalem artichoke juice containing about 150 g/L inulin. Moreover the enzymatic extract of this strain was further added to Jerusalem artichoke juice to hydrolyse the inulin producing a high fructose levels syrup to be used within a fossil fuels BDS process by Gordonia alkanivorans strain 1B. This strain wasn’t able to grow in inulin as the only carbon source, however in a SSF process with inulin and inulinases, strain 1B grew and desulphurized the dibenzothiophene (DBT), a fossil fuels model compound. Promising results are being obtained pointing out the Jerusalem artichoke hydrolysed juice as a less expensive alternative carbon source for BDS processes. Acknowledgments The authors gratefully acknowledge the financial support of the projects Carbon4Desulf - FCOMP-01-0124-FEDER-013932 and FCTCNR - Italia128585955528141 by the Fundação para a Ciência e a Tecnologia.

P78 RECENT ADVANCES IN THE BIOREFINERY OF OLIVE MILL WASTEWATER Alberto Scoma *, Lorenzo Bertin, Matilde Monti, Fabio Fava Department of Civil, Environmental and Materials Engineering (DICAM), Faculty of Engineering, University of Bologna, via Terracini 28, 40131 Bologna, Italy, Phone: (+39) 051 2090351; Fax: (+39) 051 2090322; e-mail: [email protected]

Abstract Resources availability at a reasonable price will be one of the main issue of the next years. Since biowastes have been employed as the renewable source for the obtainment of biological compounds, their extensive exploitation through multi-step integrated processes has been considered of upmost interest. In the present work, recent advances related to the case study of Olive Mill Wastewater (OMW) biorefinery are reported. Polyphenols are natural antioxidant present in high amounts in OMWs. Their recover from synthetic, as well as actual site OMWs by means of solid phase extraction processes (using Amberlite XAD16 as the solid phase) was proved to be effective. Almost complete phenols removal from OMWs, along with their subsequent recover up to 60% of the concentration occurring in OMWs (using a biocompatible extraction solvent such as ethanol), were found attainable. Recent findings showed that sustained solid phase recycling can be carried out with no significant loss of efficiency. Furthermore, the alcoholic mixture of phenols was concentrated maintaining its antioxidant and antiradical activity while allowing theoretically infinite recover of the desorbing phase. The aforementioned process produces a dephenolized OMW, whose organic matter could be employed for the production of biobased chemicals and biofuels. In particular, anaerobic acidogenic digestion could be carried out at high yields in order to produce an effluent enriched in Volatile Fatty Acids (VFAs). The importance of the latter relies on the fact that they can be used as the carbon source for polyhydroxyalkanoates (PHAs) synthesis. In this respect, it was found that by varying Hydraulic Retention Time (HRT) in continuously operated acidogenic Packed Bed Biofilm Reactors (PBBRs), biogas production and composition were found to change: in particular, a significant H2 gas production was achieved, together with a reduction in VFAs accumulation. The possibility to physically separate the hydrolytic activity leading to H2 production from the acidogenic one, thereby increasing VFAs amount in the effluent, is under way. However, VFAs concentration was also increased by means of electrodialysis. This process produced two effluents: a first with a higher VFAs amount with respect to the inlet, and a second with a lower one. The latter could be used by some photosynthetic microorganisms in the typical two-stage process, while the former outlet could be finally fed to aerobic reactors for PHAs production. In this respect, utilization of anaerobically digested OMWs for PHAs production was successfully carried out.

S70

Sub-session 2.1 - Food processing and agro-industrial waste biorefinery

P79 OLIVE-MILL WASTE AS POTTING SUBSTRATE FOR OLIVE TREE CULTIVATION: EFFECTS ON THE MICROBIOTA OF SOIL AND RHIZOSPHERE L. Fidati1, S. Scargetta1, L. Nasini2, G. Gigliotti2, P. Proietti2, G. Cenci1, E. Federici1 1 Department of Cell and Environmental Biology, University of Perugia, Italy Department of Agricultural and Environmental Sciences, University of Perugia, Italy

2

Abstract The growing attention to environmental sustainability, and particularly to the reduction of waste production and non-renewable resource consumption, has encouraged the recycling and valorization of agro-industrial by-products. The use of peat in potted plant cultivation has raised concerns because of the destruction of peat bogs. Replacing this substrate with other organic materials is thus environmentally and economically appealing. Mediterranean countries, where the production of olive oil is continuously rising, produce large amounts of olivemill waste over a short time with important environmental consequences. The humid husk generated by two-phase oil extraction systems could be used, either directly or after composting, as peat surrogate. The aim of this work was to investigate the impact on the soil and rhizosphere microbiota of these alternative substrates. Soil and rhizosphere samples were collected from potted olive trees where either peat or fresh and composted humid husk were used. Cultivation-independent methods, namely PCR-DGGE and qPCR analyses, were applied to study the microbial communities and the nitrifier bacteria populations. The composting process changed the structure of both bacterial and fungal communities present in the humid husk but didn’t seem to affect the total number of bacteria. On the contrary, fungi were more abundant in the fresh humid husk. The bacterial and fungal DGGE fingerprints were very different both in the soil and the rhizosphere when the substrates based on olive-mill waste were used in place of peat. The fresh husk seemed to have a more profound effect than the composted one. Besides modifying the community structure, the use of this substrate also showed a positive effect on the total number of microorganisms. In particular, it strongly stimulated the growth of fungi both in soil and rhizosphere. Interestingly, qPCR analysis of amoA gene abundance, showed that the number of nitrifier bacteria was significantly reduced in the rhizosphere of the olive trees potted using the husk-based substrates, particularly the fresh one, suggesting a possible negative effect on nitrogen availability for plant growth. Taken together, these data demonstrate that replacing peat with husk-based substrates modifies the soil and rhizosphere microbiota and suggest that fungi play a primary role in the degradation of the complex organic matter present in the fresh waste. The inhibition of nitrifier bacteria in the rhizosphere suggests that care should be taken when using the fresh humid husk for olive tree pot growth. In this respect, composting may represent a valuable strategy for producing peat surrogates.

P80 MORPHOLOGICAL ANALYSIS OF YEASTS BIOPROSPECTED FROM THE BRAZILIAN SAVANNAH Igor Chiarelli Perdomo1, Janina Zanoni Camargo2, Gustavo Graciano Fonseca3 1

Fcba/bioingegneria, Ufgd/polimi, Dourados/milano, Brasiliano; 2Facet, Ufgd, Dourados, Brasiliano; 3Faeng, Ufgd, Dourados, Brasiliano

Abstract The Brazilian Midwest Region has emerged due to its large biotechnological potential. The savannah is the predominant vegetation in the region and is recognized for its great microbial diversity. However it has been marginally studied so far and the on this aspect it can be said that remains still unexplored. The aim of this work was to evaluate 44 yeast strains isolated from exotic fruits found in the Brazilian savannah. In micro-morphological analysis, isolates were grew in YPD medium at 30°C for 24h and then subjected to microscopic visualization, where were analyzed the format, the presence of budding and the cell sizes compared. For the macro-morphological analysis, isolates were plated on YPD agar by exhaustion, incubated at 30°C for 72h and then performed the analysis of the formed colonies, considering the following parameters: pigmentation, border, texture, and comparison of the convexity of the colonies. Colonies were classified by similarity into five main groups, which the most important features were: group A-cell format elongated, with the presence of budding, white pigmentation, regular edges, smooth /opaque texture; group B-cell format oval, with the presence of budding, white pigmentation, regular edges, smooth/opaque texture; group C-cell format round; with the presence of budding, white pigmentation, regular edges, smooth/opaque texture; group D-with the presence of budding, smooth/opaque texture; and group E-with the presence of budding, jagged edge, wrinkled / opaque texture. The obtained groups were utilized to compare with results from molecular and biochemical tests. Morphology presented a good correlation with these other tools for microorganisms’ identification. Further studies are necessary to identify potential biotechnological applications for the yeasts

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Session 2/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S53-S74

P81 GROWTH KINETICS OF YEAST STRAINS ISOLATED FROM THE BRAZILIAN SAVANNAH Janina Zanoni Camargo1, Igor Chiarelli Perdomo2, Rosimeire Oenning da Silva, Marney Pascoli Cereda, Gustavo Graciano Fonseca3 1

Facet, Ufgd, Dourados, Brasiliano; 2Fcba/bioingegneria, Ufgd/polimi, Dourados/milano, Brasiliano; 3Faeng, Ufgd, Dourados, Brasiliano

Abstract Brazil is the world's largest ethanol producer via fermentation. Several strains of the yeast Saccharomyces cerevisiae have been industrially used for fermentation. These yeasts have been obtained by genetic modifications or by isolation in the major sugarcane mills and distilleries in the São Paulo State, Brazil. However the conditions are different in the states of the Brazilian savannah. For instance, the state presents early harvesting and higher temperatures. Moreover, it is remarkable the lack of studies on yeasts for fuel ethanol production. In this sense, the aim of this work was to compare the growth kinetics of three yeast strains isolated from a local distillery grew at different sugars (glucose and sucrose). The pre-culture was prepared by the transference of a colony of a petri dish containing the strain to a 250 ml Erlenmeyer flask with sterile mineral medium. The sugar was sterilized separately and added at a concentration of 10% (w/v). The pH was adjusted to 6.0. Growth was carried out in an orbital shaker at 30°C and 200 rpm stirring for 12 hours. An initial optical density (OD) (λ = 600 nm) of 0.1 was calculated and utilized as inoculum. The main cultures were performed in duplicate at the same conditions. Samples were taken every 30 min for pH and growth measurement. Cell growth was spectrophotometry measured (λ = 600 nm). The exponential growth phase was identified as the linear region of the plot of ln (OD) versus time. The maximum specific growth rate (μmax) was determined as the slope of this straight line. Doubling time (DT) was calculated by the quotient of ln (2) and μmax. The isolated strain BB2 presented the conversion the higher growth rates at both glucose (μmax = 0.2989 ± 0.0012 h-1) and sucrose (μmax = 0.3015 ± 0.0075 h-1) sugars. Maximum OD of 9.58 ± 0.00 and 2.27 ± 0.09 were obtained from glucose and sucrose, respectively. It can be suggested that this strain presents a good mechanism for sugar transport and probably a high production of the invertase enzyme. The lower biomass found during growth in sucrose suggest ethanol and/or organic acids production. In general, the isolated strains presented higher growth rates compared to the industrial strains, e.g. Sacharomyces cerevisiae Cat 1. It is concluded that under the studied conditions, the isolated strain BB2 is a promising candidate for ethanol production in the state.

P82 EVALUATION OF THE CAPACITIES OF ASSIMILATION AND FERMENTATION OF SEVERAL YEASTS STRAINS ISOLATED FROM EXOTIC FRUITS FROM THE BRAZILIAN SAVANNAH J. Zanoni Camargo1, F. Avelino Gonã§alves2, I. Chiarelli Perdomo3, G. Graciano Fonseca4 1

Facet, Ufgd, Dourados, Brasiliano; 2Ufrn, Ufrn, Natal, Brasiliano; 3Fcba/bioingegneria, Ufgd/polimi, Dourados/Milano, Brasiliano; 4Faeng, Ufgd, Dourados, Brasiliano

Abstract Brazilian savannah is since along time recognized as a region with a high microbial biodiversity. However it has been only marginally studied for biotechnological purposes. The aim of this work was to isolate yeasts from different Brazilian exotic fruits and evaluate their capacity to assimilate and ferment several substrates. The samples were obtained from several fruits of different tree species found in the Brazilian savannah. They were diluted in 10 mL of NaCl solution and inoculated in Petri dishes containing YPD medium added of erythromycin (32 µg/ml). The isolated yeasts were analyzed for their ability to assimilate and ferment glucose, fructose, sucrose, lactose, starch, cellobiose, mannitol, galactose, maltose, raffinose and xylose, besides their ability to assimilate nitrate, grow on YPD medium containing 10% NaCl, 50% glucose or 37 °C. Samples were considered positive for fermentation when there was gas production and positive for assimilation when the presence of microbial biomass was detected, by the tube’s turbidity, for each enumerated condition. Experiments were performed in duplicate and growth and/or fermentation analyzed after 24, 48, 72, 96 and 120 h. It was possible to obtain 44 yeast strains isolates, predominantly from samples of cherry of Rio Grande (Eugenia involucrata), jatobá (Hymenaea courbaril), and uvaia (Eugenia pyriformis). It was observed that 73.3% of the isolates were able to assimilate glucose after 24 h and 100% after 120 h. On the other hand, only 22.2% could ferment glucose after 24 h. After 120 h this number increased to 48.8%. In general, the selected isolates showed greater ability to ferment glucose, fructose and sucrose, lower ability to ferment maltose and lactose. None of them was able to ferment xylose, but 16 of them were able to assimilate this sugar. The isolated 28 was able to ferment starch. Isolates 42 and 43 showed the ability to ferment lactose. Isolate 42 presented also an interesting ability of growth in glucose at the concentration of 50 %. Both strains were isolated from pequi (Caryocar brasiliense). A more detailed study of these isolates is important to better evaluate their biotechnological potential.

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Sub-session 2.1 - Food processing and agro-industrial waste biorefinery

P83 COMPARISON OF BACTERIOCINS PRODUCTION FROM Enterococcus faecium STRAINS IN CHEESE WHEY AND OPTIMISED COMMERCIAL MRS MEDIUM Lorenzo Favaro1, Stefano Schirru2, Roberta Comunian2, Marina Basaglia1, Sergio Casella1, Antonio Paba2, Elisabetta Daga2, Bernadette Dora Gombossy de Melo Franco3, Ricardo Pinheiro de Souza Oliveira4, Svetoslav Dimitrov Todorov3 1

Department of Agronomy Food Natural resources Animals and Environment (DAFNAE), Università di Padova, Agripolis, Legnaro (PD), Italy; 2AGRIS Sardegna, Dipartimento per la Ricerca nelle Produzioni Animali, Olmedo (SS), Italy; 3Food Science and Experimental Nutrition Department, Sao Paulo University, Sao Paulo (SP), Brazil; 4Biochemical and Pharmaceutical Technology Department, Sao Paulo University, Sao Paulo (SP), Brazil

Abstract Bacteriocins production from cheap substrates could be effective for many food industrial applications. This study aimed at determining the conditions needed for the optimal production of enterocin SD1, enterocin SD2, enterocin SD3 and enterocin SD4 secreted by E. faecium strains SD1, SD2, SD3 and SD4, respectively, isolated from Sardinian goat´s milk. Cheese whey, low-cost milk by-product, was selected to be used as substrate for bacteriocins production. Skimmed milk and MRS broth were studied as reference media. Growth temperature played an important role in the bacteriocin production. Highest levels were recorded at 37oC. Growth of E. faecium strains in 0.2 and 10% skimmed milk or 0.2 and 10% cheese whey was similar to the growth observed in MRS broth. However, the levels of bacteriocins produced in skimmed milk and cheese whey were lower. Optimal enterocins production was detected when strains were grown in MRS with initial pH of 6.0 and 7.0. Growth of E. faecium SD1 and SD2 in cheese whey adjusted to pH 6.5 produced bacteriocin levels comparable to those detected in MRS broth, whereas E. faecium SD3 and SD4, once grown in cheese whey instead of MRS, showed reduced bacteriocin activity. In MRS broth, organic nitrogen sources were essential for high bacteriocin productions. Highest enterocin SD1 levels were recorded in the presence of tryptone, or with a combination of meat and yeast extract. For enterocins SD2, SD3 and SD4 the combination of meat extract, yeast extract and tryptone resulted in the highest antimicrobial activities. The growth of E. faecium strains in the presence of glucose or lactose yielded optimal bacteriocins levels while the addition of mannose in MRS supported growth and bacteriocin production only for E. faecium SD2 and SD4. Optimal bacteriocins levels were produced in the presence of K2HPO4, Tween 80 and ammonium acetate whereas the secretion of all the bacteriocins was reduced in the absence of magnesium sulphate, manganese sulphate or tri-ammonium citrate. This study showed that cheese whey, a major world-wide disposal and pollution problem for the dairy industry, could be an interesting low-cost substrate for the production of bacteriocins. However, the optimisation of MRS components seems to be more crucial towards the cost-effective production of high bacteriocins levels to be used in the food processing industry. As alternative, supplementing cheese whey with some of the MRS components may be an interesting strategy for the optimisation of bacteriocins productions from E. faecium strains.

P84 PRODUCTION OF ENZYMES FROM GRAPE STALKS AND WHEAT BRAN IN SOLID STATE FERMENTATION D.C. Masutti*, A. Borgognone, L. Setti Department of Industrial Chemistry and Materials, University of Bologna, Viale del Risorgimento, 4 - 40136 - Bologna, Italy

Abstract Many agricultural wastes could be used to produce enzymes from fungi. In 2004, the Italian production of grains was about 22,1 million tons, higher than screw about 9 million tons and olives about 4,7 million tons (www.istat.it). The hydrolytic demolition of the plant cell walls by lignocellulosic enzymes is one of the most studied approach to the valorization of agricultural wastes for the recovery of high value phytochemicals. The Pleurotus ostreatus is a white rot fungi who produce a wide range of extracellular enzymes to degrade complex lignocellulosic substrates into soluble substances that can be used as nutrients. Lignocellulosic enzymes such as cellulase, xylanase, peroxydase, laccase, and arylesterase (caffeoyl esterase and feruloyl esterase) were produced inducing through the growth of Pleurotus ostreatus in solid state fermentation using agro-food wastes (grape stalks and wheat bran) as substrates. Arylesterase activities were carried out as the prevalent on both the substrates, even if more significant values were found on grape stalks. Our findings showed that the enzymatic production was strictly dependent to the periodic removal of the produced enzymes and the arylesterase activities seem to be particularly affected by this factor. A new model for enzymatic production was discussed in order to develop new solid state bioreactor designs using Pleurotus ostreatus capable to open interesting industrial approach.

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Environmental Engineering and Management Journal

March 2012, Vol.11, No. 3, Supplement, S75-S88

http://omicron.ch.tuiasi.ro/EEMJ/

“Gheorghe Asachi” Technical University of Iasi, Romania

Sub-session 2.2. Organic waste and effluent biorefinery Main lecture BIOTECHNOLOGY FOR SUSTAINALBE SUPPLY AND USE OF PHOSPHORUS Hisao Ohtake Department of Biotechnology, Osaka University

Abstract Phosphorus (P) is one of the most critical elements in the biological building blocks and plays a crucial role in the cellular energy metabolism of all living organisms. P has been regarded as the life’s bottleneck or the ultimate limiting factor for the amount of life on Earth. P is used as a raw material in a wide variety of manufacturing industries. For instance, it is used in iron phosphate coatings in automobile industry, etching agents for aluminium line pattern substrates for liquid crystal panels and integrated circuit devices, and flame retardants for a variety of industrial products. More importantly, P is an essential element of fertilizer for agriculture and biomass production. Today, P is mostly obtained from mined rock phosphate which is a non-renewable resource. Approximately 17.5 million tons of P is mined in the world every year, and approximately 85% of the mined P is used as fertilizer for agricultural purposes. However, securing the sufficient quantity of high-quality rock phosphate at a reasonable price is becoming more and more difficult, because of the global shortage and increased P demand. For the sustainable resource management, increased attention has been paid to a closed P cycle on Earth. However, P recycling has never been realized on a large scale, mainly because of economical reasons. Among emerging issues on P recycling are the quality, quantity, cost, and market of recovered P. To tackle these issues, we have recently developed an innovative technology for P recovery using amorphous calcium silicate hydrates (A-CSHs) as inexpensive adsorbents. Importantly, the A-CSHs can be produced using unlimitedly available resources such as calcium carbonate and silicate. This technology allowed us to create a new business model for P recycling. Namely, a cement company manufactures A-CSHs at low costs, carries them to a P recovery site (e.g. wastewater treatment plant) using agitating trucks, and takes out P-binding A-CSHs to a fertilizer company. The recovered A-CSHs can be directly used as by-product phosphate fertilizer. To fully realize P recycling, the P Recycling Promotion Council of Japan (PRPCJ) was established as a nationwide association under the support of four important ministries of Japan in 2008. In this talk, our current efforts to develop phosphate refinery technology will be presented.

Session 2/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S75-S88

Oral presentations BIO-HYDROGEN AND BIO-METHANE CO-PRODUCTION BY SEQUENTIAL TWO-PHASES DARK FERMENTATION FROM AGRO-INDUSTRIAL WASTES (IMERA*) Giuseppe Lustrato, Gabriele Alfano, Giancarlo Ranalli DISTAT, Facoltà di Scienze, University of Molise, c/da Fonte Lappone, 86090 Pesche, Italy

Abstract Anaerobic digestion processes have often been applied for biological stabilization of solid and liquid wastes. These processes generate energy in the form of biogas. Recently, high-rate methane and hydrogen fermentation from renewable biomass has drawn much attention due to current environmental problems, particularly related to global warming. The aim of this paper is to study new research activities covering production of methane and hydrogen via both conventional mono and high-rate two-phase anaerobic digestion processes from LDO waste vegetable residues. At first, in order to set up and optimize several parameters related to the increase both in bio-hydrogen and methane, several aspects were considered, under lab conditions (T, type of substrate, pre-treatments of solid wastes, type of microbial inocula, HRT, biofuels recovery). Two anaerobic reactors were connected in series for the experiment: a) a working volume of 1 L and 5 L for bio-hydrogen and methane, respectively. The system operated in semicontinuous flow, at pH 5.5 and a HRT of 4 day, while the methane stage at pH 6.5-7.5 and a HRT value of 16 day. The results demonstrate the successful of an advanced biological approach to convert LDO residues (Large Distribution Organization) into renewable energy (IMERA project). The data obtained allowed to the set-up and the identification of the best conditions for fast treatment efficiency; more, permitted us the scale-up of the plant and co-production of hydrogen and methane, in a semi-pilot plant, with re-cycle between two-phases and with working volume of 300 and 1,000 l, respectively. Moreover, we proposed the use of low electric current treatment (LECT) of vegetables residues to control early stage fermentation and to improve bioH2 production. *IMERA-Bio-hydrogen and bio-methane by sequential phases dark fermentation from agro-industrial wastes. The project was supported by a contribution of MIPAAF, 2010-2013. Key words: anaerobic digestion, GDO residues, hydrogen, low electric current, two-phase anaerobic digestion

INVESTIGATION OF THE MICROBIAL COMMUNITY IN BIOGAS PLANTS BY METAPROTEOME ANALYSIS R. Heyer*1, D. Benndorf1, F.Kohrs1, A. Hanreich3, M. Klocke3, E. Rapp2, U. Reichl1,2 1

Otto von Guericke University Magdeburg, chair of bioprocess engineering; 2Max-Planck-Institute for dynamic of complex technical systems; 3Leibnitz Institute for agricultural engineering Potsdam-Bornim

Abstract Optimization of biogas plants requires a deeper knowledge about the microbial communities and their metabolism. In contrast to genetic, metabolic or cultivation- based analytical techniques, the investigation of the microbial proteome (metaproteome) is expected to detect actual biological activity and microbial interactions. Metaproteome analysis of biogas plant samples is challenging because samples contain high amount of impurities and are very complex. Furthermore, the identification of proteins is hindered due to limited availability of relevant genome sequence data. For removal of sample impurities phenol extraction showed the best results. To reduce sample complexity SDS-PAGE and 2D-PAGE coupled to LC-MS/MS were applied to biogas plant samples. Finally, proteins were identified by database search against NCBI and a metagenome, as well as by de novo sequencing. Up to 100 microbial proteins were identified including hydrolytic enzymes such as peptide hydrolases and enzymes of methanogenesis such as methyl-coenzyme M reductase and F420-dependent methylenetetrahydromethanopterin dehydrogenase. Surprisingly, plant proteins originating from the substrate maize such as photosystem type II and ribulose-1, 5-bisphosphate carboxylase/oxygenase were detected. Results show that metaproteome analysis of biogas plant samples can give valuable functional information about complex microbial communities. More comprehensive studies could be used to identify predictive biomarkers for process monitoring and control.

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Sub-session 2.2 - Organic waste and effluent biorefinery

ENHANCED ANAEROBIC DIGESTION PERFORMANCES: EFFECT OF SLUDGE ULTRASOUND PRE-TREATMENT AND ROLE OF THE MICROBIAL POPULATION DYNAMICS C.M. Braguglia, M.C. Gagliano, A. Gianico, S. Rossetti Water Research Institute, CNR, Area di Ricerca RM1-Montelibretti, Via Salaria Km 29.3- 00015 Monterotondo (RM), Italy

Abstract The biorefinery approach involves multi-step processes in which the first step typically involves biomass pre-treating to render it more amenable for further conversion steps. After the pretreatment, the components of the biomass are subject to biological and/or chemical processing, striving for energy and/or material recovery. The anaerobic digestion of sludge is an efficient and sustainable technology to stabilize sludge by means of mass and pathogen reduction, and in particular, energy recovery in the form of biogas. In this work ultrasound pretreatment aims to convert biomass as waste activated sludge, in which the particulate organics are recalcitrant to anaerobic bacterial hydrolysis, into a soluble, more biodegradable form. Anaerobic digestion occurs by the sequential co-operative action of a number of different bacterial trophic groups. Thus, the performance of an anaerobic digestion process is primarily linked to the structure of the microbial community present in the system. A deeper knowledge of the identity and function of the microbial components would allow to better control the biological processes, in particular in the case of sonicated sludge digestion, improving biogas recovery. Objective of this study was to evaluate the dynamics of the microbial population (estimated by Fluorescence in situ hybridization, FISH), during batch anaerobic digestion of either raw or sonicated waste activated sludge, at different food/inoculum ratio, comparing the performances of the “classical” 20 kHz ultrasounds with the new frequency of 200 kHz. The potential of the ultrasound pretreatment on the anaerobic digestion process was assessed by volatile solids (VS) degradation and biogas production, too. The positive effect of the high frequency ultrasound pretreatment was more evident for the low-inoculum digestion with a +35% increase of the VS degradation with respect to the +13% obtained from the test at higher inoculum fraction. The amount of Archaea increased over digestion time irrespective of F/I and of sonication treatment. The higher amount of Archaea at high inoculum content was most likely linked to the already established methanogenesis process occurring under these conditions, according to the high biogas production of F/I=0.5 digestion, where the gain due to sonication was up to 40%. Two predominant archaeal populations were identified: long Methanosaetaceae filaments, and cocci organised in sarcine, identified as Methanosarcina spp. The simultaneous presence of these genera suggests a metabolism mainly acetotrophic, whereas the entire set of experiments suggests a strict association of Methanosarcina dominance in the structure of anaerobic biomass to an efficient biogas production.

INDUSTRIAL BY-PRODUCTS AS A SOURCE OF VOLATILE FATTY ACIDS BY ANAEROBIC DIGESTION Fátima Sousa, Susana R.S. Pereira, Ana M.R.B. Xavier, Dmitry V. Evtuguin, Luísa S. Serafim CICECO, Departamento de Química, Universidade de Aveiro, Aveiro, Portugal, e-mail: [email protected]

Abstract Nowadays the economy is mainly dependent on fossil fuels. Pollution, climate changes and eminent exhaustion of natural resources (fossil fuels) are the main consequences of this dependence. Anaerobic Digestion (AD) has shown great potential in using renewable resources such as management residues from agriculture, industry and also from forestry. AD is a biological process by which organic matter is transformed into methane and carbon dioxide in the absence of oxygen. It is a complex microbiological process involving several trophic groups coordinated. The digestion process begins with bacterial hydrolysis of the input materials in order to break down insoluble organic polymers, such as carbohydrates, and make them available for other bacteria. Acidogenic bacteria then convert sugars and amino acids into carbon dioxide, hydrogen, ethanol and volatile fatty acids (VFAs). VFAs can be used to produce other compounds chemically or biologically. HSSL (Hardwood Spent Sulfite Liquor) is a by-product of the paper industry, rich in lignocelluloses that with appropriate pre-treatment provides carbon substrates as acetic acid, xylose, glucose and other sugars. HSSL has a high biochemical oxygen demand (BOD), about 2500 to 5000 ppm, which constitutes an environmental problem. HSSL can serve as substrate for yeast like Pichia stipitis to produce ethanol. At the end of this process the fermentative medium still contains a high amount of sugars that can act as substrate in an AD step to produce added value products like hydrogen, ethanol or VFAs. Therefore, the main objective in this work is not only to valorise HSSL but also apply AD (with methanogenesis suppressed) to the fermentative medium after P. stipitis ethanol production. The final goal of this project is the integration of the different processes in a biorefinery based in a pulp industry. In tests with HSSL were studied parameters such as inoculum concentration and organic load rate, verifying an increasing of 100% of the amount of formed VFAs when the load was increased from 30 to 60 gCOD/L with acetic acid as main acid formed. The metanogenesis suppression with specific inhibitors (BES) and not specific like High Pressure (HP) and temperature was also studied. An increase of 50% in total VFAs formed (acetic and butyric acid in the same proportions) and 30% (mainly acetic acid) was observed with BES and HP, respectively. In the test with HP, no propionic acid was produced.

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ANALYSIS OF MICROBIAL DIVERSITY OF INOCULA USED IN A MICROBIAL FUEL CELL A.C. Ortega Martínez1, K.Juarez-López2, O. Solorza-Feria3, N. Rinderknecht-Seijas4, H.M. Poggi-Varaldo*1 1 Environmental Biotechnology and Renewable Energies R&D Group, Dept. Biotechnology and Bioengineering, CINVESTAV del IPN, P.O. Box 14-740, 07000 México D.F., México; 2IBT-UNAM, Cuernavaca, México; 3Dept. Chemistry, CINVESTAV del IPN, Mexico; 4ESIQIE del IPN, Div. Basic Sciences, Mexico; e-mail : [email protected]

Abstract A microbial fuel cell (MFC) is a device that converts chemical energy into electrical energy with the aid of biocatalytic reactions of electrochemically active bacteria (EAB) (Kim et al., 2003). A MFC can be used to enrich a microbial consortium using wastewater as the electron donor. Molecular techniques are now widely applied to assess the diversity of microbial communities by analyzing the 16S rDNA sequence (Phung et al, 2004). Since it has been suggested that different microorganisms are enriched in MFCs fed with different inocula, this research seeks to experimentally confirm this hypothesis by characterizing the bacterial communities in MFCs fed with sulphatereducing inoculum and enriched inoculum. The sulphate inoculum was cultured in a complete mix sulfate-reducing bioreactor fed a mixture of sucrose, acetic acid, and sodium sulfate. The enriched inoculum was obtained with serial transfers of a sediment sample cultivated in a medium containing ferric citrate as electrons acceptor and sodium acetate as electron donor. The biofilm formed on the carbon cloth electrodes from the anodes was used for DNA extraction according to specific literature. Total genomic DNA was used as template for PCR amplification of approximately 1500 bp of 16S rDNA with a forward and reverse primers. The PCR products were purified, cloned, transformed into competent cells of E. coli XL1-Blue as described by in literature. Tansformants were transferred to plates containing LB broth spiked with antibiotics and the plasmids were isolated and digested for the presence of inserts. The bacterial population obtained from biofilm of the MFC loaded with sulphate-reducing inoculum and enriched inoculum was analyzed in terms of identity, abundance, phylum (class). Clostridiales bacterium was present in both inocula. There was a significant difference in community composition between both inocula. Clostridia predominated in the community of the biofilm of the MFC fed with sulphate reducing inoculum, whereas in the the biofilm of the cell loaded with enriched inocula the predominant microbes belonged to Deferribacteres class. Our results were similar to those observed in literature, with enriched electrochemically active bacteria in a MFC using glucose and glutamate (copiotrophic conditions); the enriched population consisted of γ-Proteobacteria (36.5%), followed by Firmicutes (27%) and δProteobacteria (15%). Other researchers observed that the bacterial communities that develop in MFC show great diversity, ranging from primarily δ-Proteobacteria, that predominate in sediments MFCs to communities composed of α- , β-, γ- or δ-Proteobacteria, Firmicutes and uncharacterized clones in other types of MFCs. On the other hand Geovibrio ferrireducens, Geovibrio thiophilus and Denitrovibrio acetiphilus are known to contain c-type cytochromes. Current evidence suggests that a series of c-type cytochromes associated with the inner membrane, the periplasm, and the outer membrane might interact to transfer electrons to the outer membrane surface. The bacterial population in the anodic biofilms of our cells was not as rich as found from other types of inocula. For instance, diversity given by Shanon-Weaver index was 1.27 and 1.38 and the species evenness given by Pielou´s evenness index was 0.66 and 0.71, for the sulphate reducing and enriched inoculum, respectively. These values mean that diversity of inocula was low and the evenness was low-tomoderate, respectively.

BIOTECHNOLOGICAL PROCESS FOR THE VALORISATION OF RESIDUAL GLYCEROL FROM THE BIODIESEL INDUSTRY. BROADENING THE BIOREFINERY Xavier Turon, Sergi Abad, Francesc Padrès IQS-Institut Químic de Sarrià, Ramon Llull University, via Augusta 390, Barcelona, Spain

Abstract Crude glycerol is generated in large excess by the biodiesel industry. About 10% of the feedstock is collected as contaminated byproduct, crude glycerol as an output. Purification tends to be economically unviable due to its cost and to the excess of glycerol in the market. In 2009-10 an increase of 233% was achieved in the UE. Bioprocesses using microorganisms able to metabolize the contaminated glycerol and generating valuable metabolites are explored. In this case, a marine heterotrophic microalgae or protist showed interesting yields using pure glycerol, producing very long chain poly unsaturated fatty acids (PUFA). The current work allows the characterization of the life cycle of this protist in benchtop fermenters, as potential industrial microorganisms. In terms of the metabolite of interest, docosahexaenoic acid (DHA) an omega-3 fatty acid is our main goal. But other valuable chemicals can be obtained as well (i.e: other fatty acids as the eicosapentaenoic (EPA) or arachidonic, and complex molecules as squalene). Currently the primary source is of DHA is fish-oil, carrying bioaccumulated persistent organic pollutants (POP). The current project is willing to contribute to alleviate an industrial/environmental problem and a human food/health problem, applying a biotechnology process. The added value of such molecules might substantially contribute to the economic viability of a biodiesel biorefinery, broadening the platform as includes chemicals to the liquid fuels obtained. Such large biorefinery platform might enable the economic viability of the biodiesel without any public subsidies.

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Sub-session 2.2 - Organic waste and effluent biorefinery

COUPLING WASTEWATER TREATMENT TO METHANE GENERATION IN BIOELECTROCHEMICAL SYSTEMS Marianna Villano1, Federico Aulenta2, Stefano Scardala1, Mario Beccari1, Mauro Majone1 1

Department of Chemistry, Sapienza University of Rome; 2Water Research Institute, National Research Council (IRSA-CNR)

Abstract Bioelectrochemical systems (BESs) are an innovative and attractive technology that combines bacterial metabolism with electrochemistry for wastewater treatment. In a BES, 'electro-active' bacteria engage in extracellular electron transfer reactions with solid-state electrodes, which serve as electron acceptors or donors in their energy metabolism. So far, the most studied BES is the microbial fuel cell (MFC), in which microorganisms use an anode as terminal electron acceptor for the oxidation of waste organic substrates, thereby generating electrical power. The more recent discovery that cathodes can also serve as electron donors to drive microbial reductions of oxidized compounds, has opened new perspectives for application of BESs (besides electricity generation) in the field of bioremediation and in the production of fuels and chemicals. These latter systems are typically regarded as microbial electrolysis cells (MECs) since, differently from MFCs, require the voltage generated from substrate oxidation (at the anode) to be boosted with a power supply in order to drive the target cathodic reaction, otherwise energetically unfeasible. Here, a fully biological MEC coupling acetate (as model substrate) oxidation to biomethane generation has been developed. The MEC consisted of an anodic and cathodic compartment separated by a proton exchange membrane and filled with graphite granules, which functioned as both electrodic material and support for microbial biofilm formation. An activated sludge and an anaerobic sludge from a wastewater treatment plant were used as the anode and cathode inoculum, respectively. The MEC performance, in terms of methane production, acetate removal, and coulombic and energy efficiency, has been evaluated as a function of different operating parameters, including the organic load to the anode compartment and electrodic potentials. Overall, the obtained results pinpoint a remarkable potential of BESs for electricity-driven production of biofuels from wastewater.

Posters P86 MUTAGENIC ACTIVITY OF BIOCHARS OBTAINED FROM PYROLYSIS BIOREFINERY PROCESSES IN Salmonella typhimurium TA100 AND TA 98 TESTER STRAINS WITH AND WITHOUT METABOLIC S9 MIX ACTIVATION Anna V. Piterina1,2,4, Kevin Chipman3, J.J. Leahy1,4, J. Tony Pembroke2,4, Michael H. Hayes1 1 Carbolea, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland; 2Molecular Biochemistry Laboratory, Department of Chemical and Environmental Sciences and Centre for Applied Biomedical Engineering Research (CABER), University of Limerick, Limerick, Ireland; 3University of Birmingham, Birmingham, UK; 4Materials and Surface Science Institute (MSSI), University of Limerick, Limerick, Ireland

Abstract It is growing number of recent development in technologies and procedures within industrial biorefinery and bioenergy processes that can benefit society by significant improvement in procedures within chain of smart waste disposal and sustainable agriculture. Biochar as one important by-product of biorefinery processes. Its production and applications are emerging globally as novel industrial and commercial sector, however, the quality and chemical properties of such products will depend on the processes employed and the compositions of the substrates used. It is of high importance to develop and validate series of suitable protocols for the ecological monitoring of quality and properties of pyrolysis products named as “BIOCHARs”, and to initiate an establishment regulatory constrains and procedures which required to be implemented in product characterization chain. Here, we examined suitability of Ames test to analyse mutagenic activity of DMSO biochar extracts using two bacterial models (S. typhimurium strains TA 98 and TA 100) and established which tester strain which is most sensitive to recovered mutagenic compounds and condition of metabolic activation (S9). Developed testing procotocols as described can be well suitable for further studies to ensure sensitive screening and detection of the mutagenic residuals within the other type biochar products produced on research and industrial scale.

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P87 Chlorella vulgaris GROWTH ON DIGESTED URBAN SLUDGE Alessandro A. Casazza, Bahar Aliakbarian, Patrizia Perego, Attilio Converti Chemical and Process Engineering Department “G.B. Bonino”, Genoa University, via Opera Pia 15, 16145 Genoa, Italy

Abstract The aim of this research was to investigate the effect of the addition of digested urban sludge on the growth medium of the microalga Chlorella vulgaris. The anaerobic digestion of biomass feedstock and/or the biodegradable fraction of wastes is widely recognized as a mature and cost-effective process for producing biogas, which is a valuable renewable primary-energy source. Anaerobically digested residues are effluents still rich in inorganic nutrients, with high chemical oxygen demand, which contribute to the eutrophication phenomenon. It is therefore necessary to further treat the effluent. Microalgae could potentially offer many advantages being able to use the organic carbon (heterotrophic growth), as well as inorganic nutrients such as nitrogen and phosphorus from the wastewater for their growth without an aerobic environment. In this study, an anaerobic digester (2.5 L), placed in a thermostatted water bath under mesophilic conditions (38 ± 1 °C) and pH 7.2–7.4, was employed. The mixed sludge (1.25 L) was daily fed with 30 mL of urbane sludge. Under pseudo-steady state conditions, the composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5–76.0% and 13.2–19.5%, respectively. The digested sludge was removed daily and centrifuged. The liquid phase was added in different percentages (100, 66 and 33%) into the C. vulgaris medium to perform batch cultivations. The microalga concentration at the end of the experiments reached 2.72, 2.34 and 1.22 g/L when 66, 33 and 100 % of digested sludge liquid phase were used, respectively. These preliminary results suggest that combining microalgae production with municipal wastewater treatment could be made in an environmentally effective as well as economically viable way in a not distant future.

P88 ISOLATION AND CHARACTERIZATION OF POTENTIAL BIOSURFACTANTS PRODUCED BY Bacillus STRAINS GROWING ON AGROINDUSTRIAL WASTES Anna Poliwoda1, Ewa Krzosok1, Piotr P. Wieczorek1, Paweł Kafarski1, Grażyna Płaza2 1

Opole University, Faculty of Chemistry, 11a, Kopernika str., 45-040 Opole, Poland; 2Institute for Ecology of Industrial Areas, Department of Environmental Biotechnology, 6, Kossutha str., 40-844 Katowice, Poland

Abstract Lipopeptides belong to the natural biosurfactants of bacterial origin that consist of a hydrophobic long alkyl chain linked to a hydrophilic polypeptide with cyclic or linear structure. These molecules have received considerable attention as an attractive substances used in agriculture (especially as biopesticides in plant disease control), medicine (as pharmaceuticals due to its antimicrobial, antiviral and antitumor activities and enzyme inhibition) and pollution remediation. Unfortunately, the main problem for their widespread use is the fact that these compounds have not been able to compete economically with their chemically synthesized counterparts. The best way to reduce substrate cost for biotechnology at present is to use wastes with the right balance of carbohydrates and lipids to support optimal bacterial growth and biosurfactants production, and which are either free or carry a cost credit for environmental benefit. As known, millions of tons of hazardous and non-hazardous wastes are generated each year throughout the world. There is a great need for better management of these wastes via the concept: reduce, reuse, and recycle. Therefore, the aim of this work was to isolate and characterise of lipopeptide biosurfactants produced by Bacillus spp. cultured on agroindustrial wastes (i.e. molasses and brewery effluents). The surfactants were isolated from the obtained medium (supernatants) by using solvent extraction technique with various organic extrahents (like chloroform, dichloromethane, methanol, hexane, etc.). Mass spectrometry with electrospray ionization and NMR (400 MHz) were used to characterized the purified surfactant. The type of applied solvent (-s) determined the content and type of isolated lipopeptides from analysed supernatants. The fractions rich in lipopeptides were obtained. Characteristic m/z peaks of surfactins, fengicyns and iturins families were observed. Furthermore, the novel, unknown product was isolated and its chemical structure was proposed.

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Sub-session 2.2 - Organic waste and effluent biorefinery

P89 SELECTION OF CELLULASE PRODUCER MICROMYCETES OF THE SOUTH CAUCASUS FOR THE PRODUCTION OF HIGH TECHNOLOGY SWEETENERS FROM AGRARIAN WASTES E. Kvesitadze, T. Urushadze, R. Khvedelidze, L. Kutateladze Durmishidze Institute of Biochemistry and Biotechnology of Georgian Agrarian University, Tibilisi, Georgia

Abstract One of the promising ways of producing sweeteners from non-edible raw material is enzymatic hydrolysis of cellulose. Selection of cellulases with suitable biotechnological characteristics is essential for the development of industrial technologies of this process. Active producers of high technological cellulases Aspergillus versicolor D1, Aspergillus wentii Z9-7, Sporotrichum pilverulentum S1, Aspergillus terreus 4-9, have been selected from the collection of micromycetes (2300 strains) of Durmishidze Institute of Biochemistry and Biotechnology that were isolated from different ecological zones of south Caucasus. Crude preparations of cellulases were obtained by ethanol precipitation from the cultural liquid. Ethanol precipitation allows retaining 80-85% of total activity of cellulase as compared to the total activity of the cellulase in the cultural liquid. The industrial properties of selected crude enzyme preparations: heat stability, temperature optimum of action, mechanism of cellulase inhibition by the reaction products, enzymes ability of deep hydrolysis of cellulose containing agrarian wastes (tea wastes, tobacco wastes, wine wastes, citrus wastes) forming low molecular reducing sugars and glucose have been studied. The temperature optimum of action of cellulases for A. versicolor D1 and A. wentii Z 9-7 was at 60oC, while for strains Sporotrichumpilverulentum S1 and, Aspergillus terreus 4-9 at 55oC. Cellulases from Aspergillus versicolor D1 and Aspergillus wentii Z 97 retained 50% of initial activity after 3 hours of incubation at 63°C in substrate free media. In all cases inhibition was weak and of noncompetitive type. Degree of hydrolysis of agrarian wastes was in the range of 32-72% for reducible sugars and 15- 65% for glucose. Thus, the cellulases meet the biotechnological demands of enzymatic hydrolysis of cellulose and are applicable at industrial scale.

P90 INFLUENCE OF THE TYPE OF ALKALI USED FOR PH CORRECTION OF ACID GROWTH MEDIA ON METHANE PRODUCTION BY ACETOCLASTIC METHANOGENS C. Vasmara, L. Sghedoni, V. Faeti, R. Marchetti CRA, Research unit for swine husbandry

Abstract In recent years the biological production of hydrogen and methane from organic waste and effluents in two-stage anaerobic systems has received increasing attention. The anaerobic digestion of the broth resulting from dark fermentation for hydrogen production does not always run smoothly. A critical point in methane production, when starting from a substrate already utilized for hydrogen production, is the adaptation of the methanogenic consortia to peculiar substrate conditions. In fact the organic waste used for hydrogen production has usually low pH values, due to the need to selectively favour the hydrogen producers. Moreover, high levels of volatile fatty acids (VFA) accumulate during fermentation, with a further lowering of pH. As methanogenic consortia are negatively affected by low pH values, it is necessary to provide for pH correction. The kind of alkali used to correct the pH of the broth has been invoked as a possible source of sluggish digestions sometimes observed in the methanogenic phase of the two-stage process. In this research we monitored the activity of a selected methanogenic acetoclastic consortium in a synthetic medium of composition mimicking that of a broth resulting from hydrogen fermentation. In particular, the phosphate-buffered basal medium (PBBM) for acetoclastic methanogens was modified by addition of a mixture of VFA and ethanol. The resulting pH value, equal to 4.21, was adjusted to 7 by means of different alkali: NaOH, KOH and NaHCO3. These media were compared with a medium having unadjusted pH or with a classic PBBM at pH 7. Gas production (volume and composition) was monitored during the incubation period (2 months at 35°C, in 100mL reactors). For all treatments, an initial lag-phase was observed, due either to the reduced inoculum amount (5%, v/v) or to the need for the methanogenic consortium to adapt to the new growth medium conditions. Methane production was earlier and higher when the pH of the growth medium had been adjusted with NaHCO3. Sodium bicarbonate, together with CO2, is usually utilized as a buffering agent in growth media for methanogens, where it allows for neutral conditions to be maintained. The positive effect that it showed in this experiment can be attributed to this buffering function. As no methane production was detected in the medium with unadjusted pH, pH correction seems necessary for methane production from broths resulting from hydrogen production. From a technical point of view, pH adjustment with Na bicarbonate is preferable than pH adjustment with soda.

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Session 2/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S75-S88

P91 EFFECT OF TOTAL SOLIDS CONTENT OF THE FEEDSTOCK, TEMPERATURE AND MASS RETENTION TIME ON THE PERFORMANCE OF BIOHYDROGENIC SOLID SUBSTRATE FERMENTATION OF ORGANIC WASTE C. Escamilla-Alvarado1, M.T. Ponce-Noyola2, E. Ríos-Leal3, Héctor M. Poggi-Varaldo*1 1 Environmental Biotechnology and Renewable Energies R&D Group, Dept. of Biotechnology and Bioengineering, Centro de Investigación y de Estudios Avanzados del IPN, Mexico City 07000, Mexico, Phone: 5255 5747 3800 ext 4324; 2Microbial Genetics Group, ibidem; 3Central Analítica, ibidem; e-mail: [email protected]

Abstract On the edge of oil age decline, hydrogen is regarded as an alternative to fossil fuels. An interesting technology for biohydrogen production is dark fermentation in terms of its productivities and relative ease of the process. Research performed in this field is mostly done at low solids ( temperature > MRT. Significant interactions amidst factors only occurred between TS and temperature or MRT. High total solids content affected negatively the hydrogen productivity. Variations and inhibition of hydrogen production were related to low pH and high lactic acid and solvents production. This was in agreement with reports of a strong correlation between high lactic acid concentrations and inhibition of hydrogenesis. Key words: biohydrogen, high total solids, municipal organic solid wastes

P92 A STUDY ON THE INFLUENCE OF ACTIVATED SLUDGE AS NITROGEN SOURCE ON HYDROGENIC BATCH DARK FERMENTATION OF THE ORGANIC SOLID WASTE C. Escamilla-Alvarado1, M.T. Ponce-Noyola2, H.M. Poggi-Varaldo*1 1

Environmental Biotechnology and Renewable Energies R&D Group, Dept. of Biotechnology and Bioengineering, CINVESTAV del IPN, Mexico D.F., 07000, Mexico, Phone.: 5255 5747 3800 ext 4324; 2Microbial Genetics Group, Ibidem; e-mail : [email protected]

Abstract Biohydrogen is considered a feasible option for decreasing oil dependence, and in turn, biomass from municipal wastes is a promising feedstock for its production. Carbon to nitrogen (C/N) ratio of municipal organic wastes is usually high, up to 76, yet for anaerobic fermentation the suggested C/N ratio is 25-35. Nitrogen may be supplemented as ammonia or in the form of organic nitrogen, such as manure, waste sludges, or food wastes. This research studied the addition of non sterile activated sludge as inexpensive an easily available nitrogen source for hydrogen production of organic residues. Batch, solid substrate anaerobic hydrogenogenic fermentation intermitently vented mini reactors (SSAHF-IV) were used for organic nitrogen and alkalinity supplementation studies on a 23 experimental design basis. Factors were temperature (35, 55 ºC), C/N ratio (basal 50, 30 adjusted with activated sludge), and alkalinity (none, 0.06 g CaCO3/g dry substrate with phosphate salts). Glass mini-reactors were operated as described elsewhere. During the first cycle of operation cumulative hydrogen production (PH2) and maximum hydrogenic rate (RH2) were higher in thermophilic regime than mesophilic one. No lag phase was observed: at 14 h most mini-reactors had hydrogen contents superior to 30%. Highest results were PH2 = 1575 μmolH2/gVS and RH2 = 68.3 μmolH2/gVS/h in the thermophilic mini-reactor with no addition of alkalinity nor sludge. Controls did not show significant hydrogen production. Sludge and alkalinity supplementation unexpectedly did not have a positive effect on PH2 in any of the regimes. The C/N of OFMSW was sufficient to produce high amounts of hydrogen (PH2 = 1575 umolH2/gVS, RH2 = 68.3 umolH2/gVS/h). This may infer that microorganisms introduced through supplemented sludge may have affected fermentation, particularly boosting hydrogen consumption. On the other hand, after bioreactor headspace flushing with N2, there was a second cycle of hydrogenesis in thermophilic units, but not in mesophilic ones. Gas sparging has been used previously with other substrates and 2 to 5 cycles of hydrogenesis have been obtained. Despite the few or inexistent multiple cycles, our hydrogen productions were comparable to those in literature of SSAHF-IV. It is very likely that there would be a maximum of hydrogen that might be harvested independently of the cycles. Key words: intermittent venting; municipal organic solid wastes; nitrogen supplementation

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Sub-session 2.2 - Organic waste and effluent biorefinery

P93 SERIES HYDROGENESIS-METHANOGENESIS OF OFMSW: OPERATION AND ENERGETIC FEASIBILITY OF THE PROCESS C. Escamilla-Alvarado1, M.T. Ponce-Noyola2, E. Ríos-Leal3, H.M. Poggi-Varaldo*1 1

Environmental Biotechnology and Renewable Energies R&D Group, Dept. of Biotechnology and Bioengineering, Centro de Investigación y de Estudios Avanzados del IPN, Mexico City 07000, Mexico, Phone.: 5255 5747 3800 ext 4321; 2Microbial Genetics Group, ibidem; 3Instrumental Analysis Central, ibidem

Abstract Over the last 15 years there has been an intensive research to improve the bioenergy that can be extracted from organic wastes through biotechnology. A recent option is through the series hydrogenesis-methanogenesis (H-M) process, and information on process energetic feasibility is still scarce. The aim of this work was to evaluate the effects of organic load, operation temperature on process performance and energetic feasibility of a series H-M semi-continuous process using organic fraction of municipal solid waste (OFMSW) as substrate. Four experimental cases were set-up using OFMSW 20.9% total solids intermittently fed, as reported previously. Operational variables were two thermal regimes (35 and 55°C) and hydrogenogenic bioreactors (H-stage) running at 21 and 14 d mass retention time (MRT). Fermented solids from these biohydrogenogenic units were fed to methanogenic bioreactors (M-stage) at 28 d MRT. A steady state model of the process was developed in order to estimate the energy balance for 50 000 kg/d of wet OFMSW. On the one hand, the invested energy considered pre-heating of the feed to the H-stage, heat losses to ambient air of the bioreactors, energy for mixing the contents of bioreactors, and energy for pumping. On the other hand the generated energy was based on the heats of combustion of H2 and CH4 produced. The best hydrogenic productivity was 202 mL H2/(kg d) at 21 d TRM and 55 ºC, whereas the highest methanogenic productivity was 2023 NmL CH4/(kgr d) at 55 ºC fed with fermented solids from 14 d MRT. Energy balance proved that the net power was positive in all cases; the net energy was double (or higher) the energy invested in the H-M process. The highest contribution to generated energy corresponded to the methanogenic stage, which accounted for 95-98% of the total power potential. Thermophilic was the best operating regime in terms of net power generation, despite the higher power consumption. Results confirmed earlier comparison of continuous meso and themophilic dark fermentation of OFMSW. Yet, the energetic performance was also dependent of mass retention time of the H-stage. For instance, cases #2 and #4, which operated at different thermal regime but same 14 d H-stage, had the highest Pnet and correspondingly the highest ratio Pnet/Pinvest ratio. Despite the higher combustion enthalpy of hydrogen, the main contribution of the H-stage seemed to be related to providing readily digestible organic matter to the M-stage (through hydrolysis and fermentation), rather than contributing directly the power output. This work proved that H-M process is energetically feasible, yet ways of increasing hydrogen productivities should be further studied for higher power gains. Key words: biohydrogen, energy balance, energy feasibility, methanogenesis, municipal organic solid wastes, series process

P94 BIOMETHANE PRODUCTION FROM CO-DIGESTION OF SEWAGE SLUDGE AND CRUDE GLYCEROL FROM BIODIESEL PRODUCTION C. Salomoni1, A. Caputo1, M. Bonoli1, O. Francioso2, M.T. Rodriguez-Estrada3, D. Palenzona 1

Biotec Sys Srl, Via Gaetano Tacconi, 59, 40139 Bologna, Italy; 2Dipartimento di Scienze e Tecnologie Agroambientali, V.le Fanin 40, 40127 Bologna, Italy; 3Dipartimento di Scienze degli Alimenti, V.le Fanin 40, 40127 Bologna, Italy

Abstract Crude glycerol is a major by-product of biodiesel production, which represents about 10% of the biomass input to the process. Crude glycerol is an interesting raw material for at least a couple of anaerobic digestion applications. It can be used in a dedicated facility within the biodiesel production plant itself, supplying both electric and thermal energy to the biodiesel plant; moreover, being a liquid vector with relatively high energy content, crude glycerol can be conveniently dispatched to local anaerobic digestion plants (animal farms, wastewater treatment plants, etc.) and improve their productivity. In the present study, a long-standing demonstration of crude glycerol co-digestion with sewage sludge is reported. A two-phase pilot plant was used to convert mixed sludge and crude glycerol into biomethane at the Bologna municipal wastewater treatment plant. The two-phase process was started up with sewage sludge as only substrate, as described elsewhere. Crude glycerol was introduced gradually in codigestion with sewage sludge and had no impairing effects on process parameters. Moreover, phase splitting was stable even when crude glycerol represented more than 70% of the total COD loading rate. Biomass conversion exceeded 80%, leading to a very low amount of residues. Methane production rate was highly increased, thus raising overall productivity of the plant. The significant variation of C/N ratio, due to crude glycerol addition, led to a considerable decrease in ammonia concentration in the digestate when high amounts of glycerol were added. On the basis of this result we can infer that crude glycerol addition had an important consequence on ammonia metabolism.

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Session 2/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S75-S88

P95 EFFECT OF GLYCEROL ON BIOMETHANE PRODUCTION AND VOLATILE ORGANIC COMPOUNDS IN ANAEROBIC DIGESTION OF URBAN SEWAGE SLUDGE O. Francioso1, M.T. Rodriguez-Estrada2, A. Pisi 1, S. Savioli2, C. Salomoni3, A.Caputo3, M. Bonoli3, D. Palenzona3 1

Dipartimento di Scienze e Tecnologie Agroambientali, V.le Fanin 40, 40127 Bologna, Italy; 2Dipartimento di Scienze degli Alimenti, V.le Fanin 40, 40127 Bologna, Italy; 3Biotec sys srl, Via Gaetano Tacconi, 59, 40139 Bologna, Italy

Abstract The glycerol from biodiesel industry is considered an ideal co-substrate for the anaerobic digestion and can boost biogas yields. However, in order to keep a stable digestion process, the amount of glycerol added should be controlled to avoid the risk of organic overloading. The aim of this work was to evaluate the glycerol effect used as a co-substrate in order to boost biogas production during the anaerobic treatment of urban sewage sludge in a separated, two-phase anaerobic system. The first phase was used for hydrolysis, acidification and acetogenesis of wastewater sludges, whereas the second one was employed for methanogenesis. Soluble chemical oxygen demand (SCOD), total suspended solids (TSS), volatile suspended solids (VSS), VFA, total alkalinity (TA), pH and biogas production were monitored. The effect of glycerol was also examined on the composition of volatile organic compounds (VOC) by head-space solid phase microextraction combined with gas chromatography–mass spectrometry (HSSPME-GC-MS) and on the sludges morphology by scanning electron microscopy (SEM). All results were compared with a methanogenic digester without glycerol. Glycerol addition boosted biogas yields and considerably changed the VOC composition, while no trace of volatile fatty acids (VFA) was found in the digestate. The appearance of different volatiles amines suggested that the methane formation seems to take place through the amines fermentative degradation instead of the acetate one. In conclusion, the utilization of glycerol, a residue of the biodiesel industry, can be useful to improve methane production in wastewater treatment plants with a sludge anaerobic digestion unit.

P95 CHARACTERIZATION OF A FIVE-FACE PARALLELEPIPED MICROBIAL FUEL CELL EQUIPPED WITH SANDWICH ELECTRODES A.C. Ortega Martínez 1, O. Solorza-Feria2, M.T.Ponce-Noyola3, N. Rinderknecht-Seijas4, H.M. Poggi-Varaldo *1 1

Environmental Biotechnology and Renewable Energies R&D Group, Dept. Biotechnology and Bioengineering, CINVESTAV del IPN, P.O. Box 14-740, 07000 México D.F., Méxic; 2Dept. of Chemistry, ibídem; 3Dept. of Biotechnology and Bioengineering, ibidem; 4 ESIQIE-IPN, Div. Basic Sciences, Mexico; e-mail: [email protected]

Abstract A microbial fuel cell (MFC) is a promising technology for generating electricity directly from biodegradable compounds using bacteria under anaerobic conditions.The actual voltage output of an MFC is less than the predicted thermodynamic ideal voltage due to irreversible losses; this limits MFC performance. The aim of this work was to design and characterize a novel, multiface parallelepiped MFC in the perspective of decreasing the internal resistance (Rint) and increasing volumetric power (Pv) output. The multiface MFC consisted of a parallelepiped built in plexiglass with a liquid volume of 270 mL. Five faces of this cell were fitted with ‘sandwich’ cathode-membrane-anode assemblages (CMA) and possessed a high ratio electrode surface area-to-volume ξ. The MFC was loaded with 14 mL of a model extract and 256 mL of a mixed liquor from a sulphate-reducing, mesophilic, complete mixed, continuous bioreactor as described elsewhere. The values of internal resistances were 400 and 84 Ω for the MFC connected in series and parallel, respectively. Maximum volumetric powers PV in MFC connected in series and parallel were 655 and 1800 mW/m3, respectively, and anode density powers PAn of the MFC connected in series and parallel were 18.4 and 50 mW/m2, respectively. During the connection in series, the voltage was 0.66 V; this voltage was almost double of that obtained when the MFC was connected in parallel (0.34 V). All the other response variables in MFC connected in parallel were higher than MFC connected in series. Parallel connection decreased the Rint by presumably increasing the cross sectional area for ion flow. The values of the resistances for each face of MFC were 354, 425, 321, 373, and 421 Ω. Calculations using the Ohm´s law for parallel resistance connection agrees with the the total experimental value of 84 ± 5 Ω. In effect, 1/Rint total = Σ (1/Rint,j), j = 1, ...., 5 (1). When substituting the Rint,j of each face, Eq. 1 gives Rint total = 76 ± 4 Ω, this calculated value is close to the experimental one obtained with the polarization curve method (84 ± 3 Ω). Energy loss in the series connection is known to be caused by lateral ion cross-conduction between electrodes; this phenomenon is common when fuel cell arrays sharing the same electrolyte are connected in series to increase voltage output. Parallel connection of multiple electrodes of MFC significantly increased PV-max compared to that of the MFC connected in series. Also, multiple MFC can be connected in series, forming a stacked system in order to increase the voltage. However, when this is done the stack usually undergoes voltage reversal, resulting in a dramatic decrease of stack voltage. Our results confirm the positive effect of ξ on Pv, show the advantages of the ‘sandwich’ assemblage of CMA over separated electrodes, and demonstrate the convenience of parallel connection of faces in multi-face MFC-P in order to further abate the internal resistance of the cell.

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Sub-session 2.2 - Organic waste and effluent biorefinery

P97 APPLICATION OF RUXMOYSEZ FOR OXYGEN REDUCTION REACTION IN A MICROBIAL FUEL CELL A.L. Vázquez-Larios1, O. Solorza-Feria2, E. Ríos-Leal1, N. Rinderknecht- Seijas3, R. de G. González-Huerta 3, H.M. Poggi-Varaldo*1 1 Environmental Biotechnology and Renewable Energy R&D Group, Depto. Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740, 07000 México D.F., México; 2Depto. Química, Centro de Investigación y de Estudios Avanzados del IPN, México D.F., México; 3esiqie del IPN, México D.F., México; e-mail : [email protected]

Abstract One of the actual challenges in microbial fuel cells research consists of the application of new electrochemically active catalytic materials (RuxMoySez), such as alternate electrocatalysts to replace the extensive use of the more expensive platinum. Thus, the objective of our work was to evaluate the application of RuxMoySez for oxygen reduction reaction in a new design of a single chamber microbial fuel cell (MFC). A MFC was based on extended electrode surface (larger , ratio of electrode surface to cell volume) and the arrangement of the anodePEM-cathode. The cell was built with a plexiglass cylinder, the two extreme circular faces were fitted with PEM-cathod assemblage, i.e., left and right faces. The anode consisted of 65 triangular pieces of graphite (1.4 x 1.8 x 0.5 cm, side x height x thickness). The cathode was made of flexible carbon-cloth containing 1mg/cm2 RuxMoySez catalyst. The cell was loaded with inoculum and substrate according to the specific literature. The MFC was characterized by linear sweep potential method as reported elsewhere. First, each face of the MFC was characterized by separate (left and right). Second, the MFC was characterized with the two faces connected in series and parallel. Values of EMFC, OCP obtained from the separate faces were 0.591 and 0.593 V for the left and right face, respectively. Series and parallel connection showed similar potentials. The PV-max values were 1200, 1125, 1124 and 1829 mW/m3 for separate faces, and series and parallel connection, respectively. The corresponding Rint were 146, 167, 162 and 69 Ω . The PV-max of separate face electrodes was high and comparable with the value 1010 of mW/m3 reported elsewhere. Parallel connection significantly decreased the internal resistance of the cell (69 Ω) and almost doubled volumetric power, due to increased current intensity of 1.8 mA. The results for PV-max for parallel connection can increase the volumetric power significantly. The internal resistance values in this work were in the low side of the range reported in the literature. Our results have shown that the application RuxMoySez as a cathodic catalyst for oxygen reduction reaction in a new design of a single chamber microbial fuel cell holds promise.

P98 EFFECT OF ANODIC MATERIAL ON THE PERFORMANCE OF A SINGLE CHAMBER MICROBIAL FUEL CELL A.L. Vázquez-Larios1, M.T. Ponce-Noyola1, J. Barrera-Cortés1, R. de G.González-Huerta2, O. Solorza-Feria3, H.M. Poggi-Varaldo*1 1

Environmental Biotechnology and Renewable Energy R&D Group, Depto. Biotecnología y Bioingeniería, CINVESTAV del IPN, Apdo. Postal 14-740, 07000 México D.F., México; 3ESIQIE del IPN, México D.F., México; 3Depto. Química, CINVESTAV del IPN, México D.F., México

Abstract Several groups have investigated anode materials in microbial fuel cells in attempts try to increase the power output per unit volume of reactor. Several types of materials and shapes have been used, such as carbon paper, graphite plate, carbon cloth, carbon mesh, granular graphite, granular activated carbon, carbon felt, reticulated vitrified carbon, carbon brush, stainless steel mesh. Thus, the objective of our work was to evaluate the effect of two anodic materials on the performance of microbial fuel cells (MFCs) that used RuxMoySez as a catalyst for oxygen reduction reaction in the cathode. A new design of a single chamber MFC-A was based on extended electrode surface (larger , ratio of electrode surface to cell volume) and the arrangement of the anode-PEM-cathode. The cell was built with a plexiglass cylinder, the two extreme circular faces were fitted with PEM-cathod assemblage, i.e., left and right faces. The anode consisted of 65 triangular pieces of graphite (1.4 x 1.8 x 0.5 cm, side x height x thickness) filling the anodic chamber. The single chamber MFC-B had an assemblage or ‘sandwich’ arrangement of the anode-PEMcathode. The cathodes were made of flexible carbon-cloth containing 1mg/cm2 RuxMoySez catalyst. The cell was loaded with inoculum and substrate according the specific literature. The MFC was characterized by linear sweep potential method as reported elsewhere. First, each face (left and right) of the MFCs was characterized by separate. Values of EMFC, OCP obtained were 0.591 and 0.593 V for the left and right face (MFC-A), and 0.255 and 0.260 V for the left and right face the cell B, respectively. The PV-max values for MFC-A were 1 200 and 1 125 mW/m3 and those of MFC-B were 43 mW/m3 for both separate faces. The polarization curves were very close to straight lines; the values of Rint were estimated from the slopes of corresponding regression lines as 146 and 167 Ω for MFC-A and MFC-B were 2 075 and 2 017 Ω, respectively. The proportion of Rint decreased by a factor of 27.9 and 26.1 for MFC-A. Parallel connection significantly decreased the internal resistance of the cell and almost doubled volumetric power for FMC-A and MFC-B, respectively. The results for PV-max for parallel connection can increase the volumetric power efficiently. The PV-max for MFC-A was comparable with values reported elsewhere. Finally, application of graphite anode made of small triangular pieces significantly improved the performance of a MFC-A that used RuxMoySez as a cathodic catalyst for oxygen reduction reaction. Key words: anodic material, graphite, internal resistance, microbial fuel cell

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Session 2/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S75-S88

P99 EFFECT OF ANODIC MATERIAL ON THE INTERNAL RESISTANCE OF A SINGLE CHAMBER MICROBIAL FUEL CELL G. Hernández-Flores1, O. Solorza-Feria2, M.T. Ponce Noyola3, N. Rinderknecht-Seijas4, H.M. Poggi-Varaldo*1 1 Environmental Biotechnology and Renewable Energies R&D Group, Dept. Biotechnology and Bioengineering, CINVESTAV-IPN, México; 2Dept. of Chemistry, CINVESTAV-IPN; 3Microbial Genetic Group, Ibidem; 4ESIQIE del IPN, Division of Basic Sciences. México; e-mail: [email protected]

Abstract In the search for new for alternatives to produce energy, microbial fuel cells (MFCs) have emerged as a promising. MFCs carry out at the same time pollution control of effluents and bioelectricity production. It is known that one of the important factors that reduce the electricity production in MFC is the high internal resistance (Rint) value. Thus, this research aimed at evaluating three anodic materials and their effect on Rint of a single chamber MFC. The MFC was a horizontal cylinder built in Plexiglass 80 mm long and 57 mm internal diameter with air cathode. The internal resistance was determined by the variable resistance method and polarization curve. As inoculum, a sulfate-reducing consortia was used and with a model extract (a mixture of acetic, propionic and butyric acids as well as acetone and ethanol) from spent solids generated in fermentative hydrogenogenic process, the MFC was fed. The cell with granular activated carbon (GAC) as anodic material, with total surface of 0.2 m2 had a high Rint (10 KΩ); it was associated to a low electrical conductivity of GAC (σ < 0.012 S/m). The cell equipped with an anodic graphite rod (GR, which also worked as electron collector) with a surface of 9.15X10-4 m2 displayed an Rint of 800 Ω. Finally, using small equilateral triangles of graphite (GT) of 1.4x1.8x0.5 cm (side x height x thickness, respectively) as anode with a total surface of 0.06 m2, the lowest Rint was obtained (400 Ω). Volumetric powers (Pv) were in the following order: GT ˃˃ GR ˃˃ GAC The type of anodic material and the geometric configuration had a significant effect on the Rint and Pv, the Pv increased and the Rint decreased in two materials (GR and GT). Some authors used graphite granules as anode, and the Rint was 500 Ω; others used a graphite brush as anode, but their Rint was relative high (1000 Ω) compared with this work. In this work, the best results could be explained by the high electric conductivity of GR and GT. Comparing GT with GR, GT displayed the highest Pv and the lowest Rint and this could be attributed to the relative high value of the specific surface of GT.

P100 PRODUCTION OF OXIDATIVE ENZYMES BY Trametes ochracea ON THE HIGH-MOLECULAR WEIGHT FRACTION OF OLIVE-MILL WASTEWATER M. Petruccioli, T. Stella, E. Carota, A. D’Annibale Department for Innovation in Biological, Agro-Food, and Forestry systems (DIBAF), University of Tuscia, Viterbo, Italy

Abstract Olive-mill wastewater (OMW), the incorrect disposal of which may cause serious and large-scale environmental impact, might be regarded as a possible resource due to its remarkable content in simple and complex sugars, lipids, residual oil, proteins, mineral elements and phenols with elevated biological activity (e.g., oleuropein, ligstroside and verbascoside) that could be either directly recovered and purified or utilized for fermentative production processes. The present work is part of a wider project aimed to assess the techno-economical feasibility of an OMW biorefinery that provides recovery of the phenolic substances by a membrane separation technology and the achievement of a permeate, containing a very low organic load, useful in the oil mechanical extraction process. The low-molecular weight phenols are tested as antioxidants in the animal feeding and/or in the preparation of various kinds of fresh foods while the residual high-molecular weight fraction (HMW) is used for microbial production of phenol oxidases. A preliminary screening of several white-rot fungal species led to the selection of Trametes ochracea CBS 257.74 as the most promising strain for its ability to grow on the HMW-OMW and to release phenol oxidases. Therefore, the potential of this strain to produce laccase and Mn-dependent peroxidase (MnP) activities on HMW-OMW was tested using different single and combined supplements. Levels of dephenolization and detoxification of the final effluent were also detected. The addition of carbon sources (i.e., glycerol, glucose and fructose) did not significantly affect enzyme production, dephenolization and organic load removal. On the contrary, supplementation with nitrogen sources (yeast extract, casein peptone and diammonium tartrate) resulted in a significant improvement of both phenol oxidases activities and the highest laccase and MnP activities were reached with peptone (29.3 and 5.8 IU ml-1, respectively). Among the tested putative inducers (i.e., DMSO, ethanol, Tween 80, MnSO4 and CuSO4), CuSO4 was the most effective one. In particular, laccase and MnP activity peaks reached 35.6 and 15.7 IU ml-1, respectively, in the HMWOMW medium supplemented with 0.75 mM CuSO4 and peptone. These results confirm that the high-molecular weight fraction of OMW supplemented with peptone and CuSO4 represents a good medium for phenol oxidases production by T. ochracea. A better definition of process parameters at bioreactor scale using pneumatic and mechanic systems are in progress to assess the real potential of the proposed fermentation.

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Sub-session 2.2 - Organic waste and effluent biorefinery

P101 ACIDOGENIC DIGESTION OF DEPROTEINIZED CHEESE WHEY Alberto Scoma*, Lorenzo Bertin, Fabio Fava Department of Civil, Environmental and Materials Engineering (DICAM), Faculty of Engineering, University of Bologna, via Terracini 28, 40131 Bologna, Italy, Phone: (+39) 051 2090351; fax: (+39) 051 2090322, e-mail: [email protected]

Abstract Exploitation of agroindustrial wastes for the production of biobased chemicals, high added-value compounds and/or biofuels represents a key task in the perspective of increasing the employment of renewable resources. For instance, bioconversion of the organic matter present in biowastes could be addressed towards volatile fatty acids (VFAs) production, in order to employ them as the carbon source for the production of biopolymers such as polyhydroxyalkanoates (PHAs). In this respect, a number of investigations reported the successful use of several different agroindustrial wastes. In the present contribution, anaerobic digestion of deproteinized cheese whey was carried out in order to produce a VFA-rich effluent. A wide screening of the most suitable operating conditions was conducted, by testing pH (5, 6 and 7), temperature (35 and 55°C) and presence/absence of a well characterized acidogenic inoculum together with the microflora carried by the said wastewater. Experiments were conducted in batch conditions throughout 14 days, during which reactors were sampled and pH discontinuously adjusted every 2/3 days. As a whole, highest acidogenesis yields were achieved when operating at 55°C, with a pH equal to 6 and 7, after few days of incubation (4 and 2 days, respectively). In both the latter conditions, bioconversion of the influent organic matter (evaluated as COD) into VFAs not already present in the wastewater was higher than 20%. However, the predominant VFAs produced (that is, acetic and butyric acid) accounted alone for more than 10% with respect to the influent COD. This is relevant when considering that both these VFAs are among the most suitable ones for PHAs synthesis (Dias et al., 2006). Generally, when the deproteinized whey cheese microflora was operated together with the acidogenic inoculum, VFAs production yields were improved. Nevertheless, adaptation of the microflora already present in the wastewater may establish a stronger microbial community, a key factor when moving to continuously operated reactors.

P102 IMPROVEMENT OF BUTYRIC ACID PRODUCTION IN Clostridium tyrobutyricum BY SURFACTANT FOR DETOXIFICATION OF LIGNOCELLULOSE HYDROLYSATES Kyung Min Lee1,2, Ki-Yeon Kim1, Sung Ok Han2, Byoung-In Sang*3, Youngsoon Um*1 1

Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seongbuk-gu, Seoul 136-791, South Korea; 2School of Life Science and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, South Korea; 3Department of Chemical Engineering, Hanyang University, Seongdong-gu, Seoul 133-791, South Korea

Abstract Phenolic compounds generated from lignocellulsic biomass during pretreatment and hydrolysis process inhibit the growth of Clostridia so that reduce the final concentration of fine metabolites produced by them. Here, we evaluated inhibition effects of p-coumaric acid, ferulic acid, vanillin, syringaldehyde, and soluble lignin as model phenolics on the growth and butyric acid production of Clostridium tyrobutyricum ATCC 25755, and developed a simple and inexpensive method by adding Tween 80 to the culture to eliminate the inhibition property of phenolics. The growth and butyric acid production were gradually decreased to the half levels compared with the negative control in proportion to the addition of ferulic acid, vanillin, and syringaldehyde up to 1.0 g/L as final concentration. It did not grow at all in the medium containing 0.5 g/L of p-coumaric acid even though the growth and butyric acid production showed slightly decreasing tendency in the range of 0-0.25 g/L. In case of soluble lignin, only 1.0 g/L reduced the growth and butyric acid production to the half. In the co-presence of 1.0 g/L of Tween 80 with 1.0 g/L of p-coumaric, ferulic acid, or souble lignin in the cultures, both the growth and butyric acid production were improved. In cases of soluble lignin, cell growth and butyric acid productions compared with control were totally recovered. However, the addition of Tween 80 did not improve the growth and butyric acid production at all in the vanillin or syringaldehyde containing medium. In case of rice straw and MWW hydrolysate, butyric acid production increased up to 17% and 8% in presence of the 1 g/L of surfactant, respectively. Hence, the surfactant could be applied for the detoxification of phenolic compounds towards biofuels production from lignocellulosic hydrolysates.

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P103 EFFECT OF DIFFERENT CARBON SOURCES ON EXOPOLYSACCHARIDE PRODUCTION BY Enterobacter A47 Filomena Freitas, Ana Rosa Gouveia, Cristiana A.V. Torres, Maria A.M. Reis* REQUIMTE/CQFB, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal, email: [email protected]

Abstract The bacterium Enterobacter A47 (DSM 23139) secretes a fucose-containing exopolysaccharide (EPS) when grown on glycerol byproduct from the biodiesel industry. In this work, different carbon sources, including carbohydrates (glucose and xylose) and organic acids (citrate), were tested to evaluate their impact on cell growth and EPS synthesis. Enterobacter A47 revealed to be a rather versatile bacterium, being able to grow synthesize EPS on all the tested compounds. Glucose proved to be the best source for both growth and EPS production, resulting in a high specific growth rate (~0.47 h-1) and a high specific productivity (1.70 gEPS/gCDW). The use of xylose resulted in cell growth and EPS productivity similar to values obtained previously for glycerol. Although, citrate was also used for EPS synthesis, the productivity was lower than for glycerol or the carbohydrate substrates tested. The use of different substrates has led to the synthesis of polymers with different composition in terms of sugar monomers and also acyl substituents. For all assays, the main sugar components were glucose, fucose, galactose and glucuronic acid, but their relative proportion varied. Similarly to the EPS synthesized from glycerol, in the xylose assay the EPS main sugar monomer was fucose (38%mol). On the other hand, this polymer was more enriched in glucuronic acid (18%mol) than the EPS in the glycerol assay (10%mol). The EPS obtained in the glucose and xylose assays, but the fucose content was lower in the former (29%mol). In the citrate assay the EPS main sugar monomer was glucose instead of fucose and the presence of new monomers (rhamnose, mannose and glucosamine) was detected in trace amounts (2-3%mol). In the glycerol and glucose assays, the EPS had similar acyl substituents composition that accounted for 22-23%wt of the polymers’ dry weight, being pyruvate the most abundant (14-15%wt). The EPS produced from citrate had a higher succinate content (13%wt) than all the other EPS of this study (2%wt), as well as a lower pyruvate content (6%wt). All the EPS synthesized in this study were high molecular weight biopolymers (1x106-2x106), within the range of previously reported values for EPS produced by Enterobacter A47. Overall, these results were interesting, since xylose, glucose and citrate, are present in various agro-industrial residues, such as lignocellulosic materials, citrus waste, and others wastes or byproducts. Thus, this bioprocess may be used to valorise those types of low cost materials into different value-added biopolymers.

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Environmental Engineering and Management Journal

March 2012, Vol.11, No. 3, Supplement, S89-S98

http://omicron.ch.tuiasi.ro/EEMJ/

“Gheorghe Asachi” Technical University of Iasi, Romania

Session 3 BIOLOGICAL TRANSFORMATION OF CONVENTIONAL AND BIOBASED POLYMERS IN THE ENVIRONMENT Main lecture BIOPLASTICS SCIENCE FROM A POLICY VANTAGE POINT J.C. Philp Science and Technology Policy Division, Directorate for Science, Technology and Industry, OECD, Paris

Abstract Society is fundamentally ambivalent to the use of plastics. On the one hand, plastics are uniquely flexible materials that have seen them occupy a huge range of functions, from simple packing materials to complex engineering components. On the other, their durability has raised concerns about their end-of-life disposal. When that disposal route is landfill, their invulnerability to microbial decomposition, combined with relatively low density and high bulk, means that plastics will occupy increasing amounts of landfill space in a world where available suitable landfill sites is shrinking. The search for biodegradable plastics and their introduction to the marketplace would appear to be a suitable amelioration strategy for such a problem. And yet the uptake of biodegradable plastics has been slow. The term biodegradable itself has entered public controversy, with accidental and intended misuse of the term; the intended misuse has led to accusations and instances of “greenwashing”. For this and other reasons standards for biodegradability and compostability testing of plastics have been sought. An environmental dilemma with more far-reaching implications is climate change. The need for rapid and deep greenhouse gas (GHG) emissions cuts is one of the drivers for the resurgence of industrial biotechnology generally, and the search for bio-based plastics more specifically. Bio-based has come to mean plastics based on renewable resources, but this need not necessarily imply biodegradability. If the primary purpose is GHG emissions savings, then once again plastics durability can be a virtue, if the end-of-life solution can be energy recovery during incineration or recycling. The pattern of production is shifting from the true biodegradable plastics to the bio-based plastics, and that trend is likely to persist into the future. This paper looks at aspects of the science of biodegradable and bio-based plastics from the perspective of policy advisers and makers. It is often said that the bioplastics suffer from a lack of a favourable policy regime when compared to the wide-ranging set of policy instruments that are available on both the supply and demand side of biofuels production. Some possible policy measures are discussed.

Session 3/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S89-S98

Oral presentations NOVEL ECO-FRIENDLY MULTIBLOCK COPOLYMERS POLY(BUTYLENE/TRIETHYLENE SUCCINATE): EFFECT OF BLOCK LENGTH ON MECHANICAL PROPERTIES AND BIODEGRADABILITY M. Gigli, N. Lotti*, A. Munari, A Negroni, G Zanaroli, F. Fava Dipartimento di Ingegneria Civile, Ambientale e dei Materiali, Università di Bologna, Via Terracini 28, 40131 Bologna, Italy

Abstract In the past few decades biodegradable aliphatic polyesters have attracted increasing interest owing to the huge environmental concerns about traditional non degradable plastics. Aliphatic polyesters are known to be biodegradable, being easily decomposed by the cleavage of ester bond linkage. Poly(butylene succinate) (PBS) can be considered one of the most promising one, due to its relatively high melting point and excellent processability. However, its high cost, relatively poor mechanical properties and slow biodegradation rate restrain its extensive use. There is also a great interest on polyesters based on alkylene ether glycols: the presence of ether linkages in the chain indeed increases the hydrophilic character of the polymer, favouring its dissolution under environmental conditions. It is well known that thermal and mechanical properties and biodegradability of polymers strongly depend on molecular weight, hydrophilicity, flexibility of chains, and morphology, such as crystallinity degree (Xc), size, form and number of crystallites. In this work we propose reactive blending as a simple and versatile solvent-free synthetic approach to produce new copolyether-esters, obtained by melt mixing PBS and poly(triethylene succinate) (PTES) (PBS:PTES=50:50 mol%, Ti(OBu)4 as catalyst) for different reaction times, with block length decreasing with the increase of mixing time. Their mechanical properties and biodegradation profiles were related with block length. Biodegradation test was carried out by using Candida cylindracea ([E]= 50 U/mL) at 30°C, pH=7.0. Copolymers degrade to a much higher extent than PBS. Moreover, copolymer biodegradation rate regularly increased as the block length decreased, probably due to a lowering of Xc, with exception of sample characterized by the longest sequences. ATRIR and DSC analyses confirmed that the amorphous phase is the region attacked first by enzyme. NMR analysis, indicated that enzyme hydrolysis involved preferentially ester groups of TES sequences, probably because of their higher hydrophilicity. Lastly, copolymer mechanical properties appeared to be strictly related to Xc. The random copolymer, characterized by the lowest Xc, exhibited an elastomeric behavior, having the lowest elastic modulus and the highest deformation at break (around 700%). It was demonstrated that copolymerization is an efficacious way to increase PBS biodegradability and that mechanical properties and biodegradation rate can be modulated changing block length, which affects both crystallinity degree and hydrophilic/hydrophobic ratio.

BIOBASED FEEDSTOCK VALORISATION THROUGH POLYHYDROXYALKANOATE PRODUCTION: FROM EXCESS CHEESE WHEY TO ECO-EFFICIENT BIOPLASTICS Maria G.E. Albuquerque, Rocco Adiutori, Nuno M.P. Trindade, Inês T.D. Carmo, Catarina S.S. Oliveira, Filipa Pardelha, Maria A.M. Reis* REQUIMTE - Chemistry Department, FCT/Universidade Nova de Lisboa, Portugal, Campus da Caparica, 2829-516 e-mail: [email protected]

Abstract The focus of this study was the development of a 3-stage process for cheese whey valorisation through polyhydroxyalkanoate (PHA) production by a mixed microbial culture selected under Feast and Famine conditions. The goal was to investigate all 3 stages of the process (acidogenic fermentation, PHA-storing culture enrichment, and PHA batch production) in order to identify the main process parameters implicated in the inter-dependent optimisation of the overall process and that may be used as leverages for process control. The use of a membrane bioreactor (MBR) in the acidogenic fermentation step allowed for a higher yield and volumetric productivity in volatile fatty acids (VFA) compared to continuous stirred tank reactor (CSTR) operation. The organic acids produced included lactate, acetate, propionate, butyrate, and valerate. Both reactors were successfully operated without nutrient supplementation, showing that proteins from cheese whey were used as nitrogen source. Fermented cheese whey produced in the MBR was fed to a sequencing batch reactor (SBR) operated under feast and famine conditions, with a sludge retention time (SRT) of 4 d, and a long cycle length (12 h). Due to the strong selective pressure imposed, a high enriched culture on PHA accumulating organisms was achieved. This culture was not able to use the protein, thus requiring nitrogen supplementation. Batch PHA accumulation assays performed with the enriched culture, demonstrated a stable PHA accumulation performance at a maximum PHA content of about 50%. A co-polymer of P(HB-co-HV) with an HV fraction of approximately 30% was obtained. The study fully demonstrates the feasibility of using excess cheese whey as a valuable feedstock for PHA production and, moreover, points to routes for further process development.

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INNOVATIVE VALUE CHAIN DEVELOPMENT FOR SUSTAINABLE PLASTICS IN CENTRAL EUROPE (PLASTICE) M. Scandola*, I. Voevodina “

G.Ciamician” Department, University of Bologna, via Selmi 2, 40126, Bologna, Italy, e-mail: [email protected]

Abstract The unstoppable growth in plastics use has brought with it a growing environmental burden from the perspective of resource use, emissions released during the production stage, and waste management. To control and to reduce these negative impacts on the environment it is essential to move to the production and use of plastics with a higher level of sustainability, in particular biodegradable and biobased plastics. The international project PLASTiCE “Innovative value chain development for sustainable plastics in Central Europe” is devoted to the promotion of new environmentally friendly and sustainable plastic solutions. The focus of the project is on the identification and removal of barriers to the faster and more widespread use of sustainable types of plastics, particularly biodegradable plastics and plastics based on renewable resources, in Central Europe. Although the region is lagging in the use of sustainable plastics it does possess strong centers of knowledge on biodegradable materials. Backed by information spreading activities, regulative support and a contribution of a complete value chain (producer, converter, end user) the project aims at overcoming the current obstacles experienced in Central Europe and elsewhere. Among its objectives are: raising awareness among target groups regarding biodegradable plastics, improving technology transfer and access to scientific knowledge, intensifying application-oriented cooperation between research and industry.

Posters P104 THERMOPHILIC MICROBIAL COMMUNITIES DEGRADING SELECTED SYNTHETIC POLYMERS Lucie Husarova1,2, Petr Stloukal1, Sophie Commereuc3, Vincent Verney3, Marek Koutny1,2 1

Environmental Protection Engineering, Faculty of Technology; 2Centre of Polymer Systems, Polymer Centre, Tomas Bata University in Zlin, T.G.M. sqr. 5555, 760 01 Zlin, Czech Republic; 3Clermont Université, UBP, Laboratoire de Photochimie Macromoleculaire, F-63000 Clermont-Ferrand, France

Abstract Biodegradations of three synthetic biodegradable polymers Ecoflex, PBAT, both aliphatic aromatic copolyesters and polylactic acid (PLA), an aliphatic polyester were compared under aerobic conditions at 58°C simulating composting of the mentioned materials. Rates of processes were estimated by carbon dioxide release monitoring. To get an insight into microbial community dynamics DNAs from incubations were sampled amplified with a set of primers with different specificity and analysed with temperature gradient gel electrophoresis (TGGE). A significant evolution of patterns in relation with polymer biodegradation was found with actinomycete specific primers. Our results suggest that all studied polymers were degraded by bacteria belonging to Actinobacteria under composting conditions but interesting differences were observed between both aliphatic aromatic copolyesters and polylactic acid (PLA) indicating that different microorganisms are important with different polymers. The study was complemented with attempts to isolate pure degrading strains.

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P105 SYNTHESIS OF TEXTILE DYES BY LACCASE BIOTRANSFORMATIONS Daniele Spinelli, Andrea Martorana, Maria Camilla Baratto, Riccardo Basosi, Rebecca Pogni Department of Chemistry, University of Siena, via A. De Gasperi 2, 53100 Siena, Italy

Abstract The use of biocatalysis for industrial synthetic chemistry is on the verge of significant growth. Enzymes are remarkable catalysts: capable of accepting a wide array of complex molecules as substrates, and exquisitely selective, catalysing reactions with unparalleled chiral (enantio-) and positional (regio-) selectivities. As a results, biocatalysts can be used in both simple and complex transformations without the need for the tedious blocking and deblocking steps in organic synthesis. Such high selectivity also effords efficient reactions with few by-products, thereby making enzymes an environmentally friendly alternative to conventional chemical catalysts. The chemical production of dyes is extremely complex considering the great number and the variety of the reactions, intermediate compounds and end products. Usually these reactions can present risks with respect to the workers (toxic explosion, fire hazard by raw material and intermediate handling) but also for the environment (production of toxic and recalcitrant waste by-products). Therefore, there is an increasing interest to find alternative enzymatic processes for production of both existing and new dyes in order to develop cheaper, less dangerous and more environmental friendly reactions. Laccases are excellent oxidative enzymes for aromatic compounds substituted by electro-donating groups: diphenols (ortho- and para-), polyphenols, phenols substituted by a methoxyl. The enzyme also oxydizes ortho- and para-quinones, aminophenols, aryldiamines, and polyamines. These simple molecules can serve as precursors for enzyme catalysed production of dyes under mild conditions (room temperature, absence of acids). Some interesting results on synthesis of azo, phenazine and phenoxazine dyes using free or immobilized laccase will be presented. Acknowledgements This work was funded by European project BISCOL (Bioprocessing for Sustainable production of COLoured textiles), contract n° ECO/09/256112/SI2.567273; www.biscol.unisi.it

P106 ANTIMICROBIAL ACTIVITY OF PHB BASED POLYMERIC COMPOSITIONS Svetlana Gonta1, Ludmila Savenkova1, Irina Krallish1, Elena Kirilova2 1

Institute of Microbiology and Biotechnology University of Latvia, Kronvald boulv. 4, LV-1586, Riga, Latvia, e-mail: [email protected]; 2Daugavpils University, Vienibas str. 13, LV-5401, Daugavpils, Latvia

Abstract Polyhydroxyalcanoates (PHAs) are naturally biodegradable thermoplastics produced by many bacteria as a carbon and energy reserve. The production of PHA from renewable natural resources is ecologically advantageous, compared with thermoplastics and elastomers production from fossil carbon sources. Carbon source affect not only PHB production but also the polymer extractability and its molecular weight. In our investigations, the selection of suitable row material for production of polyhydroxybutyrate (PHB) was based on the physiological requirements of the strain of Azotobacter chroococcum 23 and on the availability of the row material (row sugar, beet molasses, corn starch syrup, potato starch syrup). Starch syrups from corn and potatoes were found to be the most suitable unrefinered carbon sources for polyhydroxybutyrate (PHB) production by A. chroococcum 23. A. chroococcum 23 growth and the cell composition depend on cultivation conditions. It has been demonstrated that regulation of carbon and NH4+ concentration in cultivation medium led to lower level of polysaccharides content in A.chroococcum 23 cells that promotes PHB granule isolation. Pure PHB recovered by chloroform extraction as well as native PHB granules isolated from the biomass of A. chroococcum 23 were used for elaboration of biodegradable PHB based composed materials. Natural and chemically synthesized antimicrobial compounds were used for preparation of antimicrobial polymeric films and polymeric layers on papers surface. Various by structure plasticizers were used for preparation of PHB based polymeric compositions (both from chloroform extracted PHB and water suspension of native PHB granules). Fluorescent dye 3-(1-piperidyl)benzo[a]phenalen-7-one (P8) was incorporated in the PHB composed materials to study its diffusion rate in dependence of the material composition. Investigations showed that films mechanical properties, especially elasticity, were highly affected by films compositions. Temperature application increased composite film’s hydrophobic properties. This work was done in cooperation with Riga Technical University scientists. Antimicrobial activity of these materials was assayed by Disk Diffusion method against two Gram positive (Bacillus cereus, Staphylococcus aureus) and two Gram negative (E. coli, Pseudomonas aeruginosa) bacterial strains. Study of the antimicrobial activity showed that the most promising antimicrobial materials are both PHB and PHB/paper systems including Silbiol or benzoic acid.

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P107 BIOSYNTHESIS OF BIOTIN AND SELENOBIOTIN IN BIOTIN- INDEPENDENT S. cerevisiae STRAIN CULTIVATED IN MOLASSES MEDIUM Piotr Patelski, Katarzyna Pielech-Przybylska, Maria Balcerek, Anna Diowksz, Agnieszka Nowak, Urszula Dziekonska Lodz Technical University, Institute of Fermentation Technology and Microbiology, Wolczanska 171/173, 90-924 Lodz, Poland, email: [email protected]

Abstract Most of Saccharomyces cerevisiae strains are biotin auxotrophs, however prototrophic for biotin strains isolated from sake mashes are known. We are conducting research on enriching of yeast biomass with selenium. The aim of this study was to investigate the influence of selenium on biotin and selenobiotin biosynthesis in biotin-independent S. cerevisiae yeast. Molasses medium was supplemented with 0–1.8 mmol of Se/L. Shaken cultures of S.cerevisiae “Y” strain isolated from sake mash were kept for 48h. After biomass hydrolysis biotin and its derivatives were isolated with use of affinity chromatography and immobilized on avidin-coated matrix. Selenium was assayed with use of fluorometric method with 2,3-diaminonaphtalene. Total biotin activity ranged from 25µg/g d.m. (for 1.8 mmol Se/L) to 30µg/g d.m. (for unsupplemented biomass). Selenium dosage resulted in formation of selenobiotin up to 6.8µg/g d.m. for supplementation with 0.9mmol Se/L. The results proved selenobiotin biosynthesis in biotin-independent S.cerevisiae strain. Acknowledgements This work was financed from funds for Polish Science- R&D project N312 197838.

P108 PHOTODEGRADATION OF ALIPHATIC POLYESTERS AND THEIR COMPOSITES WITH TiO2 S. Sullalti1, G. Totaro1, H. Askanian2, A. Celli1, P. Marchese1, V. Verney2, S. Commereuc2 1

DICAM, University of Bologna, Via Terracini 28, 40131 Bologna, Italy; 2Institut de Chimie de Clermont-Ferrand Université Blaise Pascal-ENSCCFCNRS UMR 6296 Chimie 6, 24 avenue des Landais BP 80026 63171 AUBIERE Cedex

Abstract Degradability or, on the contrary, durability of polymers are very important features affecting the performances of the materials in their shelf-life and their impact on the environment. The degradation can be due to different environmental factors, including light exposure, very important for polymers used in outdoor applications. The knowledge of the photodegradation mechanisms and the possibility to modify them is a very important topic, especially for the novel biopolymers, as their durability with respect to the petro-plastics is yet an open question. Among the biopolymers, aliphatic polyesters are an emerging class of materials, thanks to the possibility of preparing them from renewable resources and their potential environmental degradability. In this study two aliphatic molecular structures, containing 1,4cyclohexylene units, have been analysed and used as matrixes for composites. The two repeating units are shown below. O

O

C

C

O

CH2

CH2

CH2

CH2

O

PBCHD

O

O

C

C

O

CH2

CH2

O

PCCD

PBCHD and PCCD were exposed to UV irradiation in an accelerated photo-ageing device and the modifications in their chemical structures were analysed by UV and IR spectroscopies. It results that the photodurability of PBCHD, containing only one cycle per repeating unit, is higher than that of PCCD. Moreover, in order to increase the durability, substances can be incorporated into the polymer. Among all the particles and nanoparticles which can be used, titanium dioxide (TiO2) is known for its capability of absorbing UV radiation. This photocatalytic effect is strictly connected to its crystalline phases. Therefore, composites containing different amount of TiO2, in the anatase and rutile crystalline phases separately, have been prepared by using a Brabender mixer. The well dispersion of the particles and the good adhesion between TiO2 and polymer have been checked by SEM observations. The composites result to have a photostability higher than those of the homopolymers. The protection of TiO2 against the photooxidation increases with the particle content and results higher for the anatase crystalline phase. As a conclusion, a good increment of the photodurability of the studied aliphatic polyesters has been obtained by preparing composites with titanium oxide. This result could be particularly significant for outdoor applications of these polymers.

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P109 PRODUCTION OF HOLLOCELLULOLYTIC ENZYMES USING AGRO-INDUSTRIAL RESIDUES: SELECTION OF TYPE OF PRETREATMENT AND SUBSTRATE A.G. López-Nevarez1, H.M. Poggi-Varaldo2, E. Cristiani-Urbina3, M.T. Ponce-Noyola1,* 1

Microbial Genetics Group, Department of Biotechnology and Bioengineering, Centro de Investigación y de Estudios Avanzados del IPN, Mexico D.F., 07360, Mexico. Tel.: 5255 5747 3800 ext 4317; 2Environmental Biotechnology and Renewable Energies R&D Group, ibidem; 3Biochemical Engineering, Escuela Nacional de Ciencias Biológicas del IPN, Mexico D.F., 11340, Mexico e-mail: [email protected]

Abstract Biofuels of second generation are produced using agro-industrial residues which are complex materials composed mainly of cellulose, hemicellulose and lignin. The aim of this work was to evaluate the acid and alkaline-pretreatment of sugarcane bagasse, agave bagasse and corn cob in terms of physical structure changes, lignin removal, production and activity of cellulases and xylanases (commonly denominated holocellulases) for the mutant PR-22 of Cellulomonas flavigena. A factorial experimental design of three factors was carried out: substrate at three levels (agave bagasse [AB], sugarcane bagasse [SCB] and corn cob [CC]); pretreatment at two levels (acid and alkali) and concentration at three levels (low [L], medium [M] and high [H]). Main experimental variables were volumetric activities of CMCase and xylanases, among others. AB, SCB and CC were pretreated in acid and alkaline dilute solutions (1-3% v/v or w/v). The effects of each pretreatment were also evaluated by scanning electron microscope (SEM) images and lignin removal. Cellulomonas flavigena mutant PR-22 was used for hollocellulase production. Experiments were carried out at 37 °C, pH 7 and 150 rpm in 125 mL shake flasks. Comparing with acid-pretreatment, alkaline-pretreatment was positive for hollocellulases production for all substrates with 23 and 53% higher volumetric xylanolytic and cellulolytic enzymatic activities, respectively. Xylanase activities were higher in substrates treated with the highest concentration of alkali; however, in these conditions CMCase activities were not improved. Concerning the type of substrate, CC had the best inducer effect of hollocellulolytic enzymes (p < 1x10-4). Alkaline-pretreatment removed among 20-50% of lignin content of residues, whereas acid pretreatment did not remove lignin at all. Moreover, SEM images showed that alkaline pretreatment was more effective in separating and thinning the fibers. Alkaline pretreatment was more suitable for lignin removal from the lignocellulosic fibers and led to higher activities of holocellulases. CC gave higher hollocellulase activities of C. flavigena PR-22 compared to agave and sugarcane bagasses. Our results indicate that use of alkali-pretreated CC and post fermented by C. flavigena mutant PR-22 is a promising substrate to obtain high titers of hollocellulases. Key words: agro-industrial residues, Cellulomonas flagivena, holocellulases, pretreatment

P110 ENZYMATIC PROCESSING OF CHITINACEOUS WASTES FOR N-ACETYL-D-GLUCOSAMINE PRODUCTION Sancharini Das1, Ramkrishna Sen1, Debasis Roy2 1

Department of Biotechnology Indian Institute of Technology Kharagpur, India; 2Department of Civil Engineering, Indian Institute of Technology Kharagpur, India

Abstract One of the major structural components of marine invertebrates and fungal biomass is chitin, which is a biopolymer ubiquitous in nature. It is an unbranched polymer of N-acetyl-D-glucosamine (GlcNAc) monomers that are linked by β-1, 4 glycosidic bonds. The annual recovery of chitin has been estimated at about 40 tones from the processing of marine invertebrates. Chitin is conventionally purified from crab or shrimp waste and is further converted into different valuable products. Among those, GlcNAc is one of the important products due to its medical application in the improvement of osteoarthritis. It has been found that sulfate and hydrochloride salts of glucosamine are commercialized for osteoarthritis disease, but these cannot be used for oral administration due to bitter taste. Alternatively, GlcNAc is being used as a supplement as it is sweet in taste. Though GlcNAc is produced by different chemical procedures such as acid hydrolysis of chitin; however, these processes suffer from limitations like production of acidic wastes, high cost and low yield. The GlcNAc could also be produced by N-acetylation of glucosamine, which also has the inherent disadvantages of a chemical process. As the enzymatic conversion of chitinaceous wastes offers an effective and environment friendly alternative to chemical methods, the present study thus involved processing of chitinaceous wastes by the chitinase from Trichoderma harzianum. In this study, shrimp wastes and fungal biomass of soil origin were used as chitinaceous wastes. The enzyme loading, amount of wastes and incubation time of enzymatic reaction were standardized for GlcNAc production. The presence of GlcNAc after enzymatic hydrolysis was detected by TLC and HPLC methods. The GlcNAc amount varied from 65% (w/w) to 80% (w/w) from these wastes.

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Session 3 - Biological transformation of conventional and biobased polymers in the environment

BIO-OIL AS FEEDSTOCK FOR THE PRODUCTION OF BIOPOLYMERS BY AEROBIC MIXED CULTURES Rita Moita, P.C. Lemos REQUIMTE/CQFB, Chemistry Department, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829 516 Caparica, Portugal, e-mail: [email protected];e-mail: [email protected]

Abstract Biomass, as an alternative energy source to fossil fuels, gained importance trough the last years due to its significant environmental advantages. Wood and other forms of biomass including energy crops and agricultural and forestry wastes are some of the main renewable energy resources available. Most processes that convert biomass into liquid fuels begin with pyrolysis, a thermal decomposition that occurs in the absence of oxygen. Bio-oils resulting from the fast pyrolysis of biomass can be converted into fuels with similar properties to the petroleum-derived ones. Several chemicals can also be extracted or derived from the bio-oil including food flavourings, specialities, resins, agro-chemicals, fertilisers and emissions control agents. Alternatively, fermentation of the bio-oil as a post-processing biological approach can be applied. Bio-oil can be use as carbon source for microbial conversion into high value products, such as bioplastics. Polyhydroxyalkanoates (PHAs) are biodegradable polyesters with promising alternatives to conventional plastics. However the significant cost difference between PHAs and petrochemical-derived plastics presents the major barrier for them to gain market share (9% among bioplastics). In order to develop more cost effective processes, low cost substrates and mixed microbial cultures have been applied for PHA production. Mixed cultures are selected by the operational conditions imposed to the biological system, allowing for savings in energy and equipment cost. Bio-oil resulting from the fast pyrolysis of chicken beds was used for the production of PHA. Selection of PHA accumulating bacteria was performed in a sequencing batch reactor operated under “feast and famine” aerobic condition. The reactor was inoculated with activated sludge from a wastewater treatment plant and fed with 60 C-mmol/day of easily biodegradable carbon contained in bio-oil. Due to the carbon complexity of the bio-oil, the effect of different carbon sources and feeding strategies in the PHA accumulation organisms selected was observed through several kinetic studies using bio-oil, or model substrates as acetate and glucose. In order to follow the culture acclimatization to this carbon source and to better understand the operating system, molecular techniques like FISH and DGGE were used. These techniques allowed observing the enrichment of PHA accumulating organisms during the acclimatization period, resulting in increased of the intracellular PHA content.

P112 HYALURONIC ACID FROM BIOFERMENTATION–MOLECULAR WEIGHT Tergitol NP10 (1.5 cmc) > Tergitol NP10 (5.0 cmc) >Triton X-100 (1.5 cmc) ≥ n–dodecane. Most AEF values were close to 1 or lower, that indicated a poor effectiveness of the desorption treatment. Best results were achieved with silicone oil and Triton X-100, with AEF values 2.2 and 2.1, respectively. In this study the AEF value of the silicone oil-treated samples suggested an improvement of the availability of lindane by 112%. Furthermore, silicone oil is known to be innocuous to most microorganisms. Despite similar results of silicone oil and Triton, we chose the first for bioremediation studies because of its reported microbial innocuousness and possibility of recovery and reuse. In our case, the system was triphasic (soil-water-silicone oil); preliminary tests of silicone oil in slurry bioreactors showed promise for increasing lindane removal from the same model soil. These results were in agreement with those of Villemur et al. (2000) who observed that 69-88% of PAHs were transferred from soil to silicone oil in the two-liquid-phase slurry systems with 15% silicone oil, and improved bioremediation of petroleum-contaminated soil. Key words: agricultural soil, desorption, lindane

PRELIMINARY CHARACTERIZATION OF TCE AND TeCA CO-METABOLISING AEROBIC CULTURES UNDER SUSPENDED AND IMMOBILIZED FORM Serena Fraraccio1, Giulio Zanaroli1, Giacomo Bucchi2, Roberta Ciavarelli2, Dario Frascari2, Fabio Fava1 1

Department of Civil, Environmental and Materials Engineering (DICAM), Faculty of Engineering, University of Bologna, Bologna, Italy; 2Department of Chemical, Mining and Environmental Engineering (DICMA), Faculty of Engineering, University of Bologna, Bologna, Italy

Abstract Chlorinated aliphatic hydrocarbons (CAHs) are toxic and environmental hazardous solvents increasingly detected in soil and groundwater for which new and most effective biormemdiation approaches are needed. In this context, the European project MINOTAURUS is aiming to develop groundwater bioremediation robust and reliable strategies based on the use of immobilized biocatalysts. This work was focused on the enrichment of aerobic trichloroethylene (TCE) and 1,1,2,2-tetrachloroethane (TeCA) co-metabolizing microbial cultures from a contaminated site in Rho (MI, Italy), their preliminary characterization both as freely suspended and immobilized biomass on different carrier materials (Biomax®, Biopearl®, Biomech®, Cerambios®) and the selection of the best performing culture to be employed in a continuous Packed Bed Reactor (PBR) process. The best performing culture was obtained through serial enrichment on filter sterilized site-water or a similar synthetic water in the presence of butane (2 mg/L) as carbon and energy source and TCE (10 mg/L) plus TeCA (3 mg/L). The structure and composition of the microbial population was carried out through denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes. The DGGE analysis outlined that the structure of the suspended microbial community changed remarkably throughout the enrichment with the exception of the most prominent phylotype. However, according to the Dice similarity coefficient, lower changes occurred during the last sub-culturing step, indicating a gradual stabilization of the community structure. When immobilized on different carriers, the most prominent phylotype previously detected represented a minor fraction of the immobilized community; conversely, several of the minor bands occurring in the suspended culture became dominant within the biofilm. Such a drastic change in the structure of the microbial community was evidenced by the separate clustering of the DGGE profiles from suspended cell cultures and biofilms. Furthermore, the high similarity indexes between the communities immobilized on different carriers (57.3% to 73.7%) suggest that the evolved consortium is quite stable and that the material and shape of the carrier do not affect remarkably its composition. Complementary information obtained from degradation kinetic tests showed that the selected culture immobilized on Biomax® retained the highest degrading activity. Identification of the community members via band sequencing and phylogenetic analysis is in progress and will allow to obtain key information on the potential TCE and TeCA co-metabolizing species both in the suspended and in the immobilized communities.

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Session 4/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S105-S114

STUDIES ON THE FATE AND BEHAVIOUR OF SOME CONTAMINANTS IN SOILS AS A PREREQUISITE IN BIOREMEDIATION Maria Gavrilescu*, Camelia Smaranda, Vasile Lucian Pavel “Gheorghe Asachi” Technical University of Iasi, Department of Environmental Engineering and Management, 73 Prof. Dr. Docent D. Mangeron Street, 700050, Iasi, Romania, e-mail: [email protected], [email protected]

Abstract Soils could be relevant receptors for contaminants as they possess the ability to bind various chemicals. Nowadays, there is an increasing concern regarding the introduction of substances, biological organisms or energy into the soil, resulting in a change of the soil quality, which is expected to affect the normal use of the soil, as well as public health and the living environment. Heavy metals and mineral oils are among the most common harmful contaminants, but the persistent organic pollutants and their degradation compounds continue to emerge in the soil. The paper deals with the fate of two types of contaminants: heavy metals and dyes, in some agricultural and urban soils with various characteristics. Sorption, desorption and bioavailability experiments were performed using six types of soils. The description of some sorption and migration phenomena for heavy metals (Cadmium and Chromium), and two dyes (Orange II and Congo Red) revealed that the extent of sorption onto soils depends on many factors, such as the type and concentration of the adsorbate, contact time, agitation speed, temperature, pH, or the surface chemistry of the adsorbent. The results indicate that soils with different physico-chemical properties have different effects on the adsorption of most contaminants, especially at higher concentration levels. As expected, differences in contaminant behaviour were observed, but their mobility was strongly pH dependent, while some soil constituents, such as clay and organic matter contents, influence the sorption capacity of the sorbent and sorption equilibrium. The presence of soil minerals also influences the sorption properties of soils by reducing sorption affinity through blocking organic matter sorption sites or by causing conformational changes in its structure. Some kinetic and thermodynamic models fitted the experimental data with good accuracy. Toxicity and phytotoxicity tests showed that important fractions of the contaminants could be bioavailable in certain conditions. The results contribute to the development of scientific databases which address these issues and the demands of decision makers and the public to understand the impact of chemicals on human health and the environment and to select the most feasible bioremediation alternatives. Acknowledgements This work was partially supported by the grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number PN-II-ID-PCE-2011-3-0559, Contract 265/2011.

MICROCOSM EVALUATION OF AUTOCHTHONOUS BIOAUGMENTATION TO COMBAT MARINE OIL-SPILLS M. Nikolopoulou1, P. Eickenbusch3, N. Pasadakis2, D. Venieri1, N. Kalogerakis1 1

Department of Environmental Engineering, Technical University of Crete, Chania, Greece; 2Department of Mineral Resources Engineering, Technical University of Crete, Chania, Greece; 3Faculty of Chemistry, University Duisburg-Essen, Germany

Abstract Bioremediation through bioaugmentation (addition of oil-degrading bacteria) and/or biostimulation (addition of nutrients N&P) constitute a promising strategy for combating oil spills following first response actions. However, bioaugmentation is one of the most controversial issues of bioremediation since nutrient addition alone had a greater effect on oil biodegradation than the addition of microbial products that are highly dependent on environmental conditions. There is increasing evidence that the best way to overcome the above barriers is to use microorganisms from the polluted area, an approach proposed as autochthonous bioaugmentation (ABA) and defined as the bioaugmentation technology that uses exclusively microorganisms indigenous to the sites (soil, sand, and water) to be decontaminated. Isolated single strains or enriched cultures, which are obtained ‘‘before’’ or ‘‘after’’ the contamination of the target sites, are administered to the sites once contamination occurs. The key idea is to conduct the enrichment of contaminant-degrading bacteria under the same or very similar conditions to those where bioaugmentation will be performed. ABA as defined in this study uses indigenous microbial consortia or isolates that are highly enriched and much better adapted to the historically or artificially contaminated sites. In this study we examined the effectiveness of a novel autochthonous bioaugmentation strategy for the successful remediation of polluted marine environments. Consortium was enriched from seawater samples taken from Elefsina Bay near the Hellenic Petroleum Refinery; a site exposed to chronic crude oil pollution. Duplicate microcosms were established in sterile 100 ml flasks containing 50 ml of seawater contaminated with 0.5% w/v weathered crude oil incubated under aerobic conditions at 20°C with continuous agitation. Three autochthonous bioaugmentation treatments were established as follows: (i) oiled seawater and pre-adapted consortium (ii) oiled seawater + KNO3, K2HPO4 and pre-adapted consortium, and (iii) oiled seawater + KNO3, K2HPO4, biosurfactant (rhamnolipids) and pre-adapted consortium. Nutrients were added to such amount to give a final concentration equivalent to a C:N:P molar ratio of 100:10:1. Growth of oil degraders was measured by most probable number (MPN) procedure and hydrocarbons were analysed with chromatographic techniques (solid-phase extraction, gas chromatography–mass spectrometry) after 0, 5, 15 and 30 days. After 30 days long experiment, MPN and plate counts showed highest growth during treatment (iii) with fertilizer and biosurfactant which correlate to chemical analysis that showed highest alkane and aromatics degradation. After 30 days treatments (ii) and (iii) were the most effective, C12-C35 n-alkanes fraction was degraded more than 70%, while in treatment (iii) degradation was 85% within 15 days. Previous work with lipophilic fertilizers (uric acid and lecithin) and biosurfactants (rhamnolipids) resulted in increased removal of petroleum hydrocarbons as well as in a reduction of hydrocarbon degraders’ lag phase. Considering the above, the use of biostimulation additives in combination with naturally pre-adapted hydrocarbon degrading consortia has proved to be the most effective treatment and is a promising strategy in the future especially when combined with lipothilic fertilizers instead of inorganic nutrients. Such an approach becomes more pertinent when the oil spill approaches near the shore line and immediate hydrocarbon degradation is needed.

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Sub-session 4.2 - Research and innovation in the ex-situ and in situ microbial remediation of contaminated soils, aquifers, sediments and marine habitats

Posters P35 CHARACTERIZATION OF A MIXED CULTURE THAT REDUCTIVELY DECHLORINATES MIXED CHLORINATED ETHENES AND ETHANES Vincenzo Maffione1, Harmien Verstraete2, Willy Verstraete1,2, Nico Boon1 1

Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium 2 Avecom NV, Industrieweg 122P, 9032 Wondelgem, Belgium

Abstract Looking for a valid bioaugmenting agent for applications in real sites, an anaerobic mixed culture able to reductively dechlorinate a mixture of C2 chlorinated aliphatic hydrocarbons (CAHs), has been enriched over a period of several years from a groundwater historically contaminated with 1,2-dichloroethane (1,2-DCA) and other C2 CAHs, such as tetrachloroethene (PCE). The culture, named Multidechlorobac, has been analysed for its microbial composition, and the effects of inoculum amount on its dechlorination capabilities have been tested on subcultures prepared in non-sterile conditions, using 1,2-DCA and PCE as final electron acceptors. Lactate, yeast extract and molasses have been used as both carbon and electron sources. Multidechlorobac consisted of both dechlorinators, such as Dehalococcoides ethenogenes strain 195 and Desulfitobacterium sp., and nondechlorinators belonging to Bacteroidetes, Proteobacteria and Firmicutes. While the former ones are directly involved in the degradation of the pollutants, the latter support the process by providing hydrogen, which acts as electron donor in the dechlorination. The initial bacterial concentration was about 108 cells mL-1, and subcultures were able to degrade the pollutants at 10 % inoculum. Higher dilutions resulted in a loss of dechlorinating capacity, leading to a slower or unsuccessful treatment, even though dechlorination was still observed for 1,2DCA. Multidechlorobac was effective in the degradation of pollutants, and it was found to be robust and easy to grow, in comparison with pure cultures. The culture might be successfully used as bioaugmenting agent in the clean-up of sites polluted with C2 chlorinated aliphatic hydrocarbons.

P36 COMPARISON OF TRIPHASIC AEROBIC AND ANAEROBIC SLURRY BIOREACTORS FOR THE BIOREMEDIATION OF LINDANE-IMPACTED SOIL W.E. Varo-Argüello1, B. Camacho-Perez1, E. Rios-Leal2, P. Vázquez-Landaverde3, M.T. Ponce-Noyola2, J. Barrera-Cortés2, N.F. Rindernknecht-Seijas4, Hèctor M. Poggi-Varaldo1∗ 1

Environmental Biotechnology and Renewable Energies R&D Group, Department of Biotechnology & Bioengineering, CINVESTAV-IPN P.O. Box 14-740, 07000, México D.F., México; 2Department of Biotechnology & Bioengineering, ibídem; 3CICATA-IPN, Qro., México; 4 ESIQIE-IPN, México; *e-mail: [email protected]

Abstract Lindane is an organochlorine pesticide that has been widely used in Mexico and other countries. Because of its toxicity, persistence and bioaccumulation potential its use has been prohibited or restricted. The use of multiphasic slurry bioreactors for ad-situ and ex-situ soil bioremediation might improve lindane availability for biological degradation. Stimulation of bioremediation by use of a co-substrate along with bioaugmentation, and electron acceptors other than oxygen, can lead to increased degradation of a variety of xenobiotics. Therefore, we evaluated the effects of co-substrate and final electron acceptor (aerobic and sulfate-reducing) on the removal of lindane in triphasic lab scale slurry bioreactors. The effect of 2 factors at 2 and 3 levels were evaluated: co-substrate sucrose (1 and 0 g/L; C or no symbol, respectively) and final electron acceptor (O2, CH4 and SO-4; or aerobic A, methanogenic M and sulfate-reducing SR, respectively). Abiotic controls were run (sterilized soil and inoculum). Bioreactors were implemented and operated as described elsewhere. Lindane and metabolites was analyzed according to literature. Lindane removal followed the order A>SR>M. Supplementation with sucrose had a positive effect for all the electron acceptors, particularly for the anaerobic regimes. The ANOVA indicated that there was a significant effect of factor ‘electron acceptors’ on removal of lindane (p< 0.0001). The order of removal A>SR>M was slightly different to that reported by some authors, who found SR>A>>M in experiments with lab scale slurry bioreactors without silicone oil. This was possibly due to the positive effect of silicone oil on oxygen transfer. Sucrose supplementation had a significant effect on the removal of lindane (p 90%). The four compounds were completely removed after 5 h with these optimal conditions, showing the high potential of laccases to improve biodegradation of environmentally persistent compounds.

Posters P48 AQUEOUS TWO PHASE MICELLAR SYSTEMS (ATPMS) APPLIED TO SEPARATE VIRUS CONTAMINATION FROM AQUEOUS SOLUTION João Vitor Dutra Molino1, Lívia Bonafini Junqueira1, Tatiana Lopes1, Daniela De Araújo Viana Marques1, Joas Lucas Da Silva2, Mario Hiroyuki Hirata2, Priscila Gava Mazzola3, Maria Silvia Gatti4, Adalberto Pessoa Júnior1 1

Department of Biochemical and Pharmaceutical Technology, FCF/USP; 2Department of Clinical Analysis and Toxicology, FCF/USP; 3Department of Clinical Pathology, UNICAMP; 4Biology Institute, UNICAMP

Abstract Water related diseases are one of the main concerns in health policies worldwide, which pressures environmental and public health policies to ensure microbiological safety. Simple separation techniques are essential to survey water source, but often lack in efficiency. Therefore, ATPMS are proposed as a separation technique to improve virus removal. Adenovirus was selected as virus model in our studies. Adenovirus suspension was obtained in Hep-2 cells in Minimum Essential Medium Eagle (MEM) at 95% humidity and 5% CO2 for 48h to 72h after infection. Quantified HAdV-5 suspension was applied in ATPMS formed by Triton X-114/McIlvaine’s buffer. Micellar systems were designed and prepared by using a factorial 23 randomized experimental design. Surfactant concentrations were 1 and 6% (w/w), McIlvaine buffer pH 5 and 7, and systems were kept at 29 and 33°C during 15h. Analysis of qPCR results demonstrated a positive effect on partition of TX-114 concentration and pH values, indicating that an increase in these variables produced a greater partition of HAd-5 to micellar poor phase. Partitioning values higher than 350 were achieved, and can still be improved by changing the system conditions by enhancing the excluded volume effect and reducing the hydrophobic interaction between virus and micelles. The results show that it is possible to concentrate Adenovirus in the micellar poor phase, and increase the separation by applying an ATPMS formed by higher TX-114 concentrations. Acknowledgements FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil; CAPES (Coordenação de Aperfeiçoamento do Ensino Superior, Brazil), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), and University of São Paulo.

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Session 6/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S121-S134

P49 EFFECT OF CELL IMMOBILIZATION ON THE PRODUCTION OF 1,3-PROPANEDIOL Mine Gungormusler, Cagdas Gonen, Nuri Azbar* Bioengineering Department, Faculty of Engineering, Ege University, 35100 Bornova, Izmir, Turkey,Phone: +90 232 3880378x138, fax: +90 232 388 495, e-mail: [email protected]

Abstract The feasibility of stainless steel wire, glass raschig ring and Vukopor®(a porous ceramic cube) as immobilization materials were investigated for the continuous production of 1,3-propanediol (1,3-PDO) from waste glycerol with locally isolated Klebsiella pneumoniae (GenBank No: 27F HM063413). In addition, the effect of hydraulic retention time (HRT) on the production of 1,3-PDO were also investigated. Continuous cultures with immobilized cells revealed that 1,3-PDO production was more effective and more stable than suspended culture system. A HRT of 0.5 h is the best one in terms of volumetric production rate. However, 1,3-PDO concentrations reached the highest values when a HRT of 12 h was used. Furthermore, cell immobilization had also obvious benefits especially for resistance of the production for extreme conditions. The results indicated that immobilized cells achieved a 2.4 fold higher productivity (4.8 g 1,3-PDO/L/h) in comparison to suspended cell system (2.0 g 1,3-PDO/L/h). The degree of cell immobilization was measured via the biomass suspended and attached on the immobilization materials. The results indicated that successful immobilization between the ranges of 70-78% was achieved for ceramic and glass materials, however a lower percentage (58%) of immobilization was observed with the immobilized bioreactor filled with stainless steel wire. This process has the ability to work in smaller reactor volumes with shorter total fermentation periods. Therefore, it allows using high dilution rates and raises 1,3-PDO productivity. As a matter of fact, HRT was found to be a significant factor. All three of the materials are good candidates for immobilization purpose; however, glass Raschig ring is a better support material than stainless steel wire and Vukopor® in terms of immobilization ratios. The results clearly indicated that 1,3-PDO production in immobilized packed-bed reactors is superior to suspended cell culture systems. The results reported in this study may help to save the cost of 1,3-PDO production since higher production in smaller bioreactors could be achieved. Therefore, continuous fermentation in a packed-bed bioreactor system is a suitable method to enhance 1,3-PDO production.

P50 DEVELOPMENT OF A BIOFILM TECHNOLOGY FOR THE PRODUCTION OF 1,3-PROPANEDIOL (1,3-PDO) FROM CRUDE GLYCEROL Silvia Casali1, Mine Gungormusler2, Lorenzo Bertin1, Fabio Fava1, Nuri Azbar2 1 Deptartment of Civil, Environmental and Materials Engineering (DICAM), Environmental Biotechnology and Biorefineries Research Unit Faculty of Engineering, University of Bologna Via Terracini 28, 40131 Bologna, Italy; 2Bioengineering Department, Faculty of Engineering, Ege University, 35100 Bornova, Izmir, Turkey; Phone: +90 232 3880378x138, fax: +90 232 388 4955, e-mail: [email protected]

Abstract Glycerol is the main by-product of transesterification of fats in the biodiesel production. 1,3-propanediol (1,3-PDO) is a valuable chemical that can be obtained from glycerol by microbial conversion. A number of Enterobacteriaceae species are able to produce 1,3-PDO from glycerol in stirred tank freely suspended cell bioreactors. Little is known about the use of crude glycerol in the production of 1,3-PDO and about the opportunity to intensify the process via strain immobilization in packed bed bioreactors. In this work, Citrobacter freundii, strain DSM 15979, and Pantoea agglomerans, strain DSM 30077, were tested for their ability to produce 1,3-PDO from crude glycerol in shaken flask batch conditions and in packed bed biofilm reactors operating under continuous conditions. Three different hydraulic retention times (HRT) were comparatively tested (8, 4 and 2 h) in order to understand its effects on 1,3-PDO production under immobilized cell conditions. The study revealed that HRT significantly influenced the process performances. The best productivities were observed when a HRT of 2 h was applied. However, both strains were found to be good candidates for 1,3-PDO production in biofilm reactors, even though P. agglomerans displayed quite higher productivities (3.6 g/(L h)) than the other strain. Using a novel microbial strain and packing material, namely, P. agglomerans and VUK, respectively, in the bioconversion of crude glycerol into 1,3-PDO in packed bed biofilm reactors. In particular, P. agglomerans appears as a promising microorganism for 1,3 PDO production than C. freundii, which however was for the first time found to produce 1,3 PDO from crude glycerol. This study provides experimental evidence of the possibility of using P. agglomerans as immobilized cells in a fixed bed bioreactor system for the continuous production of 1,3PDO from crude glycerol.

S126

Session 6- Wastewater valorisation, bioremediation, purification and reuse

P51 ADAPTATION OF IMMOBILIZED Rhodococcus CELLS TO INCREASING PETROLEUM CONCENTRATIONS IN A COLUMN BIOREACTOR M.K. Serebrennikova1, M.S. Kuyukina1,2, I.B. Ivshina1,2 1

Institute of Ecology and Genetics of Microorganisms, Russian Academy of Sciences, Perm, Russia; 2Microbiology & Immunology Department, Perm state University, Perm, Russia

Abstract Biotreatment of petroleum contaminated wastewaters is an important aspect of environmental biotechnology based on microorganisms capable of degrading petroleum hydrocarbons, and it is regarded as an effective and potentially inexpensive cleanup technology increasingly applied today. Bioreactors with immobilized bacterial cells allow enhancing hydrocarbon bio-oxidation, adapting microorganisms to toxic compounds, and maintaining cell viability at extreme contaminant loads. Rhodococcus bacteria possessing broad catabolic and unique enzymatic capabilities are of ecological and biotechnological significance and play an important role in petroleum degradation in the environment. Since crude oil is a complex substrate composed of a wide range of hydrocarbons, we used the association of strains, Rhodococcus ruber IEGM 615 and Rhodococcus opacus IEGM 249 from the Regional Specialized Collection of Alkanotrophic Microorganisms (acronym IEGM, WFCC # 768). These strains differ by the range of substrates utilized suggesting complete degradation of petroleum hydrocarbons (www.iegm.ru/iegmcol/index.html). Bacteria were adapted to increasing petroleum concentrations in a column bioreactor. Oil-contaminated water (2%, v/v) was pumped through (flow rates, 0.6, 1.2 and 2.0 mL/min) the bioreactor packed with sawdust-immobilized rhodococcal cells at room temperature for two weeks. Maximal degradation of 88-99% was obtained for C11 to C19 aliphatic hydrocarbon fraction. Longer chain aliphatics and polycyclic aromatic hydrocarbons were biodegraded to a lower extent (3055%). It was found that reuse of immobilized rhodococcal cells increased the efficiency of petroleum degradation in the reactor apparently due to Rhodococcus cell adaptation to toxic petroleum components. Moreover, the adapted bacterial cells isolated from the bioreactor after the operation cycle were more resistant to mono- (benzene, phenol) and polyaromatic (phenanthrene, anthracene) hydrocarbons and antibiotics. Currently, possible mechanisms of Rhodococcus cell adaptation to high concentrations of petroleum hydrocarbons are being investigated concerning to changes in cell-envelope lipid composition, plasmid profile and increase in the gene expression responsible for hydrocarbon biodegradation. Acknowledgement Research was funded by the Russian Academy of Sciences (MCB Program) and RF Ministry of Education and Science (contracts 16.518.11.7069, 16.513.12.3015).

P52 CHROMIUM(VI) REMOVAL FROM AQUEOUS SOLUTIONS BY Trichoderma viride FUNGAL BIOMASS Raluca Maria Hlihor1, Mariana Diaconu1, Teresa Tavares2, Maria Gavrilescu1 1 ”Gheorghe Asachi” Technical University of Iasi, Department of Environmental Engineering and Management, 73 Mangeron Street, 700050, Iasi, Romania; 2University of Minho, Department of Biological Engineering, Campus de Gualtar, 4710-057, Braga, Portugal

Abstract The bioremoval of Cr(VI) through biosorption and bioaccumulation using dead and living biomass of Trichoderma viride was investigated in batch mode, since a few studies were found in the literature on this subject. The biosorbent is an indigenous fungal strain of Trichoderma viride, isolated from a forest soil in an area from Iasi city, Romania. Effects of pH, biomass dosage, metal concentration in the initial solutions, contact time and temperature were assessed. It was found that the optimum pH for maximum removal efficiency is acidic for dead biomass and near the neutral value for living microorganisms. The biosorption process of total Cr removal by dead T. viride biomass is endothermic. The experimental data modeling and FTIR analysis showed that the mechanism of Cr(VI) removal by T. viride is based on a redox reaction. The linearized Langmuir isotherm had the best fitting compared to the other models applied showing that sorption occurs in monolayer at 25 and 40°C. Kinetic data were evaluated by the pseudo-first order and pseudo-second order adsorption kinetic models, together with pseudo-first order and pseudo-second order reduction models. The new fungal strain of T. viride has confirmed high uptakes and removal efficiencies in Chromium(VI) bioremoval.

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Session 6/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S121-S134

P53 CHROMIUM SORPTION AND Cr(VI) REDUCTION TO Cr(III) BY Litchi chinensis SEEDS D. Sánchez-García, E.A. Suazo-Madrid, E. Cristiani-Urbina* Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional. México, D.F., México, e-mail: [email protected]

Abstract Chromium [Cr] is among the most common heavy metal pollutants found in industrial wastewaters, and is exceedingly toxic to human health. Chromium is released to the environment by a large number of industrial operations, including the metallurgical, refractory and chemical industries. While hexavalent and trivalent species of chromium are prevalent in industrial wastewaters, the hexavalent form of chromium [Cr(VI)] is the most dangerous to public health due to its higher solubility in water, higher mobility in the environment, and to its toxic, mutagenic, and teratogenic traits. Therefore, the removal of Cr(VI) from the contaminated environments and industrial wastewaters is important. Biosorption has emerged as a promising bioremediation strategy for cleaning up water that has been contaminated with toxic metals by anthropogenic activities and/or by natural processes. The main purpose of this work was to evaluate the potential of Litchi chinensis seeds to remove Cr(VI) and total chromium from aqueous solutions. Furthermore, the kinetics, isotherm and thermodynamics of total chromium biosorption by Litchi chinensis seeds are described herein. It was found that acid-pretreated biomass showed higher chromium biosorption capacity than un-pretreated biomass at pH values ranging from 1 to 4, with an optimum pH of 2.5. The effects of other relevant environmental parameters, such as initial Cr(VI) concentration, shaking contact time and temperature, on chromium biosorption onto acid-pretreated Litchi chinensis seeds were evaluated. Significant enhancement of chromium biosorption capacity was observed by increasing initial metal concentration and temperature. Kinetic studies showed that the kinetic data were best described by a pseudo second-order kinetic model. Among the two-, three-, and four-parameter isotherm models tested, the Langmuir model exhibited the best fit to experimental data. Thermodynamic parameters revealed that the biosorption of chromium ions onto acid-pretreated biomass is an endothermic process. Throughout the experiments, Cr(VI) removal capacity was higher than total chromium removal capacity, and this was due to the amount of Cr(VI) which was reduced to Cr(III) by Litchi chinensis seeds and released into the aqueous solution. The high sorption capacity (168 mg g-1 at 25ºC) exhibited by acid-pretreated Litchi chinensis seeds places this biosorbent among the best adsorbents currently available for removal of chromium ions from aqueous effluents.

P54 DECOLORIZATION OF AQUEOUS EFFLUENTS USING AGRO WASTE Laura Carmen Apostol1*, Luciana Pereira2, Madalena Alves2, Maria Gavrilescu1 1 Department of Environmental Engineering and Management, Faculty of Chemical Engineering and Environmental Protection, The "Gheorghe Asachi" Technical University of Iaşi, 73, Prof. Dr. Docent Dimitrie Mangeron Str., Iaşi, 700050, Romania; 2Department of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal

Abstract In this study, the potential of agro–waste for a food dye sequestration from aqueous effluents was investigated. Initially, four local agrowaste namely pumpkin seed hull, bean null, oat straw and nut shells, were tested in natural condition. Bean hull (BH) revealed the best uptake capacity for Food Red 14 (FR14). The agro waste was characterised before and after dye sorption process using SEM and FTIR techniques. Those analyses were conducted in order to identify the principal connections contributing to the sorption process. The point of zero charge of BH surface, pHpzc was assessed at 4.6, which indicated a mostly acidic surface, favorable for dye adsorption at pH5% or >10% (depending on the kind of tissue, if TU or TF), methane production was lower than that in the control, meaning a toxic effect of high solid concentrations. Great amounts of hydrogen tended to accumulate in the reactors at high plant DM concentrations. Remarkable differences were observed in BMP, depending on the crop species. Plant biomass deriving from frequent cuttings gave rise to a better BMP when compared with than from a unique harvest, even though this behavior did not represent the rule. The BMP results are discussed in relation to plant tissue composition. On the basis of these preliminary results crop biomasses from wetlands treating wastewater appear promising sources not only of methane, but also of hydrogen.

P58 PILOT SCALE BIOAUGMENTATION FOR REMOVING NITROUS OXIDE (N2O) USING A DENITRIFYING BACTERIUM Pseudomonas stutzeri STRAIN TR2: A CASE STUDY Wakako Ikeda-Ohtsubo1, Morio Miyahara2, Sang-Wan Kim2, Akira Watanabe3, Shinya Fushinobu2, Takayoshi Wakagi2, Hirofumi Shoun2, Keisuke Miyauchi1, Ginro Endo1 1 Faculty of Engineering, Tohoku Gakuin University, Tagajo, Miyagi 985-8537, Japan; 2Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; 3Ebara Engineering Service Co., Ltd, 111 Haneda Asahi-cho, Ohta-ku, Tokyo 144-8610, Japan

Abstract Pseudomonas stutzeri TR2 is a unique denitrifying bacterium, which quickly adapts to denitrifying conditions even in the presence of oxygen and readily reduces nitrous oxide (N2O). The strain TR2 has been therefore considered to be a prospective candidate for bioaugmentation aiming to reduce N2O emission from nitrogen removal processes in the wastewater treatment systems. Here, we report a case study of bioaugmentation using a pilot-scale denitrifying reactor, where we monitored changes in N2O emission and survivability of the inoculated P. stutzeri strain TR2. In the first inoculation, the strain TR2 in the activated sludge disappeared within a few days, while there was an initial N2O reduction in appearance. Laboratory investigation showed that P. stutzeri strain TR2 is able to grow at a higher range of temperature and survives well, when 5–10 mM nitrite was provided as denitrification substrate. In light of these findings, we raised the nitrite concentration and the temperature of the reactor, which is preferable for the growth of the strain TR2, but not for other bacteria and protozoa susceptible to nitrite toxicity and higher temperature. As a result, we observed that the strain TR2 survived in the activated sludge for more than 3 weeks, and that the N2O emission from the reactor has been maintained at a lower level. This study illustrates the importance of applying an optimized condition for inculants against their competitors or predators to achieve successful bioaugmentation.

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Session 6- Wastewater valorisation, bioremediation, purification and reuse

P59 ANTI-INFLAMMATORY DRUGS REMOVAL THROUGH WASTEWATER DISINFECTION C. Noutsopoulos1, D. Mamais1, V. Samaras2, T. Bouras1, K. Antoniou1, D. Kokkinidou1 1

Department of Water Resources and Environmental Engineering, School of Civil Engineering, National Technical University of Athens; 2Water and Air Quality Laboratory, Department of Environment, University of the Aegean

Abstract Wastewater treatment plants are one of the major routes of transporting pharmaceuticals to the water bodies. Among pharmaceuticals antiinflammatory drugs ibuprofen (IBF), naproxen (NPX), diclofenac (DCF) and ketoprofen (KFN) are of significant interest due to their persistent detection in raw and treated wastewater. It is well known that these compounds are only partially removed through conventional biological wastewater treatment thus being detected in secondary effluents. Disinfection of biologically treated wastewater is the minimum additional treatment stage required in order to provide for safe wastewater reuse. As the primary goal of disinfection is to reduce wastewater microbiological content, the effectiveness of such methods to reduce pharmaceuticals is unclear. In view of the above the aim of this study was to examine whether typical disinfection methods, employed to provide for safe wastewater reuse, can lead to effective removal of IBF, NPX, DCF, KFN from wastewater and to assess the effect of critical parameters such as chlorine and UV dose and contact time in disinfection performance. Furthermore the effect of the target compounds and their metabolites generated during disinfection on the aquatic environment was assessed by the use of Vibrio fischeri as the indicative organism. According to the results for Ct values between 40-300 mgCl2·min/L, removal efficiencies were equal to 78-96%, 92-100%, 31-52% and 19-39% for NPX, DCF, IBF and KFN respectively. Removal efficiencies of NPX and DCF are practically independent of contact time whereas for both IBF and KFN removal efficiencies exhibited a polynomial dependence on the Ct dose. Therefore it can be postulated that in order to achieve a satisfactory removal of IBF and KFN significantly high chlorination Ct doses should be applied, with values higher than those required to guarantee proper disinfection for unrestricted agricultural and urban wastewater reuse. Furthermore UV disinfection at UV doses in the order of 40-150 mWs/cm2 is not effective to remove anti-inflammatory drugs. From the target compounds tested, KFN and DCF exhibited appreciable removal only at higher UV doses whereas practically no NPX and IBF removal was recorded. Based on toxicity experiments, it is anticipated that following chlorination of the target chemicals, production of more toxic metabolites is taking place. More specifically the inhibition of luminescent activity of Vibrio fischeri was significantly increased with the increase of chlorine dose. Therefore the effectiveness of both chlorination and UV to remove anti-inflammatory drugs is questionable and more research is needed to guarantee for safe wastewater reuse.

P60 MYCOREMEDIATION OF OLIVE OIL MILL WASTEWATER USING FREE AND IMMOBILIZED CELL CULTURES Mohamed Neifar1, Michel J Penninckx2, Atef Jaouani1 1

Laboratoire Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Campus Universitaire, 2092, Tunis, Tunisia ; 2Laboratoire d'Ecologie et Physiologie Microbienne, Université libre de Bruxelles 642 Rue Engeland, B-1180 Brussels, Belgium

Abstract Olive Oil Mill Wastewaters (OOMW) are recalcitrant to biodegradation for their toxicity due to high values of chemical oxygen demand (COD), biological oxygen demand (BOD), and phenolic compounds. In the present study OOMW, collected in southern Tunisia, were subjected to a biological treatment using free and immobilized Pycnoporus coccineus and Coriolopsis polyzona. Both species grew vigorously on OOMW and were responsible for color and COD reductions, which were greater in the OOMW treated with P. coccineus. This fungus was less affected by oxygen supplementation and exhibited a high tolerance to agitation in comparison to C. polyzona. However, it required a nitrogen supplementation to obtain faster and higher COD removal. The immobilized system for both fungi showed high COD decreases during three consecutive batchs without remarkable loss of performances. These results suggested that C. polyzona and particularly P. coccineus might be applicable at large scale for the treatment of OOMW. Key words: biological treatment, cell immobilization, COD removal, decolourization, olive oil mill wastewater

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P61 ISOLATION AND CHARACTERIZATION OF MICROORGANISMS FROM WASTEWATER SAMPLES COLLECTED FROM HASSA, SAUDI ARABIA Ibrahim Alshubaith1, D. James Gilmour2 1

Department of Environmental Studies, Hassa Municipality 31982, P.O. Box 60066, Hofuf, Saudi Arabia; 2Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK

Abstract As part of a study of the biodiversity of Alasfar Lake and Drainage/Irrigation Canals, Hassa, Saudi Arabia, we isolated three bacterial strains by exposing the water samples to high levels of NaCl (0.5 to 2 M). Two strains were isolated from a drainage canal prior to reaching the main lake and were identified using 16S rDNA sequencing as Staphylococcus warneri and Halobacillus blutaporensis. The third strain was isolated from the western point of Alasfar Lake and was identified by 16S rDNA sequencing as Halomonas venusta. All three species were shown to grow well at high salinities, despite their origin in a freshwater habitat. H. venusta is an unusual strain of the Halomonas genus, because it has been implicated in a human infection. Hb. blutaporensis has only been previously described as part of the microbial flora of the roots of Blutaparon portulacoides a rhizomatous herb found in sandy soil in Brazil. On the other hand S. warneri is a well known pathogenic bacterium. To better understand their presence in the freshwater Hassa habitats, the three strains have been characterized further using NMR to determine their compatible solutes and examining their susceptibility to UV light. Two important conclusions can be drawn from this work. Firstly, pathogenic bacteria (e.g. S. warneri) and potentially pathogenic bacteria (e.g. H. venusta) are present in the Hassa water samples and these strains survived isolation in high salinity media. Secondly, an unusual species of Halobacillus was isolated using the same high salinity media, which suggests that a rich diversity of bacteria may well be present in the Hassa waters which would reward a more detailed systematic study of the biodiversity of this freshwater habitat.

P62 MICROBIAL RESPONSES TO XENOBIOTIC SHOCK LOADS IN ACTIVATED SLUDGES Andrea Negroni1, Massimo Marzorati2, Fabio Fava1, Nico Boon2 1

DICAM, Faculty of Engineering, University of Bologna, via Terracini, 28, 40131 Bologna, Italy; 2Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium

Abstract In wastewater treatment, ammonia undergoes nitritation, i.e. oxidation to nitrite by Ammonia Oxidizing Bacteria (AOB). Nitrite Oxidizing Bacteria (NOB) then further oxidize nitrite to nitrate, completing the nitrification process. AOB activity, essential for the ammonia removal and COD abatement, can be disrupted by organic pollutants present in the wastewater stream, such as 3-chloroaniline (3CA). Currently the assessment of wastewater treatment plant systems’ efficiency is based on measuring basic functions (inhibited nitrification, decreased carbon removal, modification of the sludge physical properties). However the biological catalyst remains a black box. The definition of the structure and functionality of the microbial community in the sludge is a key-point to be addressed for the management of the system (Microbial Resource Management), since microbial community metabolism drives the process efficiency and stability. This work aimed at indentifying a set of microbial based parameters useful to predict the system functionality. In addition, it was also evaluated if any adaptation, resistance or resilience to 3CA shock loads are possible. WWTP sludge containing aerobic Semi-ContinuousActivated-Sludge reactors were set up. After their initial stabilization, 1.5 mg 3CA (L sludge)-1 was added either immediately or gradually (by daily adding small amounts of 3CA); non-amended reactors were incubated alongside as controls. Eight days later, each reactor was split to be either suddenly shock-loaded with 250 mg 3CA (L sludge)-1 or left under normal operating conditions. Microbial community dynamics were monitored in all the reactors throughout incubation by PCR-DGGE directed to the 16S RNA and the 16S DNA of both total bacteria and AOBs. In all the 3CA shock loaded reactors, nitritation was inhibited and accumulation of ammonia occurred, thus indicating that no adaptation was achieved. RNA-based analyses, which reflects the functionality of the targeted bacteria, showed a quick change in response to the shock load. Conversely, DNA-based analyses detected bacteria independently from their activity. The abundance of this resilient community could be used to infer the possibility of restoring the disrupted functionality. Finally, the combination of three DGGE deduced parameters ((i) the range-weighted richness (Rr) reflecting the carrying capacity of the system, (ii) the dynamics (Dy) reflecting the specific rate of species coming to significance, and (iii) Community Organization (Co) allowed to draw a user-friendly graph representing a breakthrough solution to predict possible functional failures and their management.

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Session 6- Wastewater valorisation, bioremediation, purification and reuse

P63 BIOSORPTION OF TRIVALENT CHROMIUM FROM AQUEOUS SOLUTIONS BY Pleurotus ostreatus BIOMASS I.J. Puentes-Cárdenas1, A.M. Pedroza-Rodríguez2, E.A. Suazo-Madrid1, M. Navarrete-López1, T.L. Villegas-Garrido1, E. Cristiani-Urbina1* 1

Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional. México, D.F., México, e-mail: [email protected] 2 Pontificia Universidad Javeriana. Bogotá, Colombia

Abstract Trivalent chromium [Cr(III)] is a pollutant commonly found in wastewater emanating from leather tanning, dye, wood preservation, and electroplating industries. Long-term exposure to high Cr(III) concentrations may cause allergic skin reactions, cancer and DNA damage. Therefore, the removal of Cr(III) is nowadays recognized as a key process for the detoxification of Cr(III)-contaminated water and wastewater. Biosorption of heavy metals by biomaterials has emerged as a sustainable strategy for the detoxification of wastewaters, as it is effective, cheap and eco-friendly. The main aim of this work was to study the effect of relevant environmental parameters such as solution pH, initial Cr(III) concentration, contact time, and temperature on the ability of Pleurotus ostreatus biomass to biosorb Cr(III) ions from aqueous solutions. Furthermore, the kinetics, isotherm and thermodynamics of Cr(III) biosorption by P. ostreatus are described. In addition, the functional groups on the surface of P. ostreatus involved in Cr(III) biosorption were identified by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). It was found that the optimum pH for Cr(III) biosorption was 5.5. The pseudo first-order model best fitted to the experimental data of Cr(III) biosorption. Of the ten isotherm models tested, the Langmuir model best described the Cr(III) biosorption equilibrium process. According to this model, the maximum Cr(III) biosorption capacity of P. ostreatus is 108 mg g-1. Thermodynamic parameters (activation energy, and changes in activation enthropy, activation enthalpy, and free energy of activation) revealed that the biosorption of Cr(III) onto P. ostreatus is an endothermic and non-spontaneous process. DRIFTS studies suggest that the main functional groups involved in Cr(III) biosorption by P. ostreatus are the following: C-H, OH, NH3+, C=O y CHO. Results indicate that P. ostreatus is one of the best biosorbents for Cr(III) removal from aqueous solutions hitherto reported and could therefore be effectively used to detoxify wastewaters polluted with Cr(III).

P64 TREATMENT OF A SHOPPING MALL WASTEWATER USING AN ATTACHED GROWTH ANOXIC-AEROBIC SYSTEM Cleofas O. Maceda1, Analiza P. Rollon2 1

Environmental Engineering Graduate Program, University of the Philippines Diliman, Head Corporate Environment & Pollution Control; 2Department of Chemical Engineering, University of the Philippines, Diliman

Abstract An integrated water quality management system involves both treatment of wastewater and appropriate reuse of the effluent. Treated wastewater from commercial establishments such as hotels, high-rise residential or office buildings and shopping malls are potential source of non-potable water for use as landscape watering and toilet flushing. This study aimed to determine the performance of a fullscale aerobic activated sludge system and a bench-scale attached growth anoxic-aerobic growth wastewater treatment system (WWTS) which operated at shorter HRT and higher organic loading rate (OLR) than the present full-scale system. The effect of HRT on the benchscale performance was also determined. Furthermore, this study assessed the effluent water quality for possible reuse. In the full-scale WWTS, at 2.06±0.18 days HRT and 0.396±0.123 kg COD/ OLR, the COD, BOD, FOG and TSS removal efficiency were 97.2±2.3%, 95.3±2.0%, 91.6±15.0% and 85.96±13.4%, respectively. In the bench-scale attached growth anaerobic-aerobic WWTS, the BOD, COD, FOG and TSS removal efficiency were 97.9±2.3%, 93.6±5.9%, 87.8±22.9% and 71.4±18.5%, respectively, at 6.35 h HRT and 2.42±0.40 kg COD/ OLR. The efficiencies were better at longer HRT and lower OLR. At 12.70 h HRT and 1.50±0.30 kg COD/OLR, the BOD, COD, FOG and TSS removal efficiencies were 98.1±2.4%, 94.7±4.6%, 95.0±5.1% and 91.4±2.4%, respectively. The effluent of the bench-scale WWTS after an additional tertiary treatment could be used for landscape watering and flushing toilet.

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Environmental Engineering and Management Journal

March 2012, Vol.11, No. 3, Supplement, S135-140

http://omicron.ch.tuiasi.ro/EEMJ/

“Gheorghe Asachi” Technical University of Iasi, Romania

Session 7 MICROBIAL FIXATION OF CO2 AND OTHER CLIMATE CHANGE INDUCING GASES IN SOILS AND VIA BIOTECHNOLOGICAL PROCESSES Main lectures CLIMATE CHANGE AND MICRO-ORGNISM GENETIC RESOURCES FOR FOOD AND AGRICULTURE: STATE OF KNOWLEDGE, RISKS AND OPPORTUNITIES Anna Benedetti CRA- Centro di Ricerca per lo Studio delle relazioni tra Pianta e Suolo, Roma, via della Navicella, 2-00184 Roma, Italia

Abstract Micro-organisms form the basis of most ecosystems on which agriculture and food production depend. Put simply, agriculture and food production would not exist without this “hidden” but critically important biodiversity. Micro-organisms play essential roles that we can separate into five interlinked functional domains: soil inhabitants, plant and rhizosphere inhabitants, plant pathogens, biological control agents and food production micro-organisms. The focal point of this important role played by micro-organisms in the regulation of life on the earth is the unrivalled diversity of micro-organism genetic resources for food and agriculture, and the correspondingly huge variety of functions they confer to agriculture. Many beneficial micro-organisms are linked with plants in the soil, where they induce resistance or perform biological control functions. Free-living soil-borne micro-organisms contribute to the formation and structure of soil, the storage of nutrients and carbon sequestration. Those acting in association with crop plants further regulate soil fertility and the accessibility of nutrients. Soil micro-organisms are also responsible for bioremediation of polluted sites by restoring soil fertility. Once food is produced, micro-organisms are relied upon for its conservation and transformation. Climate change will act as an additional driver of change in agricultural systems as environmental conditions are altered drastically. As a consequence of the vital functions that micro-organism genetic resources for food and agriculture confer, they can be considered as pivotal to sustainable agriculture when challenged by such drivers of change. Because plants’ performance is directly linked to interactions with micro-organisms, future strategies to counteract negative effects of climate change will need to involve more than simply deploying crop plants in environments to which they are well adapted. Micro-organism genetic resources for agriculture can be used to produce energy directly, facilitate adaptation to climate change and mitigate climate change. Novel biological control agents can be used to limit the harmful impact of pathogens and pests, obviating the need for energy-expensive pesticides. Other micro-organisms could also be used to improve the efficiency of intensified agriculture, such as those that store carbon in the soil, and hence prevent the emission of greenhouse gases. The fertility of the soil for any particular cropping system could be enhanced naturally, either by creating conducive conditions for the proliferation of beneficial micro-organisms or by introducing them directly into targeted environments. Soil-regulating micro-organisms can be used to manage soil health and ecosystem resilience. Micro-organisms also have an important role in the protection and transformation of agricultural produce, postharvest.

Session 7/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S135-S140

BIOREFINERY FOR THE PRODUCTION OF BIOGAS AND BIODIESEL BASED ON THE USE OF PLANTS AND MICROALGAE TREATING WASTEWATER Eugenia J. Olguín Institute of Ecology, Carretera Antigua a Coatepec 351, El Haya, Xalapa, Veracruz, 91070 México, e-mail: [email protected]

Abstract Biorefineries are an emergent field of research, since they offer an opportunity to improve the economic feasibility of biofuels production. In this work, a Biorefinery is envisaged as a complex in which integration of various unit operations allows the treatment of polluted river’s water, municipal or agro industrial wastewater with a simultaneous production of biomass (plants or microalgae) and its transformation into fuels and/or chemicals. Several recent Life Cycle Analysis studies have indicated that the cost-benefit of biodiesel production from microalgae may increase if produced utilizing wastewater within a Biorefinery. The aim of the present work is to discuss the design and operation of a Biorefinery combining the treatment of wastewater with aquatic plants, the production of biogas from harvested biomass and the use of treated water for cultivation of microalgae with potential for production of biodiesel. Wastewater of various sources such as municipal and agro industrial is discussed. Furthermore, the use or urban polluted rivers as source of water and nutrients, is also discussed. In particular, the use of the polluted urban “Sordo” River located in the city of Xalapa, Veracruz, México provides a good model for discussion. The use of phytofiltration for its treatment requires the selection of appropriate aquatic plants for maximum biogas production. In relation to the use of the treated water for microalgae cultivation with potential for biodiesel production, several aspects such as the availability of sufficient nutrients, the presence of contaminants, the population dynamics which is established among the inoculated and the native microalgae species, are also reviewed. Finally, some general aspects related to the major technological challenges to overcome in order to make such integrated system the most cost-effective as possible, are discussed.

Oral presentations PHOTOELECTROCHEMICAL NADH REGENERATION FOR ENZYMATIC CO2 REDUCTION: INCREASING EFFICIENCY AT METAL-MODIFIED SEMICONDUCTORS Paolo Stufano1, Zachary M. Detweiler2, Elizabeth L. Zeitler2, Angela Dibenedetto1, Andrew B. Bocarsly2, Michele Aresta1 1

University of Bari, Bari, IT; 2Princeton University, Princeton, NJ, USA

Abstract The mimicry of the photosynthetic process has inspired the enzymatic reduction of carbon dioxide into methanol through the dehydrogenase enzyme cascade: FateDH, FaldDH and ADH. Although, this process occurs under very mild conditions (water, 37°C, pH=7) and with optimal yield and selectivity (close to 100%), there still is a limitation associated with the consumption of the cofactor NADH. Although enzymatic, chemical and photo-chemical approaches have been attempted electrochemical regeneration is considered the most attractive solution. Herein we report the use of p-type semiconductor electrodes in order to utilize solar energy for photoelectrochemical NADH regeneration. While bare semiconductors were shown to produce only enzymatically inactive dimers (NAD2), modification of the surface by electro-deposition of a thin layer of Pt or Ru metal caused the formation of 1,4-NADH as the main product. In particular redlight illuminated (>600 nm) of Pt/p-GaAs showed an increased efficiency at low overpotentials (-0.75V vs Ag/AgCl) when compared to metal electrodes (> 7 fold), with no dimer detection. This study represents the first example of NADH regeneration at an illuminated semiconductor electrode. The absence of a mediator allows the direct coupling of this regeneration system with the enzymatic CO2 reduction apparatus, modeling the light and dark reactions occurring in a chloroplast.

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Session 7- Microbial fixation of CO2 and other climate change inducing gases in soils and via biotechnological processes

CARBON DIOXIDE FROM ALCOHOLIC FERMENTATION AS A CARBON SOURCE FOR FED-BATCH CULTIVATION OF Arthrospira platensis Raquel Pedrosa Bezerra1,2, Marcelo Chuei Matsudo1, Sunao Sato1, Attilio Converti2, João Carlos Monteiro de Carvalho1 1

Department of Biochemical and Pharmaceutical Technology, University of São Paulo, São Paulo, Brazil; 2Department of Chemical and Process Engineering “G.B. Bonino”, University of Genoa, Genoa, Italy

Abstract World’s production of ethanol has increased dramatically in recent years. Brazil is the world’s largest exporter of bioethanol and secondlargest producer after the United States. Considering the increasing demand for this fuel and the fact that alcoholic fermentation is responsible for a CO2 release, on weight basis, almost coincident with ethanol production, it would be interesting to develop a process for CO2 fixation able to turn it into a useful product. Photosynthetic microorganisms can fix CO2 efficiently producing biomass that contains high-value bioactive products and may provide a very promising alternative for the current CO2 mitigation strategies. Nowadays, there are numerous commercial applications of Arthrospira platensis biomass such as the enhancement of the nutritional value of foods and animal feed, bioremediation, and use in cosmetics. The objective of this work was to evaluate the Arthrospira platensis cultivation using CO2 from alcoholic fermentation and either urea or nitrate as nitrogen source at different light intensities (60 ≤ I ≤ 240 μmol photons m-2 s-1). The CO2 source (pure CO2 or from alcoholic fermentation) did not influence the maximum cell concentration (Xm), cell productivity (PX) and nitrogen-to-cell conversion factor (YX/N). On the other hand, the use of urea instead of nitrate led to higher YX/N values. Xm and PX increased when I was increased from 60 to 120-240 μmol photons m-2 s-1. Using CO2 from alcoholic fermentation, the best performance (Xm=2952 ± 35 mg L-1, PX=425±5.9 mg L-1 d-1 and YX/N=15±0.20 mg mg-1) was obtained at I = 120 μmol photons m-2 s-1 with urea. The results obtained in this work demonstrate that urea and CO2 from alcoholic fermentation could be simultaneously used in large-scale cultivations to reduce the environmental impact associated to the release of this greenhouse gas as well as to decrease the production cost of this cyanobacterium.

Posters P66 RESPONSE OF SOIL BACTERIAL COMMUNITY TO AGRICULTURAL LAND USES AND SEASONAL CHANGES Patrizia Paganin1, Maite Sampedro Pellicer1, Luigi Ledda2, Maria Cristiana Papaleo3, Renato Fani3, Anna Benedetti4, Claudia Dalmastri1, Annamaria Bevivino1* 1

Technical Unit for Sustainable Development and Innovation of Agro-Industrial System, ENEA Casaccia Research Center, Rome, Italy; 2Dept of Agronomy and Crop Genetics, University of Sassari, Italy; 3Laboratory of Microbial and Molecular Evolution, Dept of Evolutionary Biology, University of Florence, Italy; 4Research Centre for Plant and Soil System RPS–CRA, Rome, Italy

Abstract Microbial communities play a critical role in the functioning of soil and represent a very susceptible index of soil quality. Among the factors driving differences in soil bacterial community structure, anthropogenic activities and seasonal temperature changes are very important. In this work, we aimed at determining the effects of land use intensification along with seasonal variations on soil bacterial community. Five different land-use units, characterized by different use and management with growing human impact, were chosen in Sardinia (Italy): cork-oak forest (coded Su), hayland-pasture rotation (Pa), semi-natural grassland (Er), and ploughed (VL) and grass covered vineyard (Vi). The sampling sites were selected inside the same environmental unit, with neutro-acidophylous cork-oak forest as potential vegetation. At each site, five separate sub-samples were collected from the top 20 cm depth of soil in spring and autumn. Along this land-use gradient, soil physic-chemical properties and bacterial communities were analyzed. All the soils resulted to be sandy-loam, presented pH reaction sub-acid and very low organic matter content that showed the highest values in soils with a lower human impact. Cluster analysis of the banding patterns obtained by culture-dependent denaturing gradient gel electrophoresis (CD-DGGE) revealed that soil bacterial communities clearly differed depending on season. Within each season, subgroups including bacterial communities associated to different land uses with increasing level of intensification were observed. All these data are in agreement with those resulting from Terminal Restriction Fragment Length Polymorphism (T-RFLP, cultivation-independent method). Phylogenetic analysis of 16S ribosomal RNA gene revealed that cultivable bacteria included members of Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes groups, with a prevalence of certain taxonomic units in a defined season (i.e. Burkholderiales in Autumn). Investigation of the relationships between soil physic-chemical properties and microbial biodiversity is in progress. In conclusion, different microbial biodiversity among samples and shifts in soil microbial community structure can be ascribed to seasonal changes, even if anthropogenic activity can also affect soil microbial community. The positive selection of Proteobacteria and the absence of Acidobacteria indicates a good state of health for the soils examined in the present study. The high prevalence of Proteobacteria could be used as an indicator of soil health. Acknowledgements Funded by MIUR (Integrated Special Fund for Research - FISR) in the frame of the Italian National Project SOILSINK: Climate change and agro-forestry systems: impacts on soil carbon sink and microbial diversity.

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P67 MICROBIAL ACTIVITIES AND GHGs EMISSIONS FOLLOWING DIFFERENT FERTILIZATION ON MAIZE L. Bardi 1, L. Petruzzelli 3, L. Zavattaro 2, C. Bertora 2, F. Rosso 1, F. Zoppellari 1, C. Grignani 2 2

1 C.R.A. Agricultural Research Council-Research Centre for the Soil-Plant System Environment Park, Via Livorno, 60 (A2 Lab)-10144 TURIN, Italy; Department of Agronomy, Forest and Land Management-University of Turin, Via Leonardo da Vinci,44-10095 Grugliasco (TO), Italy; 3Plant Protection Service, Agrochemical Laboratory, Piedmont Region, Environment Park, Via Livorno,60 (A2 Lab)-10144 Turin, Italy

Abstract Several gases are produced in soil due to microbial activities; CO2, CH4 and N2O are considered the most important for their crucial role as GHGs. C and N availability are among the main factors affecting the production of gases in soil, along with water and O2 availability, and temperature; then fertilization, amendment, watering, tillage and other agronomical practices can act on microbial population dynamics and metabolic adaptations, affecting the metabolic pathways releasing GHGs. The choice of good practices of crops management, in order to adequately direct the soil microbial activities, could be therefore the first way to control GHGs emissions and/or favour C sequestration in soil. In this work the effect of the addition to soil of different fertilizers was investigated on a maize-cropped field trial. Treatments were maize for silage receiving farmyard manure (MS-F), slurry (MS-S) or urea (MS-U), and maize for grain receiving urea (MG-U), at 170 kg N ha-1 entirely distributed just before tillage (30 cm depth) and sowing. Soil organic C, total N, ammonium and nitrate were analyzed; microbial populations and activities were characterized by analyzing total microbial biomass C and N, cultivable fungi, aerobic and anaerobic bacteria, and nitrifying and denitrifying activities. Emissions of CO2, CH4 and N2O were monitored and their yearly cumulative emissions calculated. Manured plots (MS-F), where both the highest microbial biomass content and a high content of aerobic microorganisms were observed, induced the highest yearly CO2 emission. MS-U plots produced the lowest CO2 emissions and showed the lowest microbial biomass content, indicating that the absence of organic matter input prevents the growth and activity of microorganisms even in presence of N availability. Furthermore MS-U was the treatment revealing the lowest N2O emissions and even null cumulative CH4 emissions. The highest N2O emission was observed in MG-U, and a high emission was also observed for MS-S immediately after fertilization. MG-U and MS-S were the plots where high activities were observed for both denitrifiers and nitrifiers. Due to the high content of methanogenic microorganisms in slurries, high CH4 emission was produced in MS-S, even if the highest emission was found for MG-U, while in MS-F CH4 seemed to be oxidized. The high emission in MG-U can be ascribed to the competition between ammonia and methane as substrate for the ammonium oxidizers that can be responsible of CH4 decrease.

P68 IMPACT OF PERENNIAL ENERGY CROPS ON CHEMICAL QUALITY AND MICROBIAL COMMUNITY OF SOIL IN THE PO VALLEY F. Cattaneo*, P. Gioacchini, D. Montecchio, C. Giovannini, L. Barbanti, M. Labra, P. Di Gennaro, C. Marzadori Facoltà di agraria, University of Bologna, Bologna, Italy

Abstract Energy crops are designed for biomass production to generate bioenergy. Their recent introduction in the Po Valley (North Italy) is connected to the mitigation of environmental effects of the intensive management. Intensive management has a dramatic impact on the soil ecosystem of the area, reducing soil organic matter, degrading soil structure and altering nutrient cycles by reducing microbial biomass, and thus compromising soil productivity. Soil organic carbon (SOC) content in the Emilia-Romagna region is the lowest in the Po Valley ( fungi> bacteria. Since bacterial-based processes can show many advantages, more efforts are required to increase bacterial PUFA yields. Genetic modification could be an avenue for PUFA yield improvement. Full scale applications of microbial PUFA production, although desirable and necessary, are lacking.

P98 GH10 AND GH11 XYLANASES PRODUCED FROM THERMOTOLERANT Streptomyces sp. SWU10 WITH HIGH pH AND THERMAL STABILITY Warin Deesukon1, Yuichi Nishimura2, Naoki Harada2, Tatsuji Sakamoto*2, Wasana Sukhumsirichart1* 1

Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok 10110, Thailand 2 Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 599-8531, Japan

Abstract Thermotolerant Streptomyces sp. SWU10 isolated from rice straw produced four endo--1,4-xylanases. Three enzymes, XynSW1, XynSW2A, and XynSW2B were purified and characterized. Their apparent molecular masses were 25 kDa, 31 kDa, and 44 kDa, respectively. Optimal pH and temperature of XynSW1 were pH 5.0 and 40°C, whereas 60°C and 6.0 were for XynSW2A and XynSW2B, respectively. The enzymes were stable in a wide pH ranges, more than 80% of initial activity remained at pH 2-11 (XynSW1), pH 3-9 (XynSW2A) and pH 2-9 (XynSW2B) at 4°C for 16 h, and stable up to 50°C, 80°C and 60°C for 1 h, respectively. The xynSW1, xynSW2B and xynSW3 genes were isolated by in vitro cloning. The xynSW1 contains 1,011 bp in length and encodes a mature enzyme of 295 amino acids and 41 amino acids of signal peptide. The coding sequence of xynSW2B gene was 1,434 bp and encodes a polypeptide of 477 amino acids included 41 amino acids of signal peptide. XynSW2A seem to be the proteolytic degradation product of XynSW2B which might be cleaved at Lys-Lys at positions 330 and 331. The xynSW3 gene contains 726 bp and encodes a polypeptide of 241 amino acids. After cleavage of signal peptide, the calculated molecular mass was 20,872 Da. The amino acid sequence analysis revealed that the XynSW1 and XynSW3 belongs to glycoside hydrolase family 11 (GH11) whereas the XynSW2A and XynSW2B belongs to GH10. The xynSW1 gene was further over-expressed in Pichia pastoris using pPICZA vector. The recombinant XynSW1 exhibited some physicochemical properties that better than those of the native enzyme including higher optimal temperature (60°C), and higher specific activity, but lower optimal pH (4.0). Because of their stability in a wide pH ranges, and high temperature, these xylanases from thermotolerant Streptomyces sp. SWU10 may have potential application in several industries including food, textile, biofuel and also waste treatment.

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Sub-session 8.2- Industrial exploitation of microbes from deep seas, arid/contaminated environments

P99 HYDROCARBONOCLASTIC BACTERIA ISOLATED FROM PETROLEUM CONTAMINATED SITES IN TUNISIA: ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF THE BIOTECHNOLOGICAL POTENTIAL Mouna Mahjoubi1,2, Amel Guesmi1, Atef Jaouani1, Abdellatif Boudabous1, Ameur Cherif1,2 1

Laboratory of Microorganisms and Active Biomolecules, Faculty of Sciences of Tunis, University of Tunis El Manar, 2092, Tunis, Tunisia; 2Laboratory of Biotechnology and Valorisation of Bio-Geo resources, Higher Institute for Biotechnology, University of Manouba, Sidi Thabet Biotechpole, 2020, Sidi Thabet, Ariana, Tunisia

Abstract Petroleum hydrocarbons are important energy resources used by industry and in our daily life, whose production contributes highly to environmental pollution. In order to control this environmental risk, bioremediation constitute an environmentally friendly alternative technology that has been established and applied. Bioremediation constitute the primary mechanism for the elimination of hydrocarbons from contaminated sites by natural existing populations of microorganisms. In this work, a collection of 125 strains, adapted to grow in minimal medium supplemented with crude oil (as sole carbon source), was obtained from contaminated sediments and seawater from the Bizerte lagoon, a highly urbanized and industrialized water plan, subjected to various pollutants located in the North of Tunisia. The diversity of this collection was analyzed by amplification of the internal transcribed spacers between the 16S and the 23S rRNA genes (ITS-PCR) and by 16S rRNA sequencing. A total of 36 distinct ITS haplotypes were detected on agarose matrix. Partial 16S rRNA gene sequencing performed on 50 isolates showed high level of identity with known sequences. Strains were affiliated to Ochrabactrum, Sphingobium, Acinetobacter, Gordonia, Microbacterium, Brevundimonas, Novosphingobium, Stenotrophomonas and Pseudomonas genera. Acinetobacter and Stenotrophomons were found the most abundant species and showed an important microdiversity by ITS typing. Culture independent approach (DGGE) showed high diversity in the microbial community in all the studied samples. The biotechnological potential of different isolates revealed a significant production of biosurfactants with important emulsification activities useful in bioremediation. The highest emulsification activity was detected in Pseudomoans geniculata with 52.77% of emulsification. Our overall results suggest that the obtained bacterial isolates may constitute future candidates for bioremediation and can be useful for biotechnological applications.

P100 RHAMNOLIPID PRODUCTION BY BACTERIA ISOLATED FROM CONTAMINATED SOIL Ana C.G. Martins1, Álvaro S. Lima2, M. Rosário M. Domingues3, João A.P. Coutinho1, Luísa Seuanes Serafim1 1

CICECO, Departamento de Química, Universidade de Aveiro, 3810-193Aveiro, Portugal; 2Programa de Pós-Graduação em Engenharia de Processos, Universidade Tiradentes, Av. Murilo Dantas 300, Farolândia, Aracaju-SE, Brasil; 3Department of Chemistry, QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal; e-mail: [email protected]

Abstract The present study aims to evaluate the ability of bacterial strains isolated in situ from contaminated soil with hydrocarbons in Brazil to produce biosurfactants with the extracts from spent coffee grounds (SCGs). Surfactants are amphiphilic compounds containing hydrophobic (nonpolar) and hydrophilic (polar) moieties that confer ability to reduce the surface and interfacial tensions and forming emulsions in fluids with a wide range of applications. Many microorganisms produce this type of compounds with surface-active properties which were designated as biosurfactants. Biosurfactants bear some advantages, when compared to the conventional surfactants, such as low toxicity and high biodegradability, wide range of temperature and pH. These characteristics allow them to be applied in many different areas such as agriculture, health, food products and environmental protection. Moreover, biosurfactants can vary in terms of nature, size and properties depending on the type of producing microorganisms and each class of biosurfactant has a specific range of application. However biological production of surfactants has high production costs. A possibility to decrease these costs is the use of industrial residues of by-products and the selection of microorganisms able to use these raw materials. SCGs are rich in carbohydrates and lipids and results from the coffee drawing. Due to the worldwide popularity of coffee as a beverage, SCGs are a cheap residue and produced in high amounts. Two extracts were obtained from the SCGs, one rich in sugars and other rich in lipids (coffee oil). The oil extracted from SCGs can be used as substrate for biosurfactant production. The coffee oil was characterize by nuclear magnetic resonance (NMR) and gas chromatography with flame ionization detector (GC-FID), verifying that it was rich in palmitic acid (C16:0), oleic acid (C18:1) and linoleic acid (C18:2). The sugar solution was characterized by HPLC. After its characterization, the oil and sugars extracted from SCGs were supplied to four bacterial strains and their ability in producing biosurfactants was verified and the most efficient were selected. In order to maximize the biosurfactant production the effects of parameters, as the amount of carbon substrate, type of nitrogen source and the C:N ratio, were studied. Finally the obtained biosurfactants were characterized by mass spectrometry MALDI-TOF/TOF verifying that the selected cultures produced rhamnolipids.

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Session 8/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S141-S150

P101 INTRODUCING A NEW BACTERIAL STRAIN CAPABLE OF PRODUCING PHA FROM PETROCHEMICAL INDUSTRY WASTEWATER Hossein Motamedi1, Nasim Mayeli1, Fariba Heidari Zadeh2 1

Department of Biology, Faculty of Science, Shahid Chamran University, Ahvaz, Iran; 2Department of Chemistry, Faculty of Science, Shahid Chamran University, Ahvaz, Iran

Abstract Polyhyroxyalkanoate (PHA) is a biodegradable and environmentally friendly thermoplastic that is produced as a carbon-energy storage source by bacteria that are challenged by nutrient limitation. The major problem associated with the industrial production of PHA is their high production cost, this study was carried out using petrochemical wastewater as the major substrate to decrease the production cost in isolating and cultivation of PHA-producing bacteria. For this purpose, microorganisms were isolated from the Bandar Imam petrochemical (BIPC) wastewater of Iran. The purified colonies were screened for intracellular granules by staining with Sudan Black and Nile blue. The positive-staining strains were cultured for production of PHA in BIPC wastewater. The culture was incubated at 37ºC with shaking at 150 rpm for 5 days. Among positively stained, one novel gram positive bacteria capable of accumulating PHA was isolated and according to partial sequencing of its 16s ribosomal RNA gene with 98% similarity identified as Bacillus axaraquiensis. PHA content in 30 g/l biomass was reached 75.4% and 79.96% in synthetic medium and BIPC wastewater, respectively. This polyester was purified from freeze'-dried cells and analyzed. The growth curve of this isolate was also drawn. This curve showed that the polymer production begins from stationary stage. Interestingly, its growth curve didn't followed classical bacterial growth curve and during 80 hours showed 10 log phases followed with stationary phase. Hence that PHA production is occurs routing in stationary phase and considerable biomass is needed to obtain maximum PHA, this isolate can be a good candidate for industrial PHA production from a cheap and easily accessible carbon source such as petrochemical wastewater.

P102 EXOPOLYSACCHARIDIC MATRIX OF BIOLOGICAL SOIL CRUSTS FROM ARTIC ENVIRONMENTS Federico Rossi1, Gianmarco Mugnai1, Giovanni Colica1, Stefano Ventura2, Claudio Sili2, Cristina Mascalchi2, Roberto De Philippis1 1

Department of Agricultural Biotechnology, University of Florence, Florence, Italy; 2Institute for Ecosystem Studies, CNR, Florence, Italy

Abstract Biological soil crusts (BSC) are soil-surface communities that represent a typical example of microbial adaptability to harsh environmental conditions. They improve soil fertility and promote plant growth, representing a valid putative tool to fight desertification processes. Various mechanisms allow them to withstand constraints such as drought, high solar irradiation and wind erosion. These organisms produce extracellular polymeric substances (EPS), excreted in response to the environmental characteristics, which are known to primarily contribute in many ways to the increase of constraint tolerance. While various features of these excreted molecules have been investigated in axenic bacterial cultures, a not definitive and relatively low number of studies come from the direct study of the entire matrix of microbial aggregates, with limitative information on how they contribute to microbial survival in extreme environments. The aim of this study was to investigate the microbial composition and the characteristics, both qualitative and quantitative, of the EPS extracted from BSC collected by several sites in Ny-Ǻlesund, Svalbard Island, Norway, in a landscape mostly characterized by the high arctic tundra, where environmental conditions are extreme and where BSC play a key role in creating a favorable habitat for plant growth after deglaciation processes. A proper method for extracting EPS directly from the crust samples was optimized. Exopolysaccharidic fractions were quantified and analyzed using ion-exchange chromatography (IEC) to determine the monosaccharidic composition. Size exclusion chromatography (SEC) was also used to determine the size distribution of the EPS fractions. Abundance of phototrophic microorganisms, which are known to primarily contribute to EPS excretion, was also evaluated in all the samples. Results underlined the complexity of the polysaccharidic fractions, displaying a high number of constituent sugars; the matrix was found to be constituted by two main fractions, a higher molecular weight (MW) fraction (apparent MW around 2 106 Da) and a lower molecular weight fraction (MW < 100 103 Da). Fluctuations of these results were observed along the sampling sites suggesting the influence of environmental factors and substrate characteristics on the formation of EPS matrix. This study presents novel data concerning EPS of BSCs matrix in cold environments and the results represent a starting point to deepen the knowledge on the mechanisms at the basis of the high tolerance showed by BSC microorganisms to extreme conditions. The knowledge of these mechanisms in different extreme environments (both hot and cold) can improve the outcome of the exploitation of these communities to increase the fertility and the stability of soils in constrained environments.

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Sub-session 8.2- Industrial exploitation of microbes from deep seas, arid/contaminated environments

P103 BACTERIAL MULTICOMPONENT MONOOXYGENASES FOR THE BIOSYNTHESIS OF ANTIOXIDANTS OF INDUSTRIAL INTEREST G. Donadio1, E. Notomista1, A. Serpico1, A. Pezzella2, A. Di Donato1, V. Izzo1 1

Biologia strutturale e funzionale, Università di napoli federico ii, Napoli, Italia; 2Dipartimento di scienze chimiche, Università di napoli federico ii, Napoli, Italia

Abstract Antioxidants are compounds that reduce, neutralize and prevent the damage induced in the cells by free radicals (ROS). Tyrosol and hydroxytyrosol belong to a class of natural phenolic antioxidants whose role in the prevention of diseases such as cancer and cardiovascular diseases is rapidly emerging. Unfortunately, either the purification of these compounds from natural sources or their chemical synthesis is difficult. As a consequence much effort has been dedicated in the last years to the development of bioconversion processes to produce these molecules. In this perspective the bacterial multicomponent monooxygenase ToMO has been recently used for the biosynthesis of tyrosol and hydroxytyrosol using 2-phenylethanol, a cheap and commercially available aromatic molecule, as the starting compound. Due to the inability of the wt type enzyme to convert 2-phenylethanol into the correct tyrosol isomer, a computational model was used which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h. Based on the positive results obtained for this biocatalytic process, ToMO recombinant system was used to synthesize novel antioxidant catechols using several low-added-value aromatic compounds as starting material. The starting molecules were chosen on the basis of their similarity with the natural aromatic substrates of ToMO and were docked into the active site of ToMOA, where the hydroxylation reaction takes place. The starting compounds that have been the focus of our attention are 2-phenoxyethanol, 2,3 dihydrobenzofuran, phtalan and indanol. The different catechols obtained with the use of ToMO or its mutant E103G/F176A were purified and identified by mass spectrometry and NMR analysis. The antioxidant capacity of the dihydroxylated derivatives of 2-phenoxyethanol, 2,3-dihydrobenzofuran and 2-indanol was qualitatively evaluated with radical scavenging assays using DPPH (α, α-diphenyl-β-picrylhydrazyl). In all cases the new catechols produced were able to oxidize the stable DPPH radical. In conclusion, the ability of ToMO to produce catechols confirms the versatility of this multicomponent enzymatic system and the possibility to produce new potential antioxidants not found in nature.

P104 SOLVENT EMISSIONS CONTROLLED BY BIOREACTOR Marcello Civilini*1, Giovanni Cortella2 1

Dipartimento di Scienze degli Alimenti, University of Udine, Udine, Italy; 2Dipartimento di Ingegneria Elettrica, Gestionale e Meccanica, University of Udine, Udine, Italy

Abstract A biofilter with a working volume of 25 m3 and a max capacity to treat 16000 m3/h of exhausted air was built two years ago to treat emissions containing solvents used during the varnishing of unfinished items of the wood-working industry. The biofilter was fed with polluted air from the drying zone emissions, because their relatively steady and high concentration were advantageous to our studies After some months for optimization, the biofiltration of polluted emissions was monitored during different seasons and the behaviour was correlated with temperature and the industrial painting procedures. The biofilter treated 4000-6000 m3/h of polluted air; the resulting Empty Bed Residence Time ranged between 10 and 70 s. Organic loads of VOCs during the observed period were between 10 and 110 g C m-3h-1. The performance of the biofilter showed to be influenced by different conditions. To maintain emissions below the permissible threshold (100 mg C Nm-3), particular managing procedures were adopted especially during low temperature (first) periods. During the process, the microbial heterotrophic viable counts were performed from samples of the biofilter organic matter and internal recirculation water used to adjust the moisture content. The relative numbers of single VOC degraders were tracked and their value showed to be suitable even in the presence of VOC concentrations or air temperature harsh conditions. Environmental and safety rules require continuous monitoring to determine the yield. A thorough control of emissions from plants and their reduction by filtration requires a continuous or very frequent measurement of VOC concentration. Various methods for VOC detection can be applied, with different ability to deal with quick variations in composition and concentration which can be encountered and actually lead to a misleading interpretation of the results. For the purpose of comparing some of such methods, for about two years the behaviour of the biofilter pilot plant has been monitored by on line Photo-Ionization Detector (PID) analyzer and Gas Chromatography analyzer with Flame Ionization Detector (FID), and by discontinuous sampling with sorbent tubes. The comparison of 27 FID measurements and 34 PID measurements with the corresponding analysis of samples resulted in the evaluation of response factors for various mixture. A marked unpredictability of such factors showed that on line measurements fit for the only purpose of a rough control of the operation, while discontinuous sampling is required for an accurate evaluation of the biofilter performance.

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Session 8/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S141-S150

S164

Environmental Engineering and Management Journal

March 2012, Vol.11, No. 3, Supplement, S165-170

http://omicron.ch.tuiasi.ro/EEMJ/

“Gheorghe Asachi” Technical University of Iasi, Romania

Session 9 FP7 FUNDED PROJECTS IN THE AREA OF ENVIRONMENTAL BIOTECHNOLOGY BIOTECHNOLOGY IN FP7 AND HORIZON 2020 Alfredo Aguilar, Danuta Cichocka Unit Biotechnologies, Directorate Food, Agriculture and Biotechnology, Directorate General for Research and Innovation, European Commission, B-1049, Brussels, Belgium

Abstract Biotechnology plays an important role in addressing social, environmental and economic challenges and it is recognised as a key enabling technology in the transition to a green, low carbon and resource-efficient economy. Biotechnologies for non-health applications have received a considerable attention in the European Union's 7th Framework Programme for Research, Technological Development and Demonstration Activities (FP7). “Food, Agriculture and Fisheries and Biotechnology”, also referred as a European Knowledge-Based BioEconomy (KBBE), is one of 10 thematic areas in the FP7 Cooperation programme, and to date it has supported transnational and international projects with about 500 Million Euro on different sectors of biotechnology. In mid 2012 the final FP7 calls will be opened. They will aim at bridging the gap between FP7 and new programme for research and innovation, Horizon 2020. Running from 2014 to 2020 with a proposed budget of €80 billion budget, Horizon 2020 will combine research and innovation funding currently provided through FP7, the innovation related activities of the Competitiveness and Innovation Framework Programme (CIP) and the European Institute of Innovation and Technology (EIT). Horizon 2020 offers a coherent package of support with simplified funding rules for the best research that provides major business opportunities. Its three priorities are 1) Excellent Research 2) Industrial Leadership and 3) Societal Challenges. Biotechnology is listed among technologies which are fundamental to our economy (Key Enabling Technologies - KETs) and it will be financed under Industrial Leadership priority. Biotechnology will be also supported under the third priority, Societal Challenges. Horizon 2020 builds on the thematic research from FP7 and reflects the goals of the Europe 2020 strategy. “Food security, sustainable agriculture, marine and maritime resources and the bioeconomy” are among major concerns shared by European citizens and therefore this area is among six priority challenges selected for funding.

BACSIN FP7 (TARGETED APPLICATIONS OF BACTERIAL STRAINS FOR POLLUTANT BIOREMEDIATION) J.R. van der Meer*1,2, D. Springael2, V.de Lorenzo2, M. Fernandez2, J.L. Ramos2, H. Smid2, H. Heipieper2, E. Luye2, V. Soljan2, V. Brenner2, J.H.L. Leveau2, A. Farewell2, S. Håkansson2, W. Roling2, K. Timmis2, T. Lettieri2, D. Pieper2 1

Department of Fundamental Microbiology, University of Lausanne, Switzerland 2 www.bacsin.org; e-mail: [email protected]

Abstract Application of preselected bacterial strains for targeted remediation of environmental pollutants has been largely frustrated by the lack of reproducible success. Most likely, this was due to insufficient knowledge on the behaviour of various bacterial strains in the environment, and, in particular, the influence of diverse stresses on the catabolic activity of the bacteria. To overcome this knowledge gap, the BACSIN consortium started an ambitious program to study the catabolic and stress behaviour of five selected bacterial strains with different catabolic potential and environmental application, in order the find the key factors which determine survival and activity. The strains which the program focuses on include Pseudomonas putida, Desulfitobacterium hafniensis, Sphingomonas wittichi, Arthrobacter chlorophenolicus and Alcanivorax borkumensis. The program combines research at different levels of complexity: from genome-wide transcriptome analysis, genomics and genetics on pure isolates to their applications in the environment, in soils, on plant roots, plant leaves, or in marine systems. The consortium also addresses specific questions of formulation of biodegradation strains for optimal application under field condition. We will show various selected examples of our work in the different areas.

Session 9/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S165-170

MOLECULAR APPROACHES AND METAGENOMIC INVESTIGATIONS FOR OPTIMIZING CLEAN-UP OF PAH CONTAMINATED SITES (MAGICPAH) Dietmar H. Pieper Microbial Interactions and Processes Research Group, Helmholtz Centre for Infection Research and the MAGICPAH consortium

Abstract MAGICPAH aims to explore, understand and exploit the catalytic activities of microbial communities involved in the degradation of persistent PAHs, where the main objectives are: (i) to generate a knowledge base of the microbial aerobic catabolome, (ii) to develop concepts to quantify in situ degradation of PAHs through stable isotope analyses, (iii) to identify key players and key reactions involved in anaerobic PAH metabolism, (iv) to achieve a detailed understanding on key processes for PAH metabolism in marine and composting environments, (v) to develop methods to predict the ultimate fate and the kinetics of aerobic degradation of PAH under different conditions of bioavailability, (vi) to isolate and sequence novel key players in PAH metabolism to understand the genomic basis of niche specificities, (vii) to investigate the potential synergistic links between environmental biotechnology and medical biotechnology, (viii) to integrate detailed catabolome and reactome information to re-construct metabolic networks, (ix) to apply gathered information to improve the treatment performance of PAH contaminated sites. In the presentation we will give an overview on a subset of recent results. Extensive samplings were performed to obtain impacted materials and an Illumina-based barcoded deep sequencing strategy applied, allowing a detailed survey of community compositions and shifts. To obtain a reliable frame of reactions involved in PAH degradation, curated databases on key catabolic enzyme families were constructed, and used to design PCR primers and microarray probes to survey the spread and activity of respective genes in contaminated environments and to obtain a reliable annotation of genes observed in metagenomic and metatranscriptomic surveys. To be able to construct upgraded databases on anaerobic key steps, key steps of the anaerobic naphthalene degradation pathway were elucidated. We also exploited the enormous biodiversity through the mining for novel activities from metagenomic libraries, where active clones were now fully sequenced. To understand the distribution of aromatic degradation networks, (meta-) genomic and shotgun (meta-) proteomics were used to deduce previously unrecognized correlations between systems performance and phylogenetic, genomic and proteomic blueprints. As a step further in understanding community behaviour, mRNA sequencing has been successfully applied to obtain a metatranscriptomic overview of contaminated groundwater samples. The genome of Cycloclasticus zancles a key PAH degrader in marine environments was sequenced revealing a large repertoire of genetic determinants for the uptake of mineral nutrients that are limiting in marine environments and the strong biodegradation potential was evidenced by the exceptional multiplicity of key aromatic degradation genes.

UNRAVELLING AND EXPLOITING MEDITERRANEAN SEA MICROBIAL DIVERSITY AND ECOLOGY FOR XENOBIOTICS’ AND POLLUTANTS’ CLEAN-UP THROUGH THE FP7 EU PROJECT ULIXES D. Daffonchio1, A. Cherif2, H.I. Malkawi3, M.M. Yakimov4, Y.R. Abdel-Fattah5, M. Blaghen6, P.N. Golyshin7, M. Ferrer8, N. Kalogerakis9, N. Boon10, M. Magagnini11, F. Fava12 1

Department of Food Science and Microbiology, University of Milan, Milan, Italy; 2Laboratory of Microorganisms and Active Biomolecules, University of Tunis El Manar, Tunis, Tunisia; 3Department of Biological Sciences, Yarmouk University, Irbid, Jordan; 4Institute for Coastal Marine Environment, Consiglio Nazionale delle Ricerche, Messina, Italy; 5Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research & Technology Applications, Alexandria, Egypt; 6Laboratory of Microbiology, Biotechnology and Environment, University Hassan II - Ain Chock, Casablanca, Morocco; 7School of Biological Sciences, Bangor University, Bangor, UK; 8Institute of Catalysis, Consejo Superior de Investigaciones Científicas, Madrid, Spain; 9Department of Environmental Engineering Technical University of Crete, Chania, Greece; 10Laboratory of Microbial Ecology and Technology, Ghent University, Ghent, Belgium; 11EcoTechSystems L.t.d, Ancona, Italy; 12Department of Civil, Environmental and Materials Engineering, University of Bologna, Bologna, Italy

Abstract The pollution of a relatively restricted basin like the Mediterranean Sea is associated to socio-economic problems. Besides the effects on biodiversity and wild life, marine oil spill disasters profoundly impact the economy of the affected areas due to reduction of tourism, reduction of fishing activities and the consequent loss of jobs and heritages. The increased number of polluted coastal and marine sites is now demanding careful strategies for remediation and clean-up. The European Commission has produced numerous efforts for developing research on novel strategies for the environmental protection of the Mediterranean Sea including the funding of research project devoted to the exploitation of microbial diversity for the developing novel bioremediation strategies. The EU-funded research project ULIXES aims to unravel, categorize, catalogue, exploit and manage the microbial diversity available in the Mediterranean Sea for addressing bioremediation of polluted marine sites. The idea behind ULIXES is that the multitude of diverse environmental niches of the Mediterranean Sea contains a huge range of microorganisms and their components (e.g. catabolic enzymes) or products (e.g. biosurfactant) that can be exploited in pollutant- and site-tailored bioremediation approaches. ULIXES intends to provide the proof of concept that it is possible to establish and exploit for bioremediation site-specific collections of microbial strains, mixed microbial cultures, enzymes, biosurfactants and other microbial products. These biotechnological resources are mined by using approaches based on isolation of culturable microorganisms as well as by extensively applying advanced novel ‘meta-omics’ technologies. Three pollutant classes recognized worldwide as environmental priorities are considered: petroleum hydrocarbons, chlorinated compounds and heavy metals. Through the effort of twelve European and Southern Mediterranean partner laboratories, a large set of polluted environmental matrices from sites located all over the Mediterranean Sea are explored, including seashore sands, lagoon sediments, deep sea sediments polluted by heavy oil hydrocarbons at oil tanker shipwreck sites, hypersaline waters and sediments from polluted salty coastal lakes and natural deep hypersaline anoxic submarine basins and mud volcanoes where hydrocarbon seepages occur. The mined collections of microbial biotechnological products are exploited for development of novel improved bioremediation processes whose effectiveness is proved by ex situ and in situ field bioremediation trials. A careful dissemination action is pursued to assure capillary information of the ULIXES results and products to stakeholders and SMEs operating in the sector of bioremediation.

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Session 9 - FP7 funded projects in the area of environmental biotechnology

MINOTAURUS: MICROORGANISMS' IMMOBILIZATION: NOVEL TECHNIQUES AND APPROACHES FOR UPGRADED REMEDIATION OF UNDERGROUND – AND WASTEWATER AND SOILS P.F.X. Corvini, R. Hochstrat, T. Wintgens Institute for Ecopreneurship, School of Lifee Sciences, University of Applied Sciences and Arts Northwestern Switzerland

Abstract MINOTAURUS aims to deliver innovative bio-processes (bioaugmentation, enzyme technology, rhizoremediation with halophytes, and bioelectrochemical remediation), which are all based on the concept of IMMOBILIZATION OF BIOCATALYSTS (microorganisms and enzymes), to eliminate emerging and classic organic pollutants. The immobilization-based technologies are/will be applied to engineered (ex-situ) and natural systems (in situ) for the bioremediation of groundwater, wastewater, and soil. The selection and adaptation of modern physico-chemical, biological, and ecotoxicological monitoring tools combined to a rational understanding of engineering and enzymology/microbial physiology aspects is a pertinent approach to open the black-box of our technologies. The reliable processmonitoring constitutes a solid basis to develop and refine our biodegradation kinetics models, which will be the mean to improve the predictability of performances to be achieved with our technologies. A key strength of MINOTAURUS is the possibility of direct implementation of our technologies at five EU reference sites that are confronted with pollutants (two technologies will be tested on-site during the first year). We will deliver not only a set of tools, techniques and processes which will enhance the ability of our communities to respond to the challenges of organic pollutants but also frameworks for structuring and making evidence-based decisions for the most sustainable and appropriate bioremediation measures. MINOTAURUS consortium consists of sixteen partners from eight European and Europe-associated countries. Nine research & education institutions, five SMEs covering the whole chain of our bioremediation approaches (production/monitoring of biocatalysts, bioremediation, and engineering), one large end-user installing wastewater treatment plants, and one environmental agency will work together with the support of an advisory board mainly consisting of environmental decision-makers.

BIOTREATMENT OF DRINKING WATER RESOURCES POLLUTED BY PESTICIDES, PHARMACEUTICALS AND OTHER MICROPOLLUTANTS (BIOTREAT) J. Aamand 1, B.F. Smets 2, D. Springael 3, H.E. Kohler 4, N. Boon 5, T. Ternes 6, A. Lapanje 7, M. van Bemmel 8, M. van Wambeke 9, R. Dalgaard 10, E. Walravens 11, C.N. Albers 1 1

Geological Survey of Denmark and Greenland (GEUS), Denmark; 2Technical University of Denmark, Denmark; 3Katholieke Universiteit Leuven, Belgium; 4 EAWAG, Switzerland; 5Gent University, Belgium; 6The German Federal Institute of Hydrology (BfG), Germany; 7Institute of Physical Biology, Slovenia; 8 BIOCLEAR B.V., The Netherlands; 9AVECOM N.V., Belgium; 10 2.-0 LCA Consultants ApS, Denmark; 11Vlaamse Maatschappij Voor Watervoorziening, Belgium

Abstract BIOTREAT is a new EU project initiated 1st January 2011 aiming at developing new technologies for bioremediation of drinking water resources contaminated with micropollutants such as pesticides and pharmaceuticals. Micropollutants are organic pollutants that occur in contaminated water at extremely low concentrations ranging from 0.1 to10 μg/l, but still above the EU limit values. The basis of the proposed technologies is bioaugmentation, which in the present context is the introduction of specific degrading microorganisms or microbial consortia into existing sand filters at waterworks, mobile biofilters placed close to groundwater abstraction wells, sand barriers between surface waters and abstraction wells, and subterranean protective barriers established to prevent micropollutants from entering into aquifers. BIOTREAT brings together six research institutions and four SMEs to develop much-needed water treatment biotechnologies. These biotechnologies will be developed into prototype biofilter systems ready for subsequent commercialisation. The biofilters will contain nonpathogenic pollutant-degrading bacteria, with the bacteria being immobilised on specific carriers to ensure their prolonged survival and sustained degradative activity. Two complementary biotreatment strategies will be followed, one based on metabolic processes whereby the bacteria completely mineralise specific micropollutants and the other based on cometabolic degradation utilising the ability of methane- and ammonium-oxidising bacteria to unspecifically degrade a range of micropollutants for which specific degraders are not yet available. The biofilter systems will be carefully validated through cost-benefit analysis and environmental life cycle assessment. In addition to bringing considerable advances to water treatment biotechnology, the main outcome of BIOTREAT will thus be prototype biofilter systems (metabolic and cometabolic) ready for commercialisation in a number of highly relevant water treatment scenarios. As an example of research carried out until know sand columns have been established in the laboratory mimicking the conditions of sand filters at waterworks. The columns were packed with quarts sand and added a culture of Aminobacter strain MSH1. This strain has previously been shown to mineralise the pesticide residue 2.6-dichlorobenzamide (BAM) even at μg/l concentrations. The columns were placed at 10 °C and fed BAM (2 μg/l) polluted drinking water. Preliminary results showed 65% reduction of the BAM concentration at a hydraulic retention time of 20 minutes only. These results demonstrate that Aminobacter sp. MSH1 is a promising candidate for use in bioremediation processes aimed at cleaning natural waters polluted with BAM at the low concentrations typically found in groundwater. Acknowledgements BIOTREAT is financed by the Food, Agriculture and Fisheries, and Biotechnology Work Programme (grant. KBBE-2010-4-266039BIOTREAT).

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Session 9/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S165-170

THE GREENLAND PROJECT: GENTLE REMEDIATION OF TRACE ELEMENT CONTAMINATED LAND Jaco Vangronsveld*1, Markus Puschenreiter2, Jurate Kumpiene3, Petra Kidd4, Michel Mench5, Valerie Bert6, Andrew Cundy7, Giancarlo Renella8, Wolfgang Friesl-Hanl9, Grzegorz Siebielec10, Rolf Herzig11, Ingo Müller12, Jannis Dimitriou13, Xose Quiroga Troncosa14, Ryszard Bajorek15, Patrick Lemaitre16, Anne Serani Loppinet17 1

Hasselt University, Diepenbeek, Belgium; 2University of Natural Resources and Life Sciences, Vienna–BOKU, Vienna, Austria (project coordination); Luleå Tekniska Universitet, Luleå, Sweden; 4Consejo Superior de Investigaciones Científicas, Santiago de Compostela, Spain; 5Institut National de la Recherche Agronomique (INRA), Bordeaux, France; 6Institut National de l’Environnement Industriel et des Risques, Verneuil en Halatte, France ; 7 University of Brighton, Brighton, UK; 8University of Florence, Florence, Italy; 9Austrian Institute of Technology, Wien, Austria; 10Instytut Uprawy Nawożenia I Gleboznawstwa–Państwowy, Pulawy, Poland; 11Phytotech-Foundation, Bern, Switzerland,; 12Sächsisches Landesamt für Umwelt, Landwirtschaft und Geologie, Dresden, Germany; 13Sveriges Lantbruksuniversitet, Uppsala, Sweden; 14Tratamientos Ecológicos del Noroeste SL, Touro, Spain; 15ATON, Wroclaw, Poland; 16Innoveox, Paris, France ; 17CNRS-ICMCB, Pessac, France 3

Abstract Gentle remediation options (GRO) include various and in general plant-based approaches to remediate trace element contaminated soils at low cost and without significant negative effects for the environment. Although GRO comprise very innovative and efficient technologies, they are still not widely used as practical site solution due to several reasons of hindrance. Greenland will solve the remaining problems and make GRO ready for practical application. Contamination of soils with trace elements is worldwide still one of the major environmental problems. Conventional technologies for soil remediation are usually very expensive and may negatively affect or destroy soil structure and functions. Gentle soil remediation options, however, comprise environmentally friendly technologies that have little or no negative impact on the soil. The main technologies are phytoextraction, in situ immobilization and assisted phytostabilization. Although major progress has been achieved on the lab scale, success stories obtained on the field are still limited, in particular for phytoextraction. Also, the issue of valorization of the potentially contaminated plant biomass has insufficiently been addressed so far. Furthermore, further development is needed regarding the adequate determination of endpoints of GRO. Finally, the application of GRP as practical site solution may be hindered by legal frameworks and by insufficient knowledge of the decision makers. Therefore, an EU-FP7 (KBBE-2010-4) has been launched on January 1 2011 to address these issues and to make GRO ready for use as practical site solution. The project includes the following work packages:  Sustainable management adapted to trace element contaminated soils and deployment of GRO at field scale (WP1)  Valorisation of plant biomass produced on trace element contaminated sites (WP2)  Harmonization of methods to assess the bioavailability of trace elements and development of a tool set to monitor the sustainability of GRO (WP3)  Improving GRO through plant selection and modifications in soil trace element bioavailability (WP4)  Appraisal of current GRO practice, and development of implementation guidance and decision support frameworks (WP5)

PROSPARE: AN EUROPEAN PROJECT FOR THE VALORIZATION OF THE LEFTOVERS OF POULTRY INDUSTRY Arnaldo Dossena1, Kathy Elst2, Olga Koroleva3, Stanislas Lublinsky4, Giulio Mongia5, Yuriy Mukhin6, Vladimir Popov3, Stefano Sforza1, Luigi Sperati7, Viktor Volik8 1 Department of Organic and Industrial Chemistry, University of Parma, V.le G.P. Usberti 17/a, 43100 Parma, Italy; 2Flemish Institute for Technological Research, Separation and Conversion Technology, VITO Boeretang 200,B-2400 Mol, Belgium; 3A.N. Bakh Institute of Biochemistry of Russian Academy of Sciences, Leninsky prospekt 33, 119071 Moscow, Russia; 4JSC Mobitek M, Institut 6, 249010 Borovsk, Kaluga Region, Russia; 5CORE Biotech, Avenue Franklin D.Roosevelt 146, 1050 Brussels, Belgium; 6Symbol Ltd., Rszavki PO, VNIIPP 1, 141552 Solnechnogorsk, Russia; 7Agricola Tre Valli, Via Valpantena, 37142 Quinto di Valpantena (Verona), Italy; 8All-Russia Research Institute for Poultry Processing, Rszavki PO, VNIIPP 1, 141552 Solnechnogorsk, Russia

Abstract Food industry leftovers constitute an enormous cost and an unnecessary waste of food-grade material. In the production of meat for human consumption, up to 50% of the animal weight is discharged. In EU only, the total leftover mass from the meat industry amounts to 15 million tons, formally residues, but actually potential raw materials rich in proteins and lipids. Although this potentiality, most of this material is incinerated-only 22% is converted into feed and a bare 3% becomes food. The traditional rendering technologies, based on prolonged heating of the leftovers, ensure microbial safety and increase digestibility, but use enormous amounts of energy and induce the degradation of high biological value components. A new technological/biotechnological platform has been developed in the framework of PROSPARE (PROgress in Saving Proteins and Recovery of Energy), a joint European-Russian research project, financed under the 7th Framework Programme, aimed at the recovery of poultry industry leftovers into valuable food and feed end products, by applying new advanced enzymatic technologies for the obtainement of hydrolized meat and feather proteins of high nutritional value. In particular, by using a novel biocatalitic approach, unmarketable poultry secondary resources (feathers, bones, trimmings, etc.) were converted into added value peptide hydrolyzates, with programmable nutritional properties, and biodiesel. These hydrolyzates have been characterized in order to certificate their safety, by assessing the absence of microbial (lysteria, escherichia) as well as chemical (mycotoxins, biogenic amines, heavy metals) contaminants. Their content in free and total amino acids, the molecular weight distribution of the peptide fractions and the sequences of the main peptides present in the mixture were also determined. These parameters, together with the digestibility, allowed to tailor the nutritional properties of the mixtures obtained. The mixtures have also been characterized in order to assess their functional properties, such as antioxidant, antihypertensive, antimicrobial and prebiotic. Finally, new food and feed products were produced by using the protein hydrolizate mixtures as ingredients. The interdisciplinary character of the investigation is demonstrated by the parallel application of biotechnological, chemical, biophysical, physico-chemical, biochemical, immunological and toxicological studies, as well as the industrial trials for the evaluation of proposed technologies. The outcome of the PROSPARE project is likely to generate a significant technological breakthrough of animal by-products treatment, with an impact also on the current Regulatory Framework both in EU and Russian Federation.

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Session 9 - FP7 funded projects in the area of environmental biotechnology

SUSTAINABLE PRODUCTION OF BIOLOGICALLY ACTIVE MOLECULES OF MARINE BASED ORIGIN (BAMMBO) Daniel J. Walsh1, Olivier Thomas2, Lara Sette3, Edward McHugh4, Eric Causse5, Luis M. Botana6a, Gumersindo Feijoo6b,Maite Moreira6b, Rui Pedrosa7, Clayton Jeffryes8, Spiros N. Agathos8, Annick Verween9, Wim Vyverman9, Ivan Laptev10, Sergie Sineoky10, Fabio Ledda11, Roberto Pronzato11, Siobhan Moane1, Patrick Murray12 1

Department of Applied Science, Limerick Institute of Technology, Limerick, Ireland; 2Maître de Conférences, Métabolomique Marine Environnementale, Institut de Chimie de Nice, CNRS, Nice, France ; 3UNIVERSIDADE ESTADUAL DE CAMPINAS (UNICAMP) / Researcher of Division of Microbial Resources–CPQBA, Brazil ; 4Alage Health Ltd., Rooaunmore Lodge, Rooaunmore, Claregalway, Co.Galway, Ireland; 5GREENSEA, Parc Scientifique et Environnemental, Mèze, France ; 6aDept Farmacología, Fac. Veterinaria, University of Santiago de Compostela, Lugo, Spain ; 6bDepartment of Chemical Engineering, School of Engineering, University of Santiago de Compostela, Santiago de Compostela, Spain ; 7Grupo De Investigação Em Recursos Marinhos (Girm), Escola Superior De Turismo E Tecnologia Do Mar (PENICHE), Santuário Nossa Senhora dos Remédios, Peniche, Portugal; 8 Bioengineering Group (GEBI), Earth and Life Institute (ELI), University of Louvain (UCL), Louvain-la-Neuve, Belgium; 9Gent University, Department of Biology, Section Protistology and Aquatic Ecology, Gent, Belgium 10 Genetika, Federal State Unitary Enterprise, State Scientific Research Institute of Genetics and Breeding of Industrial Microorganisms, Moscow, Russia; 11 Dipartimento per lo Studio del Territorio e sue Risorse, Università degli Studi di Genova, Genova, Italy; 12Shannon Applied Biotechnology Centre, Limerick, Institute of Technology, Limerick, Ireland

Abstract Innovation is the most important engine of growth and jobs in knowledge-based bio-economies. The scope of BAMMBO (Biologically Active Molecules of Marine Based Origin) is ambitious. This was intentional. The BAMMBO project commenced in March 2011 with the intent to screen and identify targeted marine organisms (e.g. bacteria, fungi, sponges, microalgae, macroalgae and yeasts) from diverse global locations for their potential as sustainable producers of high–added value molecules (HVAB’s). BAMMBO is set to provide innovative solutions to overcome existing bottle-necks associated with culturing marine organisms in order to sustainably produce high yields of value-added products for use in the pharmaceutical, cosmetic and industrial sectors. Our project applies conventional and nonconventional methods for the analysis, extraction, purification and enrichment of targeted bioactive compounds. A detailed life cycle analysis of the production pathways developed in the project will be undertaken to fully evaluate the sustainability of production of biologically active products from marine organisms. BAMMBO will exploit knowledge and technologies developed during the project and effectively manage their transfer to relevant stakeholders in industry and the research community, as well as to policy-makers. BAMMBO brings together a multidisciplinary consortium of 11 specialist Research and SME partners representing 8 countries. Partners from ICPC countries Russia and Brazil provide access to White Sea and Antarctic Coasts, and those from the EU member states access to Mediterranean and Atlantic coasts. In adhering to the European Strategy for Marine and Maritime Research this three year project encourages capacity-building, integration and synergies across relevant marine sectors. Innovative technologies being developed in the project will be demonstrated with the involvement of industry partners, and the results will be of interest not only to companies directly involved in the marine sector, but to other large scale industry players such as pharmaceutical companies with interest in added-value bioactive compounds. The presentation to be delivered will provide an overview of the approach being taken by BAMMBO to contribute to the development of a knowledge-based bio-economy through the synergistic collaboration of Research and SME partners.

ECOBIOCAP ECOEFFICIENT BIODEGRADABLE COMPOSITE ADVANCED PACKAGING Nathalie Gontard INRA Research Director, Montpellier, France

Abstract EcoBioCAP will provide the EU food industry with customizable, ecoefficient, biodegradable packaging solutions with direct benefits both for the environment and EU consumers in terms of food quality and safety. This next-generation packaging will be developed using advanced composite structures based on constituents1 (biopolyesters, fibres, proteins, polyphenolic compounds, bioadhesives and highperformance bio-additives) derived from food industry (oil, dairy, cereal and beer) by-products only and by applying innovative processing strategies (blends and multilayers at different scales) to enable customisation of the packaging’s properties to fit the functional, cost, safety and environmental impact requirements of the targeted fresh perishable food (fruit and vegetables, cheese and ready-to-eat meals). Demonstration activities with SMEs and industrial partners will enable the EcoBioCAP technology to be optimised in terms stability, safety, environmental impact and cost- effectiveness before full exploitation. The development of a decision support system for use by the whole packaging chain will make the EcoBioCAP technology accessible to all stakeholders. Extensive outreach activities will not only disseminate the project results to the scientific community but also ensure that consumers and end-users are informed of the usage conditions and benefits of such bio-degradable packaging and how it should be disposed of.

1

The definition of the term” packaging constituent” used in this document is the following according to EN 13193: “constituents are parts from which packaging are made and which cannot be separated by hand or by using simple physical means”. From “Essential requirements for packaging in Europe: a practical guide to using CEN standards”. 2005. Ed. EUROPEN (the European Organization for Packaging and the Environment), Brussels, Be.

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Session 9/Environmental Engineering and Management Journal 11 (2012), 3, Supplement, S165-170

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Environmental Engineering and Management Journal

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http://www.epa.gov/Sustainability/pdfs/Biomass%20 Conversion.pdf EC Directive, (2000), Directive 2000/76/EC of the European Parliament and of the Council of 4 December 2000, on the incineration of waste, Annex V, Official Journal of the European Communities, L 332/91, 28.12.2000, Brussels. GD, (2004), Governmental Decision no. 1076/2004 surnamed SEA Governmental Decision, regarding the procedure for strategic environmental impact assessment for plans or programs, published in Romanian Official Monitor, part I, no. 707 from 5th of August, 2004. Web references The full URL should be given in text as a citation, if no other data are known. If the authors, year, title of the documents are known and the reference is taken from a website, the URL address has to be mentioned after these data. Burja C., Burja V., (2008), Adapting the Romanian rural economy to the European agricultural policy from the perspective of sustainable development, MPRA, Munich Personal RePEc Archive, On line at: http://mpra.ub.unimuenchen.de/7989/1/MPRA_paper_7989.pdf Web references must not be listed separately, after the reference list. All references must be provided in English with a specification of original language in round brackets. Citation in text Please ensure that every reference cited in the text is also present in the reference list (and vice versa). Do not cite references in the abstract. Unpublished results, personal communications as well as URL addresses are not recommended in the reference list, but may be mentioned in the text. Citation of a reference as "in press" implies that the item has been accepted for publication. Papers which have been accepted for publication should be included in the list of references with the name of the journal and the specification "in press". Reference style Text: All citations in the text may be made directly (or parenthetically) and should refer to: - single author: the author's name (without initials, unless there is ambiguity) and the year of publication: “as previously demonstrated (Smith, 2007)”; “as Smith (2007) demonstrated” - two authors: both authors' names and the year of publication: (Arnold and Sebastian, 2008; Smith and Hansel, 2006; Stern and Lars, 2009) - three or more authors: first author's name followed by "et al." and the year of publication: “As has recently been shown (Werner et al., 2005)…”, “Kramer et al. (2000) have recently shown ...." Citations of groups of references should be listed first alphabetically, then chronologically.

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DEPARTMENT OF ENVIRONMENTAL ENGINEERING AND MANAGEMENT The Department of Environmental Engineering and Management of the Gheorghe Asachi Technical University of Iasi, Romania - Faculty of Chemical Engineering and Environmental Protection offers specialization studies (diploma of engineer, M.Sc. and Ph.D.) in the field of Environmental Engineering and Management, studies that are highly requested by students and society. Since 1998, the subjects of studies for the diploma of engineer and M.Sc. programs are organized based on the transferable credit points system. The staff of the Department includes 2 Full Professors, 5 Associate Professors, 6 Lecturers and 7 Teaching Assistants, 2 Researchers and 3 Technicians. Staff members from other Faculties (Hydrotechnics and Biology) and from the Environmental Protection Agencies or Research Institutes are also co-operating within the academic curricula for specific subjects of study. Teaching and research activities in the Department of Environmental Engineering and Management  Environmental Engineering studies: Offering diploma of engineer after 4 years of study, with specialization on Engineering and Environmental Protection in Industry.  Master of Science Programs in Environmental Management Offering M.Sc. diploma in Environmental Management as well as in Environmental Management and Sustainable Energy  Ph.D. Programs in the fields of Chemical engineering (Optimization of Chemical/Environmental Processes, Environmental Impact Assessment), Chemistry, and Environmental Engineering. The major research activities develop within the Environmental Engineering and Impact Assessment Research Center, acknowledged by the Ministry of Education and Research, as a part of the Department, following the trends presented below:  Environmental impact assessment, environmental auditing,  Environmental risk assessment and management  Environmental management and the implementation of EMS or integrated systems (quality-environment, quality-safetyenvironment)  Wastewater treatment processes (conventional and advanced treatment processes)  Water treatment for drinking or industrial purposes  Technologies for treatment of gaseous fluxes  Material science and engineering  Automatic sampling, control and monitoring water and air quality (including software and implementation techniques)  Environmental biotechnology  Chemical and biological process engineering for environmental protection and control (including modeling, simulation, optimization)  Integrated pollution prevention and control, cleaner production, products and services, sustainable development  Environmental chemistry and analytic control  Environmental education The Department of Environmental Engineering and Management organizes the International Conference on Environmental Engineering and Management in Iasi, at two years intervals. The first conference (ICEEM01) was held in 2002, followed by ICEEM02 (2004), ICEEM03 (2006), ICEEM04 (2007), ICEEM05 (2009), and ICEEM06 (2011). Also, since 2002, the Department edits the Environmental Engineering and Management Journal, a quarterly peer-reviewed international publication. Starting with 2006, the journal appears in 6 issues per year, at two months intervals. In 2007, EEMJ has been included in Thomson ISI Master Journal List, Science Citation Index Expanded (SCIE), Web of Science, Chemical Abstracts as well as other international databases. Since 2010 the journal appears monthly. Scientific and Educational Programs, International Co-operation Within the Technical University of Iasi, in close cooperation with the Department of Environmental Engineering and Management acts the InterMEDIU Department for Information, Research and Consultancy for the Civil Society. InterMEDIU is integrated in the international network of Science Shops and was founded in 1999 following the Dutch science shop model (an interface between the university and civil society), based on the agreement of co-operation between the Technical University of Iasi and University of Groningen, The Netherlands. Since July 2001, InterMEDIU Department is officially recognised as the department entitled to organize and co-ordinate the Open and Distance Learning programs of the Faculty of Chemical Engineering. The members of the staff and students of the Department of Environmental Engineering and Management have founded in 1997, the NGO Academic Organization for Environmental Engineering and Sustainable Development (AOEESD-OAIMDD), which develops activities in the field of environmental protection, environmental education and awareness programs, promotion of ecological products, even within the Publishing House EcoZONE. Co-operation of the Department of Environmental Engineering and Management and the other 2 organizations mentioned previously has been achieved with various academic and research institutes, by means of bilateral agreements or for specific project co-operation. Gheorghe Asachi Technical University of Iasi Faculty of Chemical Engineering and Environmental Protection Department of Environmental Engineering and Management 73 Prof.Dr.docent Dimitrie Mangeron Street, 700050, IASI, ROMANIA Phone/Fax: +40-232-271759 http://www.ch.tuiasi.ro http://omicron.ch.tuiasi.ro/EEMJ/ http://www.ecozone.ro