Recent Advances in Bioenergy Research

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Recent Advances in Bioenergy Research Volume III

Edited by SACHIN KUMAR, A.K. SARMA, S.K. TYAGI, Y.K. YADAV Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala, India i

ISBN 978-81-927097-2-7 © Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala-2014 Electronic version published by SSS-NIRE ALL RIGHTS RESERVED

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CONTENTS Preface

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Contributors

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Part-I: Biomass and Energy Management

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Thermogravimetric characterization of wood stalks as gasification and pyrolysis feedstock Rakesh Punia, Sachin Kumar Abstract 1.1 Introduction 1.2 Materials and Methodology 1.3 Results & Discussion 1.4 Conclusion References

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Assessment of Solid Waste Management and Energy Recovery from Waste Materials in Lucknow Zoo: A Case Study Vinayak V. Pathak, Richa Kothari, A.K. Chopra, Lhaihoichong Singson Abstract 2.1 Introduction 2.2 Materials and Methods 2.3 Results and Discussion 2.4 Conclusion References

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A bioprospection of euphorbia cotinifolia for biofuel: chromatography study Punam Puri, Amita Mahajan, Anjana Bhatia, Navjot Kaur Abstract 3.1 Introduction 3.2 Objectives 3.3 Methodology 3.4 Results and discussion 3.5 Discussion 3.6 Conclusion References

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Cost effective electrical power generation in punjab using agricutural biomass Suman Abstract 4.1 Introduction 4.2 Status of Bio-energy Resources in Punjab iii

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31 31 32

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4.3 Power Consumption in Punjab 4.4 Existing Technologies for Biomass Conversion 4.5 Biomass as a Coal Substitute 4.6 Environmental Criteria 4.7 Conclusion References

34 36 37 37 38 39

Development of quality testing methodologies of fuel briquettes Madhurjya Saikia, Bichitra Bikash Abstract 5.1 Introduction 5.2 Parameters of Quality Assessment 5.3 Parameters of Combustion Characteristics of Briquettes 5.4 Conclusion References

40 40 40 41 43 45 45

Part-II: Thermochemical Conversion

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Thermal and catalytic cracking of non-edible oil seeds to liquid fuel Krushna Prasad Shadangi, Kaustubha Mohanty Abstract 6.1 Introduction 6.2 Pyrolysis and its Types 6.3 Process parameters that affect the yield 6.4 Fuel properties of seed pyrolytic oil 6.5 Catalytic pyrolysis of non-edible seeds 6.6 Conclusion References

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Evaluation of micro gasifier cookstove performance with handmade biomass pellets using region-specific fuels and assessment of deployment potential Debkumar Mandal, Vikas Dohare, Vijay H. Honkalaskar, Anurag Garg, Upendra V. Bhandarkar, Virendra Sethi Abstract 7.1 Introduction 7.2 Materials and Methods 7.3 Results and Discussion 7.4 Conclusions References

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Production of hydrocarbon liquid by pyrolysis of Camellia sinensis (tea) 68 seed deoiled cake and characterization of products Nabajit Dev Choudhury, Priyanko Protim Gohai, Bichitra Bikash, iv

Sashi Dhar Baruah, Rupam Kataki Abstract 8.1 Introduction 8.2 Materials and Methods 8.3 Results and discussion 8.4 Conclusions References 9

68 68 70 72 77 77

Comparative study of different biomass cookstove model: An experimental study K. Pal, A.K. Pandey, P Gera, S.K. Tyagi Abstract 9.1 Introduction 9.2 Mathematical Modeling 9.3 Materials and Methods 9.4 Analysis of Cookstove 9.5 Results and Discussion 9.6 Conclusions References

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79 79 81 86 91 93 95 96

Part-III: Biochemical Conversion

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10 Bioprospecting halotolerant cellulase from saline environment of Bhitarkanika National Park, Odisha Dash Indira, Sahoo Moumita, Dethose Ajay, C.S. Kar, R. Jayabalan Abstract 10.1 Introduction 10.2 Materials and methods 10.4 Results and discussion 10.5 Conclusions References

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99 100 101 103 106 107

11 Isolation and molecular characterization of cellulolytic fungi used for conversion of sugarcane biomass for bioethanol production A.M. Chetan, K.M. Harinikumar, P. Bhavani, H.B. Manoj Kumar, T. Madhu, Ningaraj Dalawai Abstract 11.1 Introduction 11.2 Material and Methods 11.3 Result and discussion References

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12 Application of thermostable cellulase in bioethanol production from lignocellulosic waste Neha Srivatsava, Rekha Rawat, Harinder Singh Oberoi

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110 111 111 114 120

Abstract 12.1 Introduction 12.2 Bioethanol production: Current production status and Challenges 12.3 Microorganism for thermostable cellulase production 12.4 Thermostable enzymes 12.5 Application of thermostable cellulases 12.6 Concluding remarks References 13 Endoglucanases: characterization and its role in bioconversion of cellulosic biomass Rekha Rawat, Neha Srivastava, Harinder Singh Oberoi Abstract 13.1 Introduction 13.2 Mechanism of cellulolysis 13.3 Effect Classification of Endoglucanases 13.4 Structure of endoglucanases 13.5 Mechanism of cellulose hydrolysis by endoglucanases 13.6 Microbial sources of endoglucanase enzyme 13.7 Application of endoglucanases 13.8 Significance of thermostable endoglucanases 13.9 Factors responsible for thermal stability 13.10Conclusion References 14 Comparative study of fermentation efficiency for bioethanol production by isolates Richa Arora, Shuvashish Behera, Sachin Kumar Abstract 14.1 Introduction 14.2 Materials and methods 14.3 Result and Discussion 14.4 Conclusion References 15 Sweet Sorghum - An ideal feedstock for bioethanol production Reetika Sharma, Gurvinder Singh Kocher, Harinder Singh Oberoi Abstract 15.1 Introduction 15.2 Origin and biology of sweet sorghum 15.3 Cultivation and harvesting of sweet sorghum 15.4 Inherent advantages of sweet sorghum 15.5 Technical hurdles 15.6 Bioethanol production from sweet sorghum 15.7 Energy ratio and environmental sustainability vi

121 122 123 124 125 128 129 129 135

135 135 136 137 137 139 139 140 142 142 144 144 149

149 149 150 151 154 154 156 156 156 158 159 160 163 163 165

15.8 Small-medium scale bioethanol production plant from sweet sorghum 15.9 Conclusions References 16 Fermentation of glucose and xylose sugar for the production of ethanol and xylitol by the newly isolated NIRE-GX1 yeast Shuvashish Behera, Richa Arora, Nilesh Kumar Sharma, Sachin Kumar Abstract 16.1 Introduction 16.2 Material and method 16.3 Results and discussion 16.4 Conclusion References 17 Comparative bioethanol production by S. cerevisiae and Z. mobilis from saccharified Sweet Potato Root Flour (Ipomoea batata L) using immobilized α- amylases and glucoamylase Preeti Krishna Dash, Sonali Mohapatra, Manas Ranjan Swain, Hrudaya Nath Thatoi Abstract 17.1 Introduction 17.2 Materials and methods 17.3 Results and discussion 17.4 Importance of enzyme Immobilization 17.5 Conclusion References 18 Genetic modifications in yeast for simultaneous utilization of glucose and xylose Nilesh Kumar Sharma, Shuvashish Behera, Sachin Kumar Abstract 18.1 Introduction 18.2 Necessity of pentose (C5) sugar fermenting organisms 18.3 Problems with pentose (C5) sugar fermenting yeast 18.4 Need of Genetic Engineering for xylose fermentation 18.5 Conclusion and future prospects References 19 To optimize the process of alcohol production from banana peel Mohit Jain, Anand Kumar Gupta, Sayan Chatterjee Abstract 19.1 Introduction 19.2 Materials used 19.3 Methodology 19.4 Results & Discussion vii

166 168 168 175

175 176 177 178 180 181 183

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194 195 196 197 198 199 202 208 208 208 210 211 212

19.5 Conclusions References 20 Commercial production of bio-CNG & organic manure from pressmud biomethanation Preetam Holkar, A.V. Mohan Rao, K.K. Meher Abstract 20.1 Introduction 20.2 Feed preparation 20.3 CSTR Digesters 20.4 Biogas cleaning process 20.5 BIO-CNG production & composition 20.6 Biogas utilization / storage devices cascades 20.7 Biogas based power 20.8 Digestate (Organic Manure) 20.9 Conclusions References 21 Feasibility of filling biogas in cylinders S.S. Sooch, Jasdeep Singh Saini Abstract 21.1 Introduction 21.2 Materials and Methods 21.3 Discussion 21.4 Conclusions References 22 Effect of pretreatment on bioconversion of wheat straw for the production of biogas Nishshesh Singh, Vivek Saini, Pranshu Gupta, Rajan Sharma, G Sanjay Kumar, Avanish K. Tiwari Abstract 22.1 Introduction 22.2 Material and methods 22.3 Results and Discussion 22.4 Conclusions References 23 Ultrasonic pretreatment to enhance biohydrogen production from food waste Abhijit Gadhe, Shriram Sonawane, Mahesh Varma Abstract 23.1 Introduction 23.2 Material and methods 23.3 Results and discussion viii

215 216 218

218 218 220 220 222 223 224 224 224 226 226 228 228 228 229 231 231 231 232

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23.4 Conclusion References

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24 Biological hydrogen production by facultative anaerobic bacteria Enterobacter aerogens (MTCC 8100) Virendra Kumar, Richa Kothari, S.K.Tyagi Abstract 24.1 Introduction 24.2 Material and methods 24.3 Results and Discussion 24.4 Conclusions References

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25 Enhanced biohydrogen production from glycerol using pretreated mixed culture Anbalagan Krishnasamy, Mohanraj Sundaresan, Kodhaiyolii Shanmugam, Pugalenthi Velan Abstract 25.1 Introduction 25.2 Materials and methods 25.3 Results and discussion 25.4 Conclusions References

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Part-IV: Chemical Conversion

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26 Isolation and characterization of freshwater microalgae Scenedesmus from contaminated field samples for bioenergy generation Mayur M. Phukan, B.K. Konwar Abstract 26.1 Introduction 26.2 Material and methods 26.3 Discussion 26.4 Conclusions References

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27 Prospects of biodiesel production from non-edible oil seeds of North East India: a review Debashis Sut, Rupam Kataki Abstract 27.1 Introduction 27.2 Non-edible vegetable oils resources 27.3 R Fatty acid profiles of the biodiesel 27.4 Properties of the biodiesel 27.5 Conclusion ix

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273 273 275 278 283 284 286

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References

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28 A critical review of enzymatic transesterification: a sustainable technology for biodiesel production Neetu Singh, M.K. Jha, A.K. Sarma Abstract 28.1 Introduction 28.2 Biodiesel 28.3 Lipases as biocatalysts in biodiesel synthesis 28.4 Lipase immobilization 28.5 Variables affecting the enzymatic transesterification 28.6 Conclusion References

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29 Single step reaction for biodiesel production of Jatropha curcus seeds Sanjaykumar N. Dalvi, Swati R. Sonawane Abstract 29.1 Introduction 29.2 Material and Methods 29.3 Result and Discussion 29.4 Conclusions References

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30 Production of biodiesel from edible and non-edible oils: a comparative study A.D. Singh, R. Rao, L.B. Reddy, H.K. Raghuvanshi, A.I. Kankia, H. Sharma, S. Srivastava Abstract 30.1 Introduction 30.2 Scope for the Study 30.3 Research Methodology 30.4 Result and Discussion References

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31 Production of biodiesel from neem oil using synthesized iron nanocatalyst 327 Mookan Rengasamy, Sundaresan Mohanraj, Krishnasamy Anbalagan, Shanmugam Kodhaiyolii, Velan Pugalenthi Abstract 327 31.1 Introduction 328 31.2 Materials and methods 329 31.3 Results and discussion 330 31.4 Conclusions 336 References 336 32 Influence of free fatty acids content in catalytic activity of [BSMIM] Cl x

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ionic liquid for biodiesel production from non edible acidic oils Subrata Das, Ashim Jyoti Thakur, Dhanapati Deka Abstract 32.1 Introduction 32.2 Materials and Methods 32.3 Results and Discussion 32.4 Conclusion References 33 Analysis of physical properties and biodiesel production from different accessions of Jatropha curcas Dheeraj Singh, Chiranjib Banerjee, Animesh Sinha, Diwaker Prasad Nirala, Santosh Prasad, Rajib Bandopadhyay Abstract 33.1 Introduction 33.2 Source: Jatropha Curcus 33.3 Different criteria which effect the biodiesel production 33.4 Material and Methods 33.5 Production of Biodiesel 33.6 Results and Discussion 33.7 Conclusion References 34 Analysis of exhaust emission from a diesel engine fueled with transesertified vegetable oils Hemanandh J., Narayanan K.V. Abstract 34.1 Introduction 34.2 Background 34.3 Methodology 34.4 Results & Discussions 34.5 Conclusion References 35 Genetic enhancement of Pongamia pinnata for bio-energy M.V.R. Prasad Abstract 35.1 Introduction 35.2 Carbon Sequestration by Pongamia 35.3 Sybiotic Nitrogen Fixation and Soil Amelioration by Pongamia 35.4 Selection of Elite Trees 35.5 Vegetative Propagation 35.6 Vayusap Plantations References xi

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347 348 349 351 351 353 354 356 357 359

359 359 360 361 364 367 368 370 370 371 372 373 373 374 374 378

Part-V: Electrochemical Processes

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36 Evaluation of electrical properties under different operating conditions of bio-electrochemical system treating thin stillage S. Ghosh Ray, M.M. Ghangrekar Abstract 36.1 Introduction 36.2 Materials and methods 36.3 Experimental results and discussion 36.4 Conclusion References

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37 Effect of salinity, acetate addition and alteration of sediment on performance of benthic microbial fuel cells D.A. Jadhav, M.M. Ghangrekar Abstract 37.1 Introduction 37.2 Materials and methods 37.3 Results and Discussion 37.4 Future perspectives 37.4 Conclusions References

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38 Biohydrogen production using single-chamber membrane-free microbial electrolysis cell with a stainless steel cathode Sundaresan Mohanraj, Krishnasamy Anbalagan, Kodhaiyolii Shanmugam, Velan Pugalenthi Abstract 38.1 Introduction 38.2 Materials and methods 38.3 Results and Discussion 38.4 Conclusions References

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39 Feasibility of interlinking two technologies for simultaneously two bioenergies generation Prashant Pandey, Vikas Shinde, S.P. Kale, R.L. Deopurkar Abstract 39.1 Introduction 39.2 Materials and methods 39.3 Results 39.4 Discussion 39.4 Conclusions References

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418 418 420 423 428 428 429

Part-VI: Hybrid Systems

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40 Development of nano based thermic fluid: rheological aspects of new energy system Vijay Juwar, Shriram Sonawane Abstract 40.1 Introduction 40.2 Materials and method 40.3 Results and Discussion 40.4 Conclusions References

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41 Conversion of plastic wastes into liquid fuels – a review Arun Joshi, Rambir, Rakesh Punia Abstract 41.1 Introduction 41.2 Target of waste plastics into liquid fuel 41.3 Plastics recycling technologies 41.4 Process technology 41.5 Advantages of process of fuel production 41.4 Conclusion and recommendation References

433 433 434 436 442 442 444 444 444 447 448 448 450 452 453

42 Kinetics of NOx reduction in BioDeNOx process water: effect of 455 temperature and iron chelate B. Chandrashekhar, Heena Tabassum, Nidhi Sahu, Padmaraj Pai, R.A. Pandey Abstract 455 42.1 Introduction 455 42.2 Materials and methods 457 42.3 Analytical methods 459 42.4 Results and discussion 459 42.4 Conclusion 465 References 465 43 Status of waste treatment, utilization and management in agro processing 467 Yogender Singh, Y.K. Yadav Abstract 467 43.1 Introduction 467 43.2 Agro processing industrial wastes treatment/utilization 470 43.3 Waste water in agriculture and food processing 472 43.4 Importance of waste management 474 43.5 Challenges in Waste management 474 43.6 Conclusions 474 References 474 xiii

Preface

Due to increasing prices of petroleum products, shortage of electricity supply, degradation of environment and availability of millions of tons of surplus biomass, R&D activities in the area of bio-energy including biodiesel, bioethanol, biomethanation, biomass gasification, biomass cookstove, etc. have received the tremendous attention all over the world. Keeping this trend in mind, the Govt. of India has already initiated the blending of 5% ethanol in the gasoline, which is likely to increase up to 10% in the coming years. While, the Jatropha Mission has been initiated for the promotion of Biodiesel in transportation and agriculture sectors and it is expected that by 2020 at least 10% of the liquid fuel used in transportation sector can be replaced by biodiesel. Similarly, MNRE has already initiated the dissemination of 10 millions of improved biomass cookstoves in the 12th five year plan through carbon revenue. The renewable energy currently has made remarkable share (12.5%) of total primary commercial energy supply of 228 GW, while the major share of around 70% of the total generation capacity is from thermal (coal, gas, oil). Since, energy security and diversification of the energy mix is a major policy driver for renewables. Growth of renewables generally contributes to energy diversification, in terms of the technology portfolio and geographical sources. Use of renewables can also reduce fuel imports and insulate the economy to some extent from fossil fuel price rises and swings. This not only increases the certainty for energy security but also symbolize the steady economic growth of a country. However, the concentrated growth of variable renewables can make it harder to balance power systems, which must be duly addressed. The electricity sector in India had an installed capacity of around 228 GW, the world's fifth largest. Non-renewable Power Plants constitute 87.55% of the installed capacity, and Renewable Power Plants constitute of around 12.45% of total installed capacity while the major share is of biomass based energy generation. After receiving the great response of first two volumes on ‘Recent Advances in Bioenergy Research’, we are introducing the third volume in the form of a book. The book is divided in six parts viz. Part-I: Biomass and Energy Management; Part-II: Thermo-chemical Conversion; Part-III: Chemical Conversion; Part-IV: Biochemical Conversion; Part-V: Electrochemical Processes; and Part-VI: Hybrid Systems. Each section includes respective chapters from

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Eminent Academician, Scientists and Researchers in the field. We are grateful for their commendable contribution for this book. Emphasis is given in such a way that the current trends of research and investigation in the bioenergy sector can easily be worked out from the in-depth study of this book. Our efforts will be successful if the readers dig up the expected gain out of these articles.

Sachin Kumar

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Contributors Arora Richa, Biochemical Conversion Division, Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala Bandopadhyay Rajib, Birla Institute of Technology, Mesra, Ranchi Banerjee Chiranjib, Birla Institute of Technology, Mesra, Ranchi Baruah Sashi Dhar, Regional Research Laboratory, Jorhat, Assam Behera Shuvashish, Biochemical Conversion Division, Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala Bhandarkar Upendra V., Department of Mechanical Engineering, Indian Institute of Technology Bombay, Mumbai Bhatia Anjana, Department of Botany, HMV College, Jalandhar Bhavani P., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru Bikash Bichitra, Assam Down Town University, Guwahati, Assam Chandrashekhar B., Environmental Biotechnology Division, CSIR-National Environmental Engineering Research Institute (CSIR-NEERI), Nagpur, Maharashtra Chatterjee Sayan, University School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi Chetan A.M., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru Chopra A.K., Department of Zoology and Environmental Sciences, Gurukula Kangri Vishwavidyalya , Haridwar Choudhury Nabajit Dev, Assam Down Town University, Guwahati, Assam Dalawai Ningaraj, Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru Dalvi Sanjaykumar N., Department of Physics, S. N. Arts, D. J. M. Commerce & B. N. S. Science College, Sangamner, Dist. Ahmednagar, Maharashtra Das Subrata, Department of Energy, Tezpur University, Tezpur, Assam Dash Indira, Food and Bioprocess Technology Laboratory, National Institute of Technology, Rourkela, Odisha Dash Preeti Krishna, Department of Biotechnology, College of Engineering and Technology, Biju Pattnaik University of Technology, Bhubaneswar Deka Dhanapati, Department of Energy, Biomass Conversion Laboratory, Tezpur University, Tezpur, Assam Deopurkar R.L., Department of Microbiology, University of Pune, Ganeshkhind, Pune, Maharastra Dethose Ajay, Food and Bioprocess Technology Laboratory, National Institute of Technology, Rourkela, Odisha xvi

Dohare Vikas, Centre for Environmental Science and Engineering, Indian Institute of Technology Bombay, Mumbai Gadhe Abhijit, Department of Chemical Engineering, Visvesvaraya National Institute of Technology (VNIT), Nagpur Garg Anurag, Centre for Environmental Science and Engineering, Indian Institute of Technology Bombay, Mumbai Gera P., Dr. B. R. A. National Institute of Technology, Jalandhar Ghangrekar M.M., Department of Civil Engineering, Indian Institute of Technology, Kharagpur Gohai Priyanko Protim, Department of Energy, Tezpur University, Napaam, Tezpur, Assam Gupta Anand Kumar, University School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi Gupta Pranshu, Chemical Engineering Department, University of Petroleum & Energy Studies, Dehradun Harinikumar K.M., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru Hemanandh J., Department of Mechanical Engineering, Sathyabama University, Chennai Holkar Preetam, Spectrum Renewable Energy Pvt Ltd, Kodoli, Warnanagar, Maharastra Honkalaskar Vijay H., Centre for Technology Alternatives for Rural Areas, Indian Institute of Technology Bombay, Mumbai Jadhav D.A., School of Water Resources, Indian Institute of Technology, Kharagpur Jain Mohit, University School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi Jayabalan R., Food and Bioprocess Technology Laboratory, National Institute of Technology, Rourkela, Odisha Jha M.K., Department of Chemical Engineering, Dr B R Ambedkar NIT, Jalandhar Joshi Arun, Department of Chemical Engineering, Doon College of Engineering and Technology, Dehradun Juwar Vijay, Department of Chemical Engineering Visvesvaraya National Institute of Technology (VNIT), Nagpur Kale S.P., Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Navi Mumbai, Maharastra Kankia A.I., School of Biotechnology, Lovely Professional University, Phagwara, Punjab Kar C.S., Office of the Principal CCF (Wildlife) & Chief Wildlife Warden, Bhubaneswar, Odisha Kataki Rupam, Department of Energy, Tezpur University, Napaam, Tezpur, Assam xvii

Kaur Navjot, Department of Botany, HMV College, Jalandhar Kocher Gurvinder Singh, Department of Microbiology, Punjab Agricultural University, Ludhiana, Punjab Konwar B.K., Department of Molecular Biology & Biotechnology, School of Science, Tezpur University, Assam Kothari Richa, Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Lucknow Krishnasamy Anbalagan, Department of Biotechnology, Bharathidhasan Institute of Technology, Anna University, Tiruchirappalli, Tamil Nadu Kumar G Sanjay, Chemical Engineering Department, University of Petroleum & Energy Studies, Dehradun Kumar H.B. Manoj, Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru Kumar Sachin, Biochemical Conversion Division, Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala Kumar Virendra, Department of Environmental Science, Babasaheb Bhimrao Ambedkar University, Lucknow Madhu T., Department of Plant Biotechnology, UAS, G.K.V.K campus, Bengaluru Mahajan Amita, Department of Bio-chemistry, RBIEBT, Kharar Mandal Debkumar, Centre for Environmental Science and Engineering, Indian Institute of Technology Bombay, Mumbai Meher K.K., Spectrum Renewable Energy Pvt Ltd, Kodoli, Warnanagar, Maharastra Mohanty Kaustubha, Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati Mohapatra Sonali, Department of Biotechnology, College of Engineering and Technology, Biju Pattnaik University of Technology, Bhubaneswar Narayanan K.V., Department of Mechanical Engineering, Sathyabama University, Chennai Nirala Diwaker Prasad, Biotechnology, Genetics and Tree Improvement Division, Institute of Forest Productivity, Lalgutwa, Ranchi Oberoi Harinder Singh, Central Institute of Post Harvest Engineering and Technology, Ludhiana, Punjab Pai Padmaraj, Environmental Biotechnology Division, CSIR-National Environmental Engineering Research Institute (CSIR-NEERI), Nagpur, Maharashtra Pal K., Thermochemical Conversion Division, Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala Pandey A.K., Thermochemical Conversion Division, Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala xviii

Pandey Prashant, School of Studies in Biotechnology, Jiwaji University, Gwalior- 474011, Madhya Pradesh Pandey R.A., Environmental Biotechnology Division, CSIR-National Environmental Engineering Research Institute (CSIR-NEERI), Nagpur, Maharashtra Pathak Vinayak V., Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Lucknow Phukan Mayur M., Department of Molecular Biology & Biotechnology, School of Science, Tezpur University, Assam Prasad M.V.R., VAYUGRID, Bangalore Prasad Santosh, Biotechnology, Genetics and Tree Improvement Division, Institute of Forest Productivity, Lalgutwa, Ranchi Punia Rakesh, Doon College of Engineering and Technology, Dehradun Puri Punam, Department of Life Sciences–Biotechnology, Punjab Technical University, Kapurthala Raghuvanshi H.K., School of Biotechnology, Lovely Professional University, Phagwara, Punjab Rambir, Department of Chemical Engineering, Doon College of Engineering and Technology, Dehradun Rao A.V. Mohan, Spectrum Renewable Energy Pvt Ltd, Kodoli, Warnanagar, Maharastra Rao R., School of Biotechnology, Lovely Professional University, Phagwara, Punjab Rawat Rekha, Central Institute of Post Harvest Engineering and Technology, Ludhiana, Punjab Ray S. Ghosh, Advanced Technology Development Centre, Indian Institute of Technology, Kharagpur Reddy L.B., School of Biotechnology, Lovely Professional University, Phagwara, Punjab Rengasamy Mookan, Department of Petrochemical Technology, Bharathidhasan Institute of Technology, Anna University, Tiruchirappalli, Tamil Nadu Sahoo Moumita, Food and Bioprocess Technology Laboratory, National Institute of Technology, Rourkela, Odisha Sahu Nidhi, Environmental Biotechnology Division, CSIR-National Environmental Engineering Research Institute (CSIR-NEERI), Nagpur, Maharashtra Saikia Madhurjya, Dibrugarh University Institute of Engineering and Technology, Dibrugarh, Assam Saini Jasdeep Singh, Department of Civil Engineering, Punjab Agricultural University, Ludhiana, Punjab xix

Saini Vivek, Chemical Engineering Department, University of Petroleum & Energy Studies, Dehradun Sarma A.K., Chemical Conversion Division, Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala Sethi Virendra, Centre for Environmental Science and Engineering, Indian Institute of Technology Bombay, Mumbai Shadangi Krushna Prasad, Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati Shanmugam Kodhaiyolii, Department of Biotechnology, Bharathidhasan Institute of Technology, Anna University, Tiruchirappalli, Tamil Nadu Sharma H., School of Biotechnology, Lovely Professional University, Phagwara, Punjab Sharma Nilesh Kumar, Biochemical Conversion Division, Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala Sharma Rajan, Chemical Engineering Department, University of Petroleum & Energy Studies, Dehradun Sharma Reetika, Department of Microbiology, Punjab Agricultural University, Ludhiana, Punjab Shinde Vikas, Department of Microbiology, University of Pune, Ganeshkhind, Pune, Maharastra Singh A.D., School of Biotechnology, Lovely Professional University, Phagwara, Punjab Singh Dheeraj, Birla Institute of Technology, Mesra, Ranchi Singh Neetu, Department of Chemical Engineering, Dr B R Ambedkar NIT, Jalandhar Singh Nishshesh, Chemical Engineering Department, University of Petroleum & Energy Studies, Dehradun Singh Yogender, Department of Food Engineering and Technology, S.L.I.E.T., Longowal, Punjab Singson Lhaihoichong, Department of Environmental Sciences, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Lucknow Sinha Animesh, Biotechnology, Genetics and Tree Improvement Division, Institute of Forest Productivity, Lalgutwa, Ranchi Sonawane Shriram, Department of Chemical Engineering Visvesvaraya National Institute of Technology (VNIT), Nagpur Sonawane Shriram, Department of Chemical Engineering, Visvesvaraya National Institute of Technology (VNIT), Nagpur Sonawane Swati R., Department of Chemistry, S. N. Arts, D. J. M. Commerce & B. N. S. Science College, Sangamner, Dist. Ahmednagar, Maharashtra xx

Sooch S.S., School of Energy Studies for Agriculture, Punjab Agricultural University, Ludhiana, Punjab Srivastava S., School of Biotechnology, Lovely Professional University, Phagwara, Punjab Srivatsava Neha, Central Institute of Post Harvest Engineering and Technology, Ludhiana, Punjab Suman, Punjab University, SSGRC, Hoshiarpur Sundaresan Mohanraj, Department of Biotechnology, Bharathidhasan Institute of Technology, Anna University, Tiruchirappalli, Tamil Nadu Sut Debashis, Department of Energy, Tezpur University, Napaam, Tezpur, Assam Swain Manas Ranjan, Department of Biotechnology, IIT Madras, Chennai Tabassum Heena, Environmental Biotechnology Division, CSIR-National Environmental Engineering Research Institute (CSIR-NEERI), Nagpur, Maharashtra Thakur Ashim Jyoti, Department of Chemical Sciences, Tezpur University, Tezpur, Assam Thatoi Hrudaya Nath, Department of Biotechnology, College of Engineering and Technology, Biju Pattnaik University of Technology, Bhubaneswar Tiwari Avanish K., Centre for alternate energy research, University of petroleum & energy Studies, Dehradun Tyagi S.K., Thermochemical Conversion Division, Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala Varma Mahesh, Department of Chemical Engineering, Visvesvaraya National Institute of Technology (VNIT), Nagpur Velan Pugalenthi, Department of Biotechnology, Bharathidhasan Institute of Technology, Anna University, Tiruchirappalli, Tamil Nadu Yadav Y.K., Sardar Swarn Singh National Institute of Renewable Energy, Kapurthala

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Recent Advances in Bioenergy Research Vol. III 2014

Part I Biomass and Energy Management

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 1 THERMOGRAVIMETRIC CHARACTERIZATION OF WOOD STALKS AS GASIFICATION AND PYROLYSIS FEEDSTOCK Rakesh Punia, Sachin Kumar

Abstract Energy is an integral part of a society and plays an important role in its socio-economic development. A nation economic development can be assessed by the pattern of consumption and quality of energy availability. Energy and chemicals from agriculture and regional available wood residue can be efficiently produced by gasification and pyrolysis methods. Indian has abundance of biomass sources. Selected physical and chemical properties of biomass are related to thermochemical conversion, which has been observed to determine the thermochemical conversion. The main objective of the present work is to investigate the comparison of use of different biomass fuels and their parameters on the performance of small scale downdraft gasifier, biomass fuels selected are locally available plant stalks such as Prosopis juliflora (Kikkar), Eucalyptus (Sefeda), Pigeon pea (Arhar Dal), Albizia procera (Surash), Melia sp. (Bakain) and Mulberry sp. (Sahatoot). Fuel feedstock characterisation of selected biomass has been carried out at macroscopic as well as microscopic levels such as determination of dry density, calorific value, proximate & ultimate analysis, and thermogravimetric analysis (TGA). On the basis of characteristics, it is found that Melia sp., and Eucalyptus could be energy efficient feedstock for small scale downdraft gasifier possess high fixed carbon content and calorific value as compared to other selected wood stalks. Key Words: Wood stalks , Gasification, Thermogravimeter analyser, Reaction kinetics. 1.1

Introduction To maintain the ecology, sustainable and equitable development become the critical

issues in most parts of world. The alarming population coupled with developmental activities based on decisions for resource scarcity in many parts of India. A judicious choice of energy utilization is required to achieve growth in a sustainable manner (Keyhani et al., 2010). Gasification and pyrolysis can convert lignocellulosic materials to synthesis gas (syngas) without the need for delignification. For different applications, the syngas from gasification 2

Recent Advances in Bioenergy Research Vol. III 2014 can be further converted and separated to other chemicals (by various reforming processes) or fuel gas or hydrogen for fuel cell (Anjireddy et al., 2011). Gasification is a thermochemical process by which any carbonaceous feed can be converted to gaseous products with useable heating value (primarily carbon monoxide and hydrogen in a controlled oxidizing atmosphere). Pyrolysis in particular converts biomass into high energy content biofuels provided that the adequate temperature and heating rate are reached and may be used to fuel internal combustion engines and gas turbines after an intermediate process that converts the feedstock into a liquid or gaseous biofuel (Goyal et al., 2008; Fantozzi et al., 2003) Pyrolysis is one of the first step of all thermochemical processes occurring in an inert atmosphere (Fantozzi et al., 2010). Energy generation from biomass is environmental friendly and does not increase the CO2 in the atmosphere. Biomass energy can be generated locally and can make any country energy self-sustainable and less dependent on foreign petroleum resources (McKendry, 2002). Interest in bioenergy has been enhanced because it also manages the biomass wastes. The advantage of gasification has ability to utilize a wide range of feedstocks ranging from any plant residue, organic by-product (with protein, lignin or oil) of industry or even municipal wastes , as compared with other bioenergy generation techniques. Gasification and pyrolysis are efficiently viable options for processing biomass feed stocks, which cannot be fermented to ethanol technically or economically (Kumar et al., 2008). Mathematical modeling to predict the product gas qualities during gasification and pyrolysis requires the reaction kinetics knowledge of biomass volatilization and its subsequent reactions. Thermogravimetric analysis (TGA) is very useful in determining the reaction kinetics of gasification and pyrolysis. It has been used extensively for the characterization of various feedstocks. This method have been used by many researchers to determine the kinetics parameter for bagasse in a nitrogen atmosphere (Nassar et al., 1996), rice husk in an oxygen atmosphere (Mansaray and Ghaly, 1999), rapeseed straw and stalks in a nitrogen atmosphere (Karaosmanoglu et al, 2001), forestry wastes in a nitrogen atmosphere (Lapuerta et al., 2004) and poplar wood in a nitrogen atmosphere (Katarzyna et a.l, 2012). However, there is a lack of kinetics information on gasification or pyrolysis of different wood stalks. This research study surrounds to the thermochemical conversions properties of biomass and determine its reaction kinetics in inert and oxidizing atmospheres using a thermogravimetric technique.

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Recent Advances in Bioenergy Research Vol. III 2014 1.2

Materials and Methodology Variety of biomass samples were collected from various accessible locations. Prosopis

juliflora, Eucalyptus, Albizia procera, Melia sp. and Mulberry sp. wood stalks were purchased from Kapurthala timber market. Pigeon pea (Arhar Dal) stalks were collected from SSS-NIRE (Kapurthala) campus. Biomass samples were collected from gasifier feed stock in such a manner to represent homogeneity of biomass characteristics. Collected biomass samples were sun dried for five days. Wood stalk dust/flakes were prepared on saw mill and ground in grinder for analysis purpose. Ground wood samples were screened using vibratory screen for attaining particle size of about 425 µm. Moisture content was removed from screened samples by drying in oven Broadly, biomass characterization are categorized in two types ; macroscopic and microscopic property. Macroscopic analysis of biomass fuel properties are proximate analysis, ultimate analysis, particle size, bulk density, calorific value, ash fusion temperature, etc. Biomass fuel properties for microscopic analysis includes thermal properties, chemical kinetics, and mineral data, etc. 1.2.1

Proximate analysis The proximate analysis of the sample was done as per ASTM standards. The

parameters namely moisture content (MC) (ASTM D3172-73), volatile matter (VM) (ASTM D3175-73) and ash content (ASTM D3174-73). 1.2.2

Ultimate analysis The ultimate analysis of the biofuel is done with CHNS Analyzer. The modern

elemental analyzers of Elementor (Vario MICRO Cube), analysis samples from 1 mg to 800 mg solid or liquid samples. 1.2.3

Calorific value This important characteristics of a fuel were determined by bomb calorimeter

(Toshniwal -CC01-M3). The crushed sample was compressed from the ground sample was compacted from an original average density of 460 to 1200 kg/m3. The pellet was then combusted to determine energy content. 1.2.4

Biomass density The size and density affects the burning characteristics of biomass fuel by heating and

drying rate during combustion. It also dictates how the material is likely to behave during subsequent thermo-chemical or biological processing as a fuel or feed stocks. Average mass 4

Recent Advances in Bioenergy Research Vol. III 2014 of is measured by Micro-balance and volume of dry biomass samples is determined by water displacement method. 1.2.5

Kinetic study (activation energy) In general, the microscopic characterisation of gasifier feedstock is done by

Thermogravimetric analysis (TGA), which is used to determine the kinetics parameter in presence of oxygen or inert atmosphere. Pyrolysis is a sub-category of gasification, the difference being this process takes place in an inert atmosphere (generally nitrogen) Thermogravimetric analysis experiment is performed using on Perkin-Elmer (STA 6000) equipment. The temperature of furnace and weighting system of TGA were calibrated according to the manufacturer’s recommendation. Temperature calibration was performed by measuring Curie points of alumel, nickel, perkalloy and iron. Depending on the density of biomass, samples weight is placed in the pan of the TGA microbalance. Approximately 20-42 mg of the wood powder were taken in crucible pan for TGA experiment. Air is used as purge gases and all TGA experiments were conducted at a constant purge flow rate of 20 ml/min. Residual weight of the sample and the derivative of weight, with respect to time and temperature (differential thermogravimetry analysis, DTG), were recorded. A program is prepared and saved in which samples were held at 30oC for 1 min and heated to 850oC at the rate of 10oC/min and then held at 850oC for 1min. 1.2.5.1 Procedure to determine parameters of reaction kinetics To calculate chemical kinetics of reaction using the procedure of Duvvuri et al. (1975) as applied by Mansaray et al. (1999) and Karaosmanoglu et al. (2001) which is explained as bellow : Global kinetics of the vitalization reaction can be written as −

= k xn

(1)

Where, x is the sample weight, k the reaction constant and n the order of the reaction. Applying the Arrhenius equation, k = Ae-E/RT

(2)

The combined form of the above two equations (1) and (2) can be written in linear form as ln [



] = ( ) − ( ) + ln (

) ,

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{ Put x =



}

(3)

Recent Advances in Bioenergy Research Vol. III 2014 where, wo is the initial weight at the starting stage, wf is the final weight at the end of TGA and w is the weight at any temperature, dw/dt the ratio of change in weight to change in time, A the pre-exponential factor and R the universal gas constant (gas value). Eq. (3) is of the form y = B + Cx + Dz,

(4)

where y = ln[ B = ln(A);



],

x= ,

C = (- ),

z = ln (

) ,

D=n

The constants B, C, D were estimated by multi-linear regression. The determination of activation energy form selected data is done by regression calculation with Analysis Tool of MS-Excel. 1.3

Results & Discussion Analysis results for selected biomass samples which includes Proximate Analysis,

Ultimate Analysis, Density Measurement, Calorific Value, Activation Energy are contained in this section. 1.3.1

Proximate Analysis Proximate analysis data (Table-1) shows that the moisture contents of analyzed

biomasses were in between 5 to 8% (by weight) which is under the range of Downdraft Gasifier biomass feedstock. Volatile Matter and Fixed Carbon has major role in heating value of biomass fuels. Maximum VM of Melia was found (83.79%). Maximum Fixed carbon content was of Eucalyptus and Pigeon pea has lowest (7.44%) FC value. Ash in the biomass was in the range of 0.7 to 4% which plays a catalyst role during biomass gasification. Table-1: Proximate analysis data of biomass feed stocks. Proximate analysis (Wt.%) Biomass

Moisture Content

Volatile Matter

Fixed Carbon

Ash

Prosopis juliflora

6.32

82.46

9.02

2.52

Eucalyptus

5.93

80.53

10.82

2.67

Pigeon pea

8.27

82.53

7.44

1.53

Albizia procera

6.72

83.58

8.07

0.79

Melia (Bakain)

5.03

83.79

8.73

1.37

Mulberry

6.24

81.93

10.04

1.69

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Recent Advances in Bioenergy Research Vol. III 2014 Table-2: Results of biomass calorific value and Dry Density Biomass Samples

Calorific value (HHV)

Dry Density

MJ/ kg.

(Kg/m3)

Prosopis juliflora (Kikkar)

17.8038

496.711

Eucalyptus

17.4344

443.109

Pigeon pea (Arhar)

16.0138

272.509

Albizia procera

16.8509

326.300

Melia (Bakain)

17.8643

378.561

Mulberry (Sahatoot)

16.4012

251.785

On observing dry density data, it is evident that P. juliflora has substantially highest density than other biomasses. Pigeon pea and Mulberry wood stalks have comparatively lower dry density than other biomass samples. 1.3.2

Ultimate Analysis Among all the biomass samples, Melia and Eucalyptus have comparatively higher

fixed carbon wt. % which indicates that they could produce syngas of high calorific value. Nitrogen for analyzed biomass was less than 1% (by weight) of biomass. As evident from data (Table-3), sulfur content is missing in all the samples. Table-3: Ultimate Analysis of Biomass Samples Ultimate analysis (wt.%) Biomass Samples

Carbon

Hydrogen

Nitrogen

Sulphur

Oxygen

Prosopis juliflora

47.391

6.117

0.400

0.000

45.92

Eucalyptus

48.193

5.958

0.335

0.000

45.457

Pigeon pea

47.314

5.837

0.450

0.000

46.073

Albizia procera

46.431

6.851

0.470

0.000

47.044

Melia (Bakain)

48.738

6.463

0.550

0.000

44.247

Mulberry(Sahatoot)

45.285

5.963

0.095

0.000

48.221

1.3.3 Thermogravimeter analysis (TGA) Wood samples were characterized by thermogravimeter analyser under Nitrogen (Inert) and Air atmosphere. The reduction in weight of biomass with steady state rise in the reactor temperature were analysed. 7

Recent Advances in Bioenergy Research Vol. III 2014 1.3.3.1 TGA in Nitrogen (inert) atmosphere TGA plots of selected biomass in nitrogen atmosphere are similar to (Fig. 1). The first stage of weight loss ranged from 30oC to around 125oC was clearly distinct from the other stages of weight loss. It may correspond to the loss of water and light volatile compounds in the biomass sample (Mansaray and Ghaly, 1999). Following the first stage, there was negligible weight loss (< 0.5%) in the temperature range of 160–250oC. The second phase of weight loss started around 250oC. The derivative plot of the region between 250 and 850oC showed only one observable peak (Fig. 1).

Fig. 1: Typical TGA and DTG diagram of biomass in nitrogen atmosphere When the data between 250 and 850oC were used for determining parameters of reaction kinetics, the r2 values for the multiple-regression were less than 0.80, and the predicted values deviated from the experimental data. This suggested that there may have been two different reaction stages of weight loss occurring in this region (250–850oC). Total of three distinct stages (Fig. 1) represented the global kinetics of weight loss occurring during TGA of biomass in inert atmosphere. 1.3.3.2 TGA in Air (Oxidizing) Atmosphere In an air atmosphere, the TGA plots similar to (Fig. 2) clearly suggested that there were three stages of weight loss. The first stage in the oxidizing atmosphere ranged from 25 to 115–140 oC. It was very similar to the first stage in the inert atmosphere. The weight loss 8

Recent Advances in Bioenergy Research Vol. III 2014 between the end of the first stage (130oC) and the start of the next stage (240oC) was much less (10%/oC) as compared with the inert atmosphere. Separation between the second and third stages in the oxidizing atmosphere was very clear. During the third stage, which ranged from 400 to 560oC, there was a small amount of weight loss (~10%) at a slower rate. The weight loss in the third stage was very much lower as compared with the second stage and also as compared with weight loss during the third stage in an inert atmosphere. Also, the third stage in the oxidizing atmosphere had a very narrow temperature range as compared with the third stage in the inert atmosphere. This suggests that the third stage in the inert and oxidizing atmospheres were different. The amount of oxygen in the air atmosphere in our experiment was sufficient for oxidation of a small amount of biomass particles (~15–20 mg).

Fig. 2 -Typical TGA and DTG diagram of biomass in air atmosphere

1.3.3.3 Parameters of Reaction Kinetics Kinetics of weight loss in air atmosphere at a heating rate of 10oC/min was similar to that in nitrogen atmosphere . But, at higher rates of 30 and 50 oC/min the reaction during the second stage occurred very rapidly and activation energies were higher than activation energies in nitrogen atmosphere.

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Recent Advances in Bioenergy Research Vol. III 2014 Collected data is studied according stages from I to III and ash point of biomass. The first stage of weight loss corresponds to the loss of water and light volatile compounds in analysed biomass. The low moisture content in biomass samples resulted in low weight loss during this stage of weight loss. The second stage may correspond to the major loss (65 to 80% wt.), due to the decomposition of cellulose and hemicellulose components and partial loss of the lignin component of biomass. Following this stage, there was a continuous and slow weight loss from 450–550 to 850oC, due to the thermal degradation of lignin or complex high-molecular weight components of biomass. TGA stages shows that the performance of eucalyptus and melia as gasifier feedstock are better than other selected biomasses. The data between 250 and 450oC were used for determining parameters of reaction kinetics. Biomass weight percent at the interval of 25oC are collected from main TGA data (Table 4). Table-4

Activation Energy during second stage in an air atmosphere and N2 Atmosphere Activation Energy

Air Atmosphere

N2 Atmosphere

E (kJ mol-1)

E (kJ mol-1)

Prosopis juliflora

86.34

64.31

Eucalyptus

88.62

75.34

Pigeon pea

85.34

68.29

Albizia procera

86.74

66.74

Melia

78.53

68.53

Mulberry

76.35

59.57

Biomass

1.4

Conclusions TGA of wood stalks in nitrogen atmosphere indicates distinct three stages of weight

loss. A comparative study of different regionally available gasifier feed stocks i.e. Prosopis juliflora, Eucalyptus, Albizia procera, Melia sp. and Mulberry sp.was done on the basis of determination of dry density, proximate analysis, ultimate analysis and thermogravimetric analysis. Proximate analysis data shows highest fixed carbon content in Eucalyptus as compared to Mulberry, which indicates high heating value. Ultimate analysis of all the selected biomasses was found that all the samples were free from sulfur. The carbon percentage was higher in Melia, and Eucalyptus as compared to other biomasses. Dry density of Prosopis juliflora and Eucalyptus were found to be highest among all the selected biomasses. On the compilation of TGA data, it was found that activation energy of Eucalyptus

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Recent Advances in Bioenergy Research Vol. III 2014 is highest in both the cases (inert / air) which indicates that more external energy is required to initiate the combustion process. Acknowledgments One of the authors (Rakesh Punia) would like to thank to Dr. A.K. Jain, the Director of SSS-NIRE for their permission to perform experiments at SSS-NIRE, Kapurthala.

References 1. Bhavanam A. and Sastry R.C. (2011) Biomass Gasification Processes in Downdraft Fixed Bed Reactors: A Review. Int. J. Chem. Eng. App., 2:425-443. 2. Duvvuri M.S., Muhlenkamp S.P., Iqbal K.Z. and Welker J.R. (1975) The pyrolysis of natural fuels. J. Fire Flamm., 6:468-477. 3. Fantozzi F., D’Alessandro B. and Bidini G. (2003) IPRP – Integrated pyrolysis regenerated plant – gas turbine and externally heated rotary-kiln pyrolysis as a biomass waste energy conversion system. Influence of thermodynamic parameters. Proc. Inst. Mech. Eng. A- J. Pow., 217:519-527. 4. Fantozzi F., Laranci P. and Bidini G. (2010) CFD simulation of biomass pyrolysis syngas vs. natural gas in a microturbine annular combustor. Proc. ASME Turbo Expo: Power for Land, Sea and Air, 14-18, Glasgow, UK. 5. Goyal H., Seal D. and Saxena R. (2008) Bio-fuels from thermochemical conversion of renewable resources: a review. Renew. Sust. Energy Rev., 12:504517. 6. Karaosmanoglu F., Cift B.D. and Ergudenler A.I. (2001) Determination of reaction kinetics of straw and stalk of rapeseed using thermogravimetric analysis. Energy Sources, 23:767-774. 7. Keyhani A., Ghasemi-Varnamkhasti M., Khanali M. and Abbaszadeh R. (2010) An assessment of wind energy potential as a power generation source in the capital of Iran, Tehran. Energy, 35:188-201. 8. Kumar A., Lijun W., Dzenis Y.A., Jones D.D. and Hanna M.A. (2008) Thermogravimetric characterization of corn stover as gasification and pyrolysis feedstock. Biomass Bioenergy, 32:460- 467. 9. Lapuerta M., Hernandez J.J. and Rodriguez J. (2004) Kinetics of devolatilisation of forestry wastes from thermogravimetric analysis. Biomass Bioenergy, 27:385-391.

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Recent Advances in Bioenergy Research Vol. III 2014 10. Mansaray G.K. and Ghaly A.E. (1999) Determination of kinetic parameters of rice husks in oxygen using thermogravimetric analysis. Biomass Bioenergy, 17:19-31. 11. McKendry P. (2002) Energy production from biomass (Part I): overview of biomass. Bioresour. Technol., 83:37-46. 12. Nassar M.M., Ashour E.A. and Wahid S.S. (1996) Thermal characteristics of bagasse. J. App. Polymer Sci., 61: 885-890. 13. Slopiecka K., Bartocci P. and Fantozzi F. (2012) Thermogravimetric analysis and kinetic study of poplar wood pyrolysis. App. Energy, 97: 491-497

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 2 ASSESSMENT OF SOLID WASTE MANAGEMENT AND ENERGY RECOVERY FROM WASTE MATERIALS IN LUCKNOW ZOO: A CASE STUDY Vinayak V. Pathak, Richa Kothari, A.K. Chopra, Lhaihoichong Singson

Abstract Zoo is the facility in which animals are captured within enclosures, displayed to the public and they may breed also. Thus zoo is the centers which provide entertainment, education and protecting endangered species. The animals and the visitors inside the zoo generate large amount of solid waste. The present work is a case study of Lucknow zoo in order to identify the sources of solid waste generation and their sustainable management with an approach of energy recovery. According to findings number of mammals (468), birds (100) and reptiles (378) were present which generated 482 kg of fresh animal waste per day. On the other hand various plant species such as Madhuca longifolia, Aegle marmelos, Poltalthia longifolia, Cycas circinnalis, Ficus benhalensis etc were also observed which were produce 6.5 to 7 kg of biomass per day. The selected zoo attracts more than 900,000 to 1000,000 of tourists and visitors annually which contribute in solid waste generation. Various suggestive measures for treatment of animal waste, plant waste and anthropogenic waste were identified for conversion of this high organic waste in to energy rich biofuel. Key words: Solid waste, Sustainable, Energy recovery, Biomass, Biofuel 2.1

Introduction Wastes have been recognized as valuable sources whether it is organic or inorganic.

Various sources have been identified for the generation of waste like industrial, domestic, agricultural or commercial etc. Animal waste generated from agricultural and commercial practices primarily used for plant nutrient (feedstuff) and feedstock for energy production (methane) (Morse, 1995; Williams, 1995; Gunaseelan, 1997). Animal waste are mainly produced either in operations like dairy farming, poultry farming or in Zoo areas. Zoos are the areas in which animals are confined within a definite boundary, where they breed and

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Recent Advances in Bioenergy Research Vol. III 2014 displayed for the tourists, hence Zoo provide education and entertainment with protection of endangered species (Gibson, 1980). All animals and tourists produce large amount of waste inside the zoo premises which consist of both organic as well as inorganic waste. Zoos having access of millions of people have the ability to educate and communicate a number of people for sustainable lifestyles. A serious contribution towards sustainable future can be made by the Zoo by following the sustainability in their policies, strategies and management. A number of criteria such as expenses, environmental impact, profitability, complexity and sponsorship are considered in order to develop zoo as eco-development. In India Central Zoo Authority (CZA) is the organizing body of Central government for all zoos which is also associate member of world Association of Zoo and Aquarium (WAZA). CZA classify zoos depending on area, number of animals and annual attendance of visitors. CZA provide financial support to zoo and evaluate it time to time in order to provide recognition. Lucknow zoo at present has about 468 mammals, 378 reptiles, and many different species of wild animals. The main attraction of this zoo is Royal Bengal tiger, White Tiger, Lion, Wolf, Barking deer, Hog Deer, Asiatic Elephant, Giraffe, Zebra, Hill Mynahs, Giant squirrels, Great pied hornbills, Golden pheasant, Silver Pheasant etc. Zoo not only protect these animals but also achieve successful breeding of Swamp deer, Black buck, Hog deer, barking deer, White Tiger, Indian wolf, and several pheasants.. A number of plants are also protected inside the zoo such as Madhuca longifolia,Aegle marmelos, Poltalthia longifolia, Cycas circinnalis, Ficus benghalensis, tectona grandis, Eucalyptus hybrid, Santalum album, Acacia nilotica, mangifer indica, Tamaridus indica, cassia siamia, Delonix regia etc. Apart from being a conservation centre of various animals it also provide various facilities such as an aquarium , nocturnal house, Botanical garden, Museum, Jurassic Park,

Solar power house etc. The Lucknow zoo attracts 9,00,000 to

1,00,00,000 of tourists due to the above-mentioned facilities which resulted in large amount of solid waste generation. This consists of complex waste such as polythene bags, wrappers, papers, plastic bottles and waste from cafeteria. On the other hand animal present inside the zoo produces large amount of organic waste. This is why the main objective of the present study is based on how

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Recent Advances in Bioenergy Research Vol. III 2014 to manage solid waste problems in zoo premises with identification of conversion techniques for waste to energy on theoretical assessment of data collected. .

Fig.1 Month wise total visitors in Lucknow Zoo (Data collected from the official website of Lucknow Zoo). 2.2

Materials and Methods

2.2.1 Preparation of questionnaire In order to obtain technical information infor regarding management of Zoo, Z amount of solid waste generation, its utilization questionnaire method is adapted. The questionnaire was categorized mainly in three parts: parts i.

Animal waste

ii.

Biomass waste

iii.

Anthropogenic waste

2.2.2 Identification of different routes for fo conversion of waste to energy Various routes of conversion for waste to energy have been identified. The routes may vary depending on the type of waste. According to the waste collected from the zoo premises, it can be reduced or manage by using various advanced and technical processes. processes predication is done on the basis of data collected by the questionnaire method.

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The

Recent Advances in Bioenergy Research Vol. III 2014

Fig. 2 Questionnaire for the animal waste

Fig. 3 Questionnaire for the Biomass waste 2.3

Results and Discussion

2.3.1 Data collection by questionnaire method According to the data collected from the questionnaire it was found that Zoo has 468 mammals, 378 reptiles and 100 birds. These animals are the source of large amount of organic waste which is valuable for the bio-energy bio energy production. Apart from the animal waste w big

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Recent Advances in Bioenergy Research Vol. III 2014 amount of plant derived waste is also collected in the Zoo as there is more than 1000 small and big trees were present. Anthropogenic waste was mainly consisting of non-biodegradable non biodegradable materials such as plastic wrappers, bottles, polythene and food waste as a biodegradable waste. On the basis of the data collected from the questionnaire different alternative of energy sources can be opted from the different type of waste materials. Thus the solid waste can be utilized in sustainable way without harming ing the environment.

Fig. 4 Questionnaire for the anthropogenic s waste Table-1. Quantitative measurement of the Fresh animal waste in the Zoo premises Name of the animals

No. of animals

Waste generated per day

Himalayan black bear Sloth bear Giraffe Rhinoceros Swamp dear Barking dear Hog deer Samber deer Spotted deer Rabbit Total

3 3 2 1 57 23 30 15 198 10 342

1 Ibs×3 (1 Kg. =2.2 Ibs ) 1Ibs ×3 5 Ibs ×2 1000 Ibs×1 3500 gm 3500 gm 3500 gm 3500 gm 3500 gm 2800 gm 482 kg/day 17

Recent Advances in Bioenergy Research Vol. III 2014 Table-2.Quantitative measurement of plant derived waste inside the zoo premises Sources

Composition

Twigs Dry leaves Total

Approx. 2 Kg/day Approx. 3.5-4.0 Kg/day Approximate 6.5-7.0 Kg/day

2.3.2 Potential alternative approaches for utilization of waste material The suggestive measures based on data collected from the questionnaire can be taken for the best use of solid waste generated inside the Zoo premises. These alternatives routes of conversion may vary depending upon the type and composition of organic waste. Following conversion routes are identified for the energy recovery from the waste collected from the Zoo. 2.3.3 Animal and biomass waste Animal and biomass waste having organic content are utilized as a feedstock in process of biological conversion (Li et al., 2010) and thermo chemical conversion. Both waste vary in chemical composition, physical form and quantity produced. The main factor involve the variation of animal waste are i.

Digestive physiology of various species

ii.

Composition and form of the diet

iii.

Stage of growth and productivity of animal On the other hand plant derived waste mainly differs in respect to their composition and

nature of degradability.

Fig. 4 Conversion platform for animal and biomass waste in to energy

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Recent Advances in Bioenergy Research Vol. III 2014 a) Biological conversion This process mainly involves biological treatment of animal and biomass waste by (i) anaerobic digestion with full scale production of combustible biogas by using phototrophic microorganism such as Algae, (ii) fermentative process for production of Bio-hydrogen and (iii) Plant nutrient to support crop production. Anaerobic digestion involves breakdown of complex organic materials and the biogas is formed by following hydrolysis, acidogenesis, acetogenesis and methanogenesis

(Van

Haandel and Van der Lubbe, 2007). The solid compound gets liquefy in acidogenesis and converted in to acid, alcohol and volatile fatty acids. Bio-hydrogen production is carried out by following three steps such as photosynthetically by algae in two stage photosynthesis and H2 production, photobiologically by photofermentataive bacteria and by anaerobic fermentative bacteria.

Fig.5 Flow Diagram of Anaerobic Digestion Process and End Points of products

b) Thermo-chemical conversion This is a high temperature chemical reforming process that breaks apart the bond of organic matter and converts these intermediated in to char, syngas and highly oxygenated biooil. The advantages of thermo-chemical conversion involve small footprints, efficient nutrients recovery, no fugitive gas emission, short processing time on the order of minutes

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Recent Advances in Bioenergy Research Vol. III 2014 and high temperature elimination of pathogens and pharmaceutically active compound (Cantrell et al., 2007). Pyrolysis, gasification and direct liquefaction these three processes are mainly identified for the thermo-chemical conversion. Pyrolysis process mainly drive out in absence of oxygen and it converts organic waste in to a mixture of char and volatile gases. Slow pyrolysis has benefits of energy production and carbon credit generated from carbon sequestration (Tri et al., 1996; Budenheim et al., 1994). Direct liquefaction process includes hydrolysis of organic waste (lignocellulosic materials) in to bio-oil. It proceeds in a pressurized environment (5-20 Mpa) and typically occurs at low temperature. This process convert organic portion of dry weight or biomass in to the minor byproduct and primarily non condensable permanent gases, CO, CO2, H2 and low molecular hydrocarbon gases with the help of air, oxygen or stem as a reaction medium (McKendry, 2002). 2.3.4 Anthropogenic waste These wastes are produced due to human activities and mainly consist of fraction of degradable and non-degradable wastes. These wastes may create serious hygienic problem as it contains high amount of organic matter and pathogens. Vermicompsting can be applied for the degradable waste, which enhance the soil fertility and increases crop yield. Non biodegradable waste can be managed by incineration. This process involves burning of solid waste in a properly designed furnace under suitable temperature and operating conditions. Incineration provide reduction of waste to one tenth of their volume without producing offensive gases this process not only help in the elimination of odor but also protect the wall of incinerator. 2.3.5 Evaluation of the Biogas and Electricity production The total amount of organic waste collected inside the zoo premises was found to be 490 kg/day (approx.). This amount of organic waste is capable to produce 25.48 m3/day biogas and 25 kWh of electricity.

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Recent Advances in Bioenergy Research Vol. III 2014

Fig. 6 Main thermo Chemical conversion Processes, their Intermediate products and suggested End use 2.4

Conclusion Waste management is highly important issue for Zoos as it poses as a good source of

various types of waste like animal waste, biomass and anthropogenic waste. These can be managed using the 3 R’s (Recycling study guide, 1998) (Reduce, Reuse and Recycle of the reduction principal of sustainability). The visitors of the zoo should be strictly prohibited from throwing unwanted materials within the zoo premises in order to reduce the volume of waste to great extent. From the present study area it has been found that of all the three wastes (animal waste, biomass and anthropogenic waste) can be managed properly as per the suggestive measures identified in the present study.

References 1.

Budenheim D.L. and Wydeven T., 1994. Advances in Space Research (ISSN 0273-1 177), 14:113-123.

2.

http://www.lucknowzoo.com/list_of_visitors1.html

3.

Keri B. Cantrell, Thomas ucey, Kyoung S., Ro, Patrick G. hunt- Livestock waste to energy generation opportunities, United states Department of Agriculture, ARS, coastal Plains soil, Water and plant research center, 2611 W, lucas St. Florance, Sc29501, USA.

4.

Li, R., Chen, S., Li, X., 2010. Biogas production from anaerobic co-digestion of food waste with dairy manure in a two-phase digestion system. Appl. Biochem. Biotechnol. 160, 643–654.

21

Recent Advances in Bioenergy Research Vol. III 2014 5.

McKendry, P., 2002. Energy production from biomass (part 1): overview of biomass. Bioresource Technol., 83: 37–46

6. Morse, D. 1995. Environmental considerations of livestock producers. J. Anim. Sci. 73:2733−2740. 7. P. W. Gibson (Bienergy Organizers, Inc., Baltimore, MD), Baltimore Zoo Digester Project: Final 8. Recycling Study Guide, Wisconsin Department of Natura Resources, January, 1988. 9. Report, DOE/R3/06058–T1, U.S. Department of Energy, 1980. 10. Tri, T.O., Edeen, MA., and Henninger, D.L., SAE 26th International Conference on Environmental Systems, Monterey, CA. Paper #961592, 8p. July 8-1 1, 1996. 11. V. N. Gunaseelan, “Anaerobic Digestion of Biomass for Methane Production: A Review,” Biomass and Bioenergy 13, 83–114 (1997). 12. Van Haandel, A.C., van der Lubbe, J., 2007. Handbook biological waste water treatment: design and optimisation of activated sludge systems, first ed. Quist, Leidschendam. 13. Williams, P.E.V. 1995. Animal production and European pollution problems. Anim. Feed Sci. Technol. 32:106−115.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 3 BIOPROSPECTION OF EUPHORBIA COTINIFOLIA FOR BIOFUEL: CHROMATOGRAPHY STUDY Punam Puri, Amita Mahajan, Anjana Bhatia & Navjot Kaur

Abstract Biodiesel have been receiving attention in recent years to overcome energy crisis. India is rich in biodiversity and known for vast treasure of knowledge about use of plants for various purposes. Many Euphorbiaceae species have been proposed as potential biofuel crops. E.cotinifolia as one of the potential biofuel crop in future. In this research paper the focus will be on the chemical constitutions that have been identified from Euphorbia cotinifolia species. With different modes of chromatography, different lipids were determined. TLC in the adsorption mode (silica gel), the principle application in lipid analysis is for the separation of different lipid classes from plant tissues. It is a relatively easy matter to resolve each of the main simple lipids from a tissue in one step, i.e. cholesterol esters, triglycerides, free fatty acids, cholesterol and diacylglycerols, using mobile phases consisting of a mixture of hexane and diethyl ether, with a little formic acid to ensure that the free acids migrate successfully. The aim of the study was to investigate the biochemical constituents and thin layer chromatography (TLC) of the ethanol and acetone extracts of Euphorbia cotinifolia. Keywords: - Euphorbia cotinifolia, chromatography, lipids analysis, complex lipids. 3.1

Introduction Renewable energy is an alternative solution for fossil fuel because it is clean and

environmentally safe.

Professor Melvin Calvin (Calvin, 1977) revived the idea that

hydrocarbon-producing plants could be used as future oil and other chemical sources. He also gave the energy farming concept. The plant families mainly Euphorbiaceae and Asclepiadaceae were screened for assessing their suitability as a source of low molecular weight (mw) and non-polar petroleum-like hydrocarbons. Air-dried plant materials were successively extracted with acetone and benzene, and the extracts were analyzed spectroscopically for yield of rubber, wax, glycerides, isoprenoides and other terpenoides. In the Euphorbiaceae family, the genus Euphorbia comprises 2000 species ranging from annuals 23

Recent Advances in Bioenergy Research Vol. III 2014 to trees and is subdivided into many subgenera and sections. This family is one of the largest families in the plant world. All contain latex and have unique flower structures (Barla et al., 2006; Chaudhry et al., 2001; Jassbi et al., 2006). In the search for new plants with a high potential for the production of chemicals and liquid fuels as alternative energy sources, several Euphorbia species have been previously examined for possible economic utilisation (Zarrouk and Cherif, 1983; Hemmers and Gülz, 1986; Villalobos and Correal, 1992). This genus has been investigated in view of different specialties, like more energy content, as alternative source of hydrocarbons, laticifers, phytochemicals and systematic (4-10). Plants of the genus Euphorbia are well known for their chemical diversity of their isoprenoid constituents. An analytical screening program has been conducted by the USDA [11–15] to evaluate and identify plant species as source of high energy, easily extractable compounds suitable for fuel, chemicals and petroleum-sparing chemical feedstock. Plant families that yielded

more

than

one

promising

species

were

Anacardiaceae,

Asclepiadaceae,

Caprifoliaceae, Compositae, Eupforbiaceae and Labiaceae. This research emphasized on phytochemical screening of latex extract of E.Cotinifolia by chromatography techniques. Chromatography is a method for separating the components of a mixture by differential adsorption between stationary phases and a mobile (moving) phase. Thin-layer chromatography and column chromatography are different types of liquid chromatography. A column (or other support for TLC, see below) holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster. As the components elute from the column they can be quantified by a detector and/or collected for further analysis. An analytical instrument can be combined with a separation method for online analysis (16-18). 3.2

Objectives The general project objective is to evaluate the potential of integrating biofuel raw

material production in wasteland, adapted technology and their implications on energy access, ecological sustainability, food security, economic and social efforts for wellbeing for future generation.The present proposal deals with a plant of Euphorbia cotinifolia which will be taken up for fatty acid and hydrocarbon estimation. In this research paper main objectives are:-

24

Recent Advances in Bioenergy Research Vol. III 2014 i)

Extraction of oil and different lipids polar as well as non polar lipids with respective solvents by column chromatography technique.

ii)

To study phyto-chemical screening of plant extracts of E.cotinifolia.

iii)

To find out the same compound in the known and unknown sample.

iv)

Further analysis of biocrude oil (viscosity, density, cetane number, distillation range water content, discussion of fuel properties of triglycerides etc.).

3.3

Methodology

3.3.1 Materials and methods Biocrude of E. cotinifolia of acetone extract had determined through soxhlet extraction method. 3.3.2 Phytochemical screening of plant extracts E.Cotinifolia The latex acetone extract of E.Cotinifolia was analyzed by thin layer chromatography (TLC) and column chromatography. 3.3.3 Method for Thin layer chromatography TLC on silica gel G plates, on Silica Gel G plates (Loba chemie Pvt.Ltd.107, Wodehouse road, Mumbai, India).Prepared silica gel slurry .Silica gel plate of layer thickness 0.25 mm kept in oven activation for 2-3 hrs at 700 C. Sample of acetone extract of E.Cotinifolia and standard oleic acid were loaded and allowed to run in developing solvent Petroleum Ether: Diethyl Ether: Acetone (7:3:0.1), spray reagent concentrated sulphuric acid, p-anisaldehyde and glacial acetic acid (2:1:100). After development, the phytocompounds plates were transferred into iodine chamber (resublimed iodine, Avarice laboratories Pvt. Ltd., India). 3.3.4 Method for Column chromatography Weighed 5gm silica gel 60-120 (Avarice laboratories Pvt. Ltd., India) and made slurry in water. Poured the gel into the column tightly so that no air spaces were left. This left a space of 4-5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column securely to a ring stand .5ml sample of 3 mg acetone extract was loaded in the silica gel filled column. Once the sample was in the column, fresh eluting solvent was added to the top and we were ready to begin the elution process. Only force the solvent to the very top of the silica:

25

Recent Advances in Bioenergy Research Vol. III 2014 do not let the silica go dry. Add fresh solvent as necessary. The following solvent system ran in the column chromatography were:1) Hexane: petroleum ether (90:10) respectively. 2) Hexane: petroleum ether (50:50) resp. 3) Petroleum ether: diethyl ether (50:50) resp. 4) Diethyl ether: acetone (50:50) resp. 5) Acetone: methanol (50:50) resp. 6) Methanol 100% The first solvent running in the column was hexane and petroleum ether in (90:10 ratios) respectively and the total quantity was 100 ml. There was appearance of 3 colored bands in the column. The colored bands were travel down the column as the compound was eluted. As soon as the colored compound began to elute, the collection beaker was changed. The colored fractions were collected separately. Similarly all the above solvents ran in the column and collected different fractions from the running column. 3.4

Results

Results of TLC

Fig. 1 Biocrude of plant extract spp. E.Cotinifolia

Fig. 2 Performing TLC

26

Recent Advances in Bioenergy Research Vol. III 2014

Fig 3. Results of TLC

Fig 4. Results of TLC

Results of column chromatography

Fig 6. Shows band formation by using Fig. 5. Shows loaded sample in coloumn

different solvents running in the column

chromatography

chromatography.

27

Recent Advances in Bioenergy Research Vol. III 2014

Fig 7. Shows different colored band Fig 8. Shows band is travelling down in the formation

by

using

solvent

hexane: column.

petroleum ether (90:10) respectively.

Fig 10. Shows five fractions in eppendorf vials were obtained from Fig 9. Shows oil sample was obtained by running first solvent i.e. hexane: petroleum ether (90:10)

28

respective solvents.

Recent Advances in Bioenergy Research Vol. III 2014 3.5

Discussion The results obtained from TLC and column chromatographies were good. In TLC

standard sample was oleic acid travel with acetone extract of biocrude of E. Cotinifolia.in the results arrows shows that there was a same compound in E. Cotinifolia which were present in the known sample. In fig. 4, TLC results shows that the standard oleic ran with oil sample extracted during column chromatography. The good results were obtained. In column chromatography the results were also good and surprising we got oil in very small fractions from 3 mg sample during running the solvent hexane and petroleum ether (90:10) and other five fractions were collected from respective solvents. In future we will do the triglycerides analyses (viscosity, density, cetane number, distillation range water content discussion of fuel properties of triglycerides etc.). The sample will be forward to SAIF Centre Chandigarh for IR, GC/MS AND NMR data. 3.6

Conclusion The bio-energy system makes a significant contribution to the world’s growing energy

needs. The renewable sources would only be able to compete with the fossil fuel resources, if special plant crops containing energy-producing, hydrocarbon-like material are breed and cultivated. A great advantage of utilization of such plants is by replacing the current use of the traditional food crops for fuel production and providing the biodiesel industry with a more consistent "green" supply. Large scale experiments would be required to analyses of the different classes of secondary metabolites isolated from this plant. In this paper more emphasize on phyto-chemical analysis of compounds and extraction of components from different running solvents system and all these extractions further analysed by different techniques. Hope this will be the one of the future petrocrop in the world.

References 1. Calvin M. Chem Eng News 1978;20:31–6. 2. Barla A, Biraman H, Kultur S, et al. Secondary metabolites from Euphorbia helioscopia

and their Vasodepressor activity.Turk J Chem, 30, 2006, 325- 332.

3. Zarrouk M. and Cherif A. (1983), Lipid contents of Biol. Plant. 42, 417Ð422. 4. Kalita D, Saikia CN (2004). Chemical constituents and energy content of some latex bearing plants, Bioresource Technology 92 (3), 219-227.

29

Recent Advances in Bioenergy Research Vol. III 2014 5. Monacelli B, Valletta A, Rascio N, Moro I, Pasqua G (2005). Laticifers in Camptotheca acuminata Decne: distribution and structure. Protoplasma 226 (3-4), 155-161. 6. Bruni, R, Muzzoli, M, Ballero, M, Loi, MC, Fantin,0G, Poli, F, Sacchetti, G. (2004). Tocopherols,Fatty Acid acids and sterols in seeds of four Sardinian wild 7. Mallavadhani UV, Satyanarayana KVS, Mahapatra A, et al. (2006). Development of diagnostic, microscopic and chemical markers of some Euphorbia latexes Journal of Integratıve Plant Biology 48 (9), 1115-1121. 8. Shi HM, Williams ID, Sung HHY, et al. (2005). Cytotoxic diterpenoids from the roots of Euphorbia ebracteolata, Planta Medıca 71 (4), 349-354. 9. Jiao, W, Mao, ZH; Dong, WW, et al. (). Euphorbia factor L-8: a diterpenoid from the seeds of Euphorbia lathyris. Acta Crystallographica Section E-Structure Reports Online, 64, (03). 10. Suarez-Cervera M, Gillespie L, Arcalis E, L Thomas A., Lobraeau –Callen D., Seoane– Camba JA., (2001). Taxonomic significance of sporoderm structure in pollen of Euphorbiaceae: Tribes Plukenetieae and Euphorbiceae. Grana 40 (1-2), 78-104. 11. Abbott TP, Patterson RE, Tjark LW, Palmer DM, Bogby MO. Econ Bot 1990;44:278– 84. 12. Bagby MO, Buchanan RA, Otey FH. In: Klass DL, editor. Biomass as a non fossil fuel source. ACS Symposium. Series, vol. 144. 1981. p. 125–36. 13. Campbell TA. Econ Bot 1983; 37:174–80. 14. Carr ME, Bagby MO, Roth WB. J Amer Oil Chem Soc 1986;63:1460–4. 15. Seiler GJ, Carr ME, Bagby MO. Econ Bot 1991;45:4–14. 16. Archer J. P. Martin (1952). "The development of partition chromatography". Nobel Lecture, December 12, 1952. Nobel Lectures, Chemistry 1942-1962, Elsevier Publishing Company, Amsterdam, 1964. 17. Ettre, L. S. (2001). "The Predawn of Paper Chromatography". Chromatographia, vol. 54, pp. 409-414. 18. Frederick Sanger (1988). "Sequences, Sequences, and Sequences". Annual Review of Biochemistry, vol. 57 (1988), pp. 1-28

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 4 COST EFFECTIVE ELECTRICAL POWER GENERATION IN PUNJAB USING AGRICUTURAL BIOMASS Suman

Abstract Energy is the key input to drive and improve the life cycle. The impact of the traditional fossil fuels on our environment and the fact that these are fast depleting sources of energy, have encouraged the need to find alternative energy sources to fossil fuels. Biomass can be burned for fuel by itself or co-fired with other fuels. But in recent years Biomass and Coal co-fired based systems are receiving more attention due to high Power Generation Efficiency and reduced Green House Gas (GHG) emissions. This paper critically analyzes the scope, potential and implementation of agricultural -Biomass conversion to Energy in Punjab context. Brief descriptions of potential conversion routes have been included, with their possible and existing scope of implementation. As far as possible, the most recent statistical data have been reported from various sources. The discussion reveals that a large potential exists for the Biomass feed-stocks from the various kinds of waste Biomass. The analysis to identify irreversibility and the ways to improve the performance of Power Generation systems is discussed. The Energy generated from various kinds of Biomass products is analyzed and its role to improve the Power Generation systems is also presented. Keywords: Biomass, Electrical energy, Efficiency, Greenhouse gas (GHG), Green Economy, Power Generation 4.1

Introduction In recent years, the World is facing Energy crisis, Economic, Green & clean

Environmental problems. A lot of research efforts are put to find economically viable and sustainable energy resources to reduce this energy crisis with green and clean environment. With growing population, improvement in the living standard of the humanity, industrialization of the developing countries, the Global demand for energy is expected to increase. India rank fifth in the world in total energy consumption (with installed capacity 228.722 GW up to September 2013). Coming to Power production in the country, India ranks 31

Recent Advances in Bioenergy Research Vol. III 2014 sixth in the world with increased installed power capacity from 1362MWh to 855.3 billion kWh (up to 2012) since independence [6]. This achievement is impressive but not sufficient. The country still encounters peak and energy shortage of 9 % & -8.7 % respectively (up to 2013). The major sources which meet the energy requirement of India are coal and oil. The use of these fuels is a problem because of the reasons: a) The natural formation of Coal and Oil is a very slow process which takes long time .b) Emission of Green house gases. c) These are fast depleting sources of Energy. Moreover, India is dependent on the imports for Oil requirements. In 2004–05, 72% of India’s total oil consumption was dependent on the imports [2]. This figure reached to 76.5% during 2009–10, 78% for 20010–11, and the tentative figure for 2011–12 is 80.5% [3]. These imports are increasing year after year with the growing economy of the country and contribute in continuous increase of the import bills. By 2025, it will be importing 90% of its crude oil from OPEC countries. Therefore, Utilization of renewable energy sources is one of the best ways to meet the objectives as : a) These are the energy sources that will never run out. b) These sources are Environmental friendly means reduce Green house effect and provide clean Environment. c) Social –cost benefits. The major Renewable sources of Energy available freely are Solar energy, Wind energy, Small Hydropower, Biomass, Biogas, and Energy recovery from Municipal and Industrial wastes. 4.2

Status of Bio-energy Resources in Punjab India’s energy basket has a mix of all the resources available including renewable.

Biomass contributes as the world’s fourth largest energy source up to 14% and in developing countries it can be as high as 35% of the primary energy. Punjab the “Grain Bowl” of India is the major agriculture state of the country. Agricultural biomass has immense potential for power production in Punjab. Punjab has made tremendous progress not only in the agriculture sector but in the industrial, transport and household sectors. This has increased energy demand significantly. This state does not have its own resources of conventional fuels such as coal, petroleum products for electricity energy. The state has to depend on neighboring states for petroleum products and on the far-off states for coal. But the state has plenty of renewable energy sources, such as biomass, wind and solar energy, which can be exploited to provide sustainable energy base for socio-economic development. Table 1: shows the various type of biomass available in Punjab [3]. Punjab "Granary of India” is historically considered to be one of the most fertile areas on Earth. The region is ideal for growing rice, wheat, cotton, sugarcane, maize and

32

Recent Advances in Bioenergy Research Vol. III 2014 vegetables covering nearly 1.5 percent of India's land. Today the state produces nearly India’s 11% rice, 22% wheat, 18 % cotton and 3 % sugarcane .In worldwide terms; this represents 1/30th or 3% of the world's production of these crops, so Punjab produces world’s 1% of cotton, 2% of its wheat, 2% rice and 0.3% of sugarcane. Table2: Shows production of major crops during the recent years with area in Punjab & it is clear that from the last few years’ production of major crops increases which result in increase in the bio waste product [5]. Table 1: Type of Biomass S.No.

Type of biomass

Name of crop

1

Straw

Wheat ,Paddy, Barley, Pulses

2

Stalk

Cotton, Maize, Rapeseed &mustard

3

Bagasse Tops & leaves

Sugarcane Sugarcane

4

Cobs

Maize

5

Husk

Paddy

Table-2: Production of Major Crops during the Recent Years 2009- 10

2010-11

2011-12

2012-13

2013-14(E)

Year Type of BioMass

Area

Production Area

Production Area

Production Area Production Area Production

(lac

(lac MT)

(lac MT)

(lac MT)

ha.)

(lac ha.)

(lac ha.)

(lac (lac MT)

(lac

ha.)

ha.)

(lac MT)

Rice

28.02

112.36

28.31

108.37

28.18

105.42

28.45

113.69

27.50

110.00

Sugar

0.60

40.56

0.70

49.04

0.80

56.53

0.82

56.73

0.95

66.50

Wheat

34.02

151.69

35.10

164.72

32.03

125

34.52

140.60

35

161.02

cotton

5.11

20.06

4.83

18.22

5.15

16.21

4.81

16.44

5.20

19.58

Maize

1.39

4.75

1.33

4.91

1.26

5.02

1.29

4.71

1.50

5.40

cane

33

Recent Advances in Bioenergy Research Vol. III 2014 On the basis of survey of ratios of various major crop residues for the year 2012-2013, the net production of residue could be around 481 Lac MT ,as described in greater detail in Table 3. Table -3: Estimation of Biomass Production in Punjab (Crop Wise Data) Crop

Rice

Main Crop Production

Type of

Crop to

Residue

(Lac MT)

Residue

Residue

Quantity

ratio

(Lac MT)

Husk

0.3

34.107

Straw

1.3

147.797

113.69

Wheat

140.60

Straw

1.5

210.9

Sugarcane

56.73

Bagasse

0.3

17.019

Tops & leaves

0.09

5.105

Stalk

3.5

57.54

Gin Waste

0.1

1.644

Stalk+Cobs

1.5

7.065

Cotton

Maize

16.44

4.71

Grand Total

481.177

Further studies indicate that about 15-20% of the agriculture residue is available for power generation rest is used for other purposes such as cooking & cattle feed. So we have nearly 100 Lac MT crop residue is available for Power Generation [1]. 4.3

Power Consumption in Punjab The Total Demand of Electricity in Punjab is 48724 MU. The availability of Energy in

Punjab is approximately 46119 MU, facing energy shortage of 5.2%. Total energy in Punjab state is provided by the PSPCL with its own Thermal Plants and Hydro Plants. Electricity demand of Punjab will vary with changes in weather. On an average, the demand of power in Punjab will vary between 1,039LU to 2,072LU while the availability of power will also vary between 873LU to 1,584LU during different months of the year [4]. The variation in annual demand and energy requirement for the year April 2012 to March 2013 are given in Table 4. The common pool projects are the Bhakra Nangal Complex, the Dehar Power Plant & the Pong Power Plant. Punjab shares about 51% of the Power generated from the Bhakra Nangal Complex. 48% from the Power generated at the Pong

34

Recent Advances in Bioenergy Research Vol. III 2014 Project. By including this share of generation Punjab is still deficit with 2600 MU Power. According to PSPCL estimates, the power-supply gap will vary between 4% to 31% the entire year.

Table -4: Month Wise Power Supply Position of Punjab in 2012-13 Year

Demand

Energy

Peak

Demand

Surplus

(%)

Energy

Demand

Met

(+) /

Surplus

requirement

Deficit (-)

/Deficit

Availability

Surplus(

(%)

+)

Surplus/

/Deficit

Deficit

(-) April -12

6391

5246

-1145

-17.9

3031

2948

-83

-2.7

May -12

7236

6091

-1145

-15.8

3763

3651

-112

-3.0

June -12

10474

8452

-2022

-19.3

5437

5053

-384

-7.1

July -12

11520

8073

-3447

-29.9

6611

5867

-744

-11.3

Aug-12

9114

8751

-363

-4.0

5923

5374

-549

-9.3

Sep-12

8147

8147

0

0.0

4745

4622

-123

-2.6

Oct-12

8441

6860

-1581

-18.7

3813

3671

-142

-3.7

Nov-12

5676

4502

-1174

-20.7

3040

2941

-99

-3.3

Dec-12

5336

5336

0

0.0

2517

2362

-155

-6.2

Jan-13

5797

5197

-600

-10.4

3055

2938

-117

-3.8

Feb-13

5197

5018

-179

-3.4

3917

3844

-73

-1.9

Mar -13

5264

5264

0

0.0

2872

2848

-24

-0.8

Table -5: Anticipated Power Supply Position in the Punjab during 2013-14 Energy

Peak

Requirement

Availability

Surplus(+)/Deficit(-)

Requirement

Availability

Surplus(+)/Deficit

(MU)

(MU)

(MU)

(MU)

(MU)

(-) (MU)

50850

40819

-10031

-19.7

12200

35

9075

-3125

-25.6

Recent Advances in Bioenergy Research Vol. III 2014 The studies carried out for formulating the anticipated power supply in Punjab for the next year 2013-14, as shown in Table 5. indicate that there would be Energy shortage of 19.7% and Peak shortage Of 25.6% in Punjab. 4.4

Existing Technologies for Biomass Conversion Due to technological developments and cost reductions, renewable solar, hydro, wind

and biomass energy are gaining momentum across the globe. There are a variety of processes and technologies that convert biomass into heat, steam, electricity, and other types of fuel & products. Some of them are depicted in Table 6 [2]. Table 6: Waste Agricultural Biomass to Energy – Technology S.No Type of

1

Examples of Types of

Byproducts

Applications

Technology

Waste Handled

Direct

Crop residues such as

Carbon Dioxide, Water

Power Generation ,

Combustion

wheat straw, rice straw,

& Heat

Heating , Cooking

Crop residues such as

Syngas, Heat, Some

Power Generation ,

wheat straw, rice straw,

CO2 and H2O

Heating , Cooking ,

rice husk, Bagasse 2

Gasification

rice husk 3

Pyrolysis

Transportation

Crop residues such as

Bio- Ethanol

wheat straw, rice straw,

Power Generation , Transportation

rice husk 4

Fermentation

Sugarcane & starch

Solids (charcoal),

Power Generation ,

substrates like wheat,

Liquids (Pyrolysis oils)

Transportation ,

maize, sugar beet

and a mix of

Heating

Combustible gases 5

Esterification

Rape-seed

Glycerine and

Power Generation ,

RME(RapeMethyl

Transportation

Ester)

Above mentioned technologies which are already installed must be upgraded keeping requirements in mind while those which are presently running Global like Fermentation, Esterification. Brazil recovered from oil crisis because of development of Cars powered by

36

Recent Advances in Bioenergy Research Vol. III 2014 100% Ethanol or Petrol or combination of both, such technologies are awaited to be modeled for Punjab’s Energy Policy. 4.5

Biomass as a Coal Substitute Biomass Power technologies compete in niche applications as well as in direct

competition with Conventional Electricity sources in Centralized Electricity supply. In large scale grid based applications, cost is the primary determinant of competitiveness. A power plant with the capability of producing 8MW of electricity could cost up to 40 crore INR. While annual maintenance (done for 1week twice a year) costs 50 Lakhs INR. Variable expenses are related to price of Biomass cost (approx. 3500-5500 INR per metric ton) is highly variable, depending upon the source, location etc while other expenses include manpower wages [1]. Operating life of Biomass Power plant lies between 25 to 30 years. since the cost of setting a biomass plant is high as compared to thermal plant but it has many advantages over thermal power plant such as--a) Biofuels can be transported and store and allow for heat and power generation on demand. b) The energy balance of biomass plants indicates that biomass energy is 10 to 30 times greater than the energy input for fuel production and transport. c) Accessibility in rural areas where commercial fuels and centralized electric grid are not available. d) Greater employment for local populations. 5) Restoration of deforested and degraded lands by energy plantations. e) Near-zero fuel costs (paid in local currency), commercial use of a waste product, decentralized supply and increased fuel efficiency leading to an increase in the economic. f) Cost of electricity per unit from biomass power plant is lower than coal plan. 4.6

Environmental Criteria Expanding the share of Renewable Energy in its Energy mix is one of the key pillars of

India’s low-carbon development strategy. The Biomass fuels are more suitable & promising than coal due to its low carbon, sulphur and nitrogen content as depicted in Table 7. Since CO2 and acidification of SO2 & NO2 are primarily responsible for global warming & coal contain maximum value of these elements (Carbon, Nitrogen & Sulphur) as compared to other Biomass.[1] So coal contributes more towards the Global warming. Moreover depending upon the content of Carbon & Sulphur there is Environmental taxes (High Tax & Low Tax). High tax scenario with $50 per ton of carbon tax and $400 per ton of Sulphur dioxide tax. Low tax scenario with $25 per ton of carbon tax and $200 per ton of Sulphur 37

Recent Advances in Bioenergy Research Vol. III 2014 dioxide tax. The cost of delivered Electricity under the Low tax and High tax cases for Coal Power increases by 1.4 and 2.8 cents/kWh, respectively. Table -7: Estimated Analysis:(Where C-carbon, H-hydrogen,O-oxygen, N-nitrogen,Ssulphur) C

H

O

N

S

Ash content (%)

75

5

8

1.5

0.5

10

Rice husk

38.35

5.08

36.24

0.56

0.16

14.9

Wheat straw

48.54

5.73

40.71

0.81

0.17

8.5

Rice straw

43.36

5.44

39.03

0.87

0.10

19.2

Maize(stalk+cob)

49

4.9

-

0.6

-

Bagasse(dry basis)

49

6.5

42.7

0.2

0.1

1.5

Cotton stalk

51

4.9

43.87

1.00

-

6.68

Coal

Further Biomass fuels have less reactive character as compared to Coal & Cogeneration applications in agriculture processing industries typically achieve fuel efficiency of 40 to 45% compared to 30% efficiency of the conventional technologies . Although the conversion of Biomass to Electricity in itself does not emit more CO2 than is captured by the Biomass through photosynthesis. This analysis suggests that under, these advantages, together with more efficient and versatile Biomass Electricity Generation with Modern Technologies, have led to the transition of re-emergence of Biomass as a competitive and Sustainable Energy option in the Future Energy Scenarios. 4.7

Conclusion

Significant conclusions of this paper are as follows: a) Punjab has abundant capacity to produce reliable, price competitive and ecologically sustainable Bio-energy to meet the energy demand of domestic and commercial sector. A number of such Power Generation project have not only solved the rural electrification problem for the remote villages, where infrastructural costs could have been quite high for conventional electrification, but also the power generation cost has also been relatively low.

38

Recent Advances in Bioenergy Research Vol. III 2014 b) In Biomass, Carbon, Nitrogen & Sulphur contents are low, which favours in lesser or Zero Global warming. Moreover their quantity decides the Environmental tax, so for biofuel we have to pay less tax as compared to Coal. Further, it is analyzed that replacement of each KWh of Coal –based electricity by Biomass-based electricity is likely to reduce CO2 by 1Kg. c) Biomass based decentralized generation is likely to generate direct or indirect & skilled or unskilled employment to about 84 people in rural areas. d) Biomass based power plants helps in reducing the bio-waste disposal problem in effective way. e) Cogeneration applications in agriculture processing industries achieve fuel efficiency up to

40 to 45% as compared to 30% efficiency of the conventional technologies.

Apart from having so many Economical & Environmental benefits, it also opens a new door for future innovations in our Country.

Refereences 1. Buljit Buragohain, Pinkeswar Mahanta & Vijayanand S. Moholkal (2010) Biomass for decentralized power generation :The India perspective . Science Direct ,14:73-92 2. Sara Schuman & Alvin Lin(2012)China’s Renewable Energy Law & its impact on renewable power in China. Energy policy ,51:89-101 3. Jagtar Singh , B.S Panesar & S.K. Sharma (2008) Energy Potential through agricultural biomass using geographical information system-A case study in Punjab. Science Direct , 14:301-307 4. Load Generation Balance report 2013-2014 by Central Electricity Authority 5. Department of Agricultural Punjab (2013) National Conference on Agricultural for Kharif 6.

Compaign.

K.S.Sidhu .(2006) Director of Punjab state Electricity Board. Non Conventional energy resources

39

Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 5 DEVELOPMENT OF QUALITY TESTING METHODOLOGIES OF FUEL BRIQUETTES Madhurjya Saikia and Bichitra Bikash

Abstract Biomass material such as rice straw, banana leaves and teak leaves (Tectona grandis) could be densified by means of wet briquetting process. Wet briquetting is a process of decomposing biomass material up to a desired level under controlled environment in order to pressurize to wet briquettes at a lower pressure. Upon drying these wet briquettes could be used as solid fuels. This study is aimed at to develop methodologies to measure quality and handling characteristics of these briquettes and burning characteristics as well. Key words: Biomass, briquettes, durability, shear strength, impact resistance. 5.1

Introduction India produces yearly a large amount of agro residue such as rice straw, rice husk,

coffee husk, jute stick, coir pith, bagasse, groundnut shells etc. Some part of this agro residue is used as animal fodder. A large amount of agro residue is left on the paddy fields to be burnt or decomposed [Ponnamperuma et al., 1983]. Both ways are means of pollution to environment, as field burning of agro residue emits a lot of GHGs to environment and decomposition of the agro residue too produces methane gas which is considered of the GHGs [Campbell et al., 2002; Sokhansanj et al., 2006]. Instead of letting these agro residues to be burnt or decomposed, these could be converted to densified forms by briquetting method [Stanely, 2003]. This will mitigate the problems of pollution while at the same time we would be successful to trap this energy resource for industrial purposes. Briquettes are far better to handle rather than loose biomass. Biomass briquetting is the densification of loose biomass material to produce compact solid composites of different sizes with the application of pressure. Three different types of densification technologies are currently in use [Saikia and Baruah, 2013]. The first, called pyrolizing technology relies on partial pyrolysis of biomass, which is added with binder and

40

Recent Advances in Bioenergy Research Vol. III 2014 then made into briquettes by casting and pressing. The second technology is direct extrusion type, where the biomass is dried and directly compacted with high heat and pressure (Grover and Mishra, 1994). The last type is called wet briquetting in which decomposition is used in order to breakdown the fibers. On pressing and drying, briquettes are ready for direct burning or gasification. These briquettes are also known as fuel briquettes [Stanley, 2003, Chou et al., 2010, Saikia et al., 2013]. These fuel briquettes are the newest edition of briquettes manufactured at low pressure unlike the others. As these briquettes are produced at relatively low pressure, a series of quality test such as durability, compressive strength, shear strength, impact test and combustion tests are needed to be done so that they could be more competitive with existing types. 5.2

Parameters of Quality Assessment Durability, shear strength and impact resistance of briquettes are the basic parameters to

assess the quality of briquettes in terms of handling and transportability of fuel briquettes (Grover and Mishra, 1994). . 5.2.1 Durability Durability of briquettes gives the mechanical handling of the solid fuel (Chou, 2009). This is measured by standard procedure ASAE S269.4 [Kaylyan et al]. To measure durability, 100 g of sample is taken and sample is tumbled at 50 rpm for 10 minutes in a dust tight enclosure of size 300mm×300mm. Metal cloth of aperture size 4mm is used to retain crumbled briquettes after tumbling. Briquettes durability index in % given by=

!" #$ ,"

+&)

%&'() ) *" #$

&

" $'*) + , %&'() ,

×100

Durability test set up: Working principle: The test set up is fabricated according to specification of ASAE S269.4 (Temmerman et al., 2006). The set up consists of a box 300 mm× 300 mm × 125 mm mounted on a frame. The box can be rotated by wooden handle or a motor at 50 rpm for 10 minutes which simulates the probable conditions of briquettes under transport by truck or conveyer belt into furnace. Figure below shows a durability measuring test set up made according to specification. 5.2.2 Shear Strength Test To measure shear strength, a simple test is done. Briquette is sheared between two planes and shear force developed is the shear strength of briquette (Saikia and Baruah, 2013). 41

Recent Advances in Bioenergy Research Vol. III 2014

Fig. 1 Durability test set up

Fig. 2 Shear strength test set up

Shear strength test set up: To measure shear strength, shearing test set up has been fabricated. The instrument consists of three wooden plates. The middle plate with a central cylindrical hole of 45 mm diameter and 30 mm thickness slides over the bottom plate. In the top plate, a cylindrical hole of same diameter as that of moving plate with 20 mm thickness is being provided in such a way that it coincides with the one that is provided in the movable plate when this plate is fully inserted between top and non moving bottom plate. The movable plate is connected to dead weights. Shear strength, kPa =

2F π D2

Where F= Force causing shear in briquette, kN D= diameter of briquette, m 5.2.3 Impact Resistance Test This test simulates the forces encountered during emptying of densified products from trucks onto ground, or from chutes into bins. Drop tests can be used to determine the safe height of briquette production during mass production (Debdoubi A. et al., 2005). ASTM D440-86 method is used to determine impact resistance index (Kaliyan N. and Morey R.V., 2008).. In the drop test, briquettes are dropped twice from a height 1.83 m onto a concrete floor. An impact resistance index (IRI) is calculated. IRI =

100×N n

Where, N= Number of drops, n= Total number of pieces. The upper limit of IRI is 200 which would result if the briquettes are not broken even after dropping twice.

42

Recent Advances in Bioenergy Research Vol. III 2014 5.3

Parameters of Combustion Characteristics of Briquettes Proximate analysis and combustion rates of briquettes were done to assess effectiveness

of briquettes as cooking fuel. 5.3.1 Proximate Analysis Moisture Content: The moisture content is determined according to the method described in the Forestry Hand Book (Wenger, 1984). 10 g of sample is taken immediately upon sampling and then air dried. This air- dried sample is taken immediately in an aluminum moisture box and kept in an oven heated at 105º ± 3 ºC until constant weight is obtained. The difference of the oven dry weight of the sample and the fresh weight of the sample is used to determine the percentage of moisture content as follows: Moisture content, % =

-" %

,. + /"0 -" %

× 100

Ash content: For determination of ash content, ASTM Test No. D-271-48 is followed. At first, an empty 25 ml. silica crucible as well as the sample is heated in a moisture oven. Sample is weighed accurately to 2 g. The sample in the crucible is kept in muffle furnace with the lid cover. Muffle furnace temperature is set at 550º ± 50ºC and kept for 6 hours. After 6 hours of burning crucible is removed from the furnace and placed in a desiccators and weighed accurately. Percentage of ash content was calculated as follows: Weight of ash

Ash content, % = Weight of sample × 100 Volatile matter: Volatile matter of samples is determined by ASTM Test No. D- 271- 48. A clean platinum crucible of 10 ml. capacity is taken and heated in a furnace at 950ºC for 2 minutes and cooled in desiccators for 15 minutes. Crucible weighed to nearest 0.1 mg. Sample filled crucible is weighed and heated in a furnace at 950ºC for 2 minutes. After volatile matter escaped, the crucible is removed from furnace and cooled in air 2 to 5 minutes and then in desiccators for 15 minutes. The percentage of weight loss of the samples is reported as volatile matter as follows: VM= VM=

Wt. loss of dry sample Net wt.of dry sample

Wt. loss of wet sample ×100 Gross wt. of wet sample

100- percent moisture

× 100

× 100 (Wet samples)

Determination of fixed carbon content Fixed carbon (FC) is determined by ASTM Test No. D- 271- 48 FC (on dry basis) = 100- (% of volatile matter +% of ash) 43

Recent Advances in Bioenergy Research Vol. III 2014 FC (on wet basis) = 100 – (% of volatile matter + % of ash + % of moisture) 5.3.2 Calorific Value Calorific value is determined by using Automatic Microprocessor Calorimeter (5EAC/ML) (make- Changsha Kaiyuan, China) (Saikia M. and Baruah D., 2013). The briquette’s material is nicely ground and pellets of 10 mm diameter are prepared from grounded material. The pellets are placed inside a crucible so that tungsten wire touches the pellet. The calorific value is analyzed by the Automatic Microprocessor Calorimeter or auto bomb calorimeter. The system has facilities such as water cycling system, automatic water feeding, temperature adjusting with a PT 500 temperature sensor, zero drift bridge temperature circuit to ensure that temperature resolutions reach 0.0001

, auto-diagnose, remote data transfer and auto-

weight entry.

Fig. 3 Auto bomb calorimeter for proximate analysis 5.3.3 Combustion Rate Combustion rate or burning rate is the mass loss per unit time. The briquettes are dried at 105ºC so that it does not affect on combustion or burning. Dried briquette is placed on a steel wire mesh grid resting on three supports allowing free flow of air (Chaney J. O.et al. , 2010) Now the whole system is placed on mass balance. Briquette is ignited from top and mass loss data is taken at an interval of 30 seconds.

44

Recent Advances in Bioenergy Research Vol. III 2014

Briquette

Steel wire mesh Three leg support

Electronic mass balance

Stop watch

Fig. 4 Combustion rate determination set up for briquettes 5.4

Conclusion With the help of this study we aim to standardize the practice of briquette making for

the commercial purpose. Generally, there are very few literatures on the standardization of quality testing methodologies of briquettes and burning characteristics as well. Durability, shear strength and impact resistance of briquettes could be determined easily as discussed in the section II in order to assess the quality of briquettes in terms of handling and transportability. Higher the durability, impact resistance and shear strength, higher will be the handling characteristics. But we need to reach at an optimum value of these indexes in order to produce quality briquettes at a cost effective way as quality always adds cost to production. Similarly, we can also asses the burning rates of briquettes in room condition as discussed in the section III. This generally helps to build a rough idea of performance of briquettes in actual condition. Moreover, with the knowledge of burning rates, we can further manipulate parameters of briquette manufacturing such a density and porosity of briquettes in order to obtain a desired level of burning rate. Apart from that proximate analysis and calorific value tests will so help us to give answer to some of the questions regarding the fuel such as whether it produces too much harmful fly ash and unwanted gases which are general indoor air pollutants many households and effectiveness of the fuel in terms of heat value. A fuel without heat value would be useless as a lot of fuel will be needed during use for its lower heat value.

References 1. Stanley R. (2003). Fuel Briquette making, Legacy Foundation. 45

Recent Advances in Bioenergy Research Vol. III 2014 2.

Kaliyan N. and Morey R.V. (2008). Factors affecting strength and durability of densified of biomass products, Biomass and Bioenergy, Vol 33, pg 337-359.

3. Wenger (1984). Forestry Hand Book. 4. Chaney J. O., Clifford M. J., Wilson R. An experimental study of the combustion characteristics of low-density biomass briquettes. Biomass magazine 2010, Vol 1. 5. Grover P.D. and Mishra S.K. (1994). Development of an appropriate biomass briquetting technology suitable for production and use in developing countries Energy for Sustainable Development, Vol 1. 6. Faborod M. 0 (1987).Optimizing the Compression/Briquetting of Fibrous Agricultural materials, Journal of agricultural. Engineering Resources, Vol 38, pg 245-262. 7. Chen L. (2010). The development of agro-residue densified fuel in China based on energetics analysis, Waste Management, Vol 30, pg 808–813. 8. Suramaythangkoor T. and Gheewala S.H. (2010). Potential alternatives of heat and power technology application using rice straw in Thailand, Applied Energy, Vol 87, pg 128–133. 9. Chou CS, Lin S.H., Peng C.C. and Lu W.C. (2010).The optimum conditions for preparing solid fuel briquette of rice straw by a piston-mold process using the Taguchi method, Fuel Processing Technology, Vol 90, pg 1041–1046. 10. Mania S., Tabil L.G. and Sokhansanj S. (2006). Effects of compressive force, particle size and moisture content on mechanical properties of biomass pellets from grasses, Biomass and Bioenergy, Vol 30, pg 648–654. 11. Chou C. S. (2009). Preparation and characterization of solid biomass fuel made from rice straw and rice bran, Fuel Processing Technology, Vol 90, pg 980–987. 12. Singh R.N. (2004). Equilibrium moisture content of biomass briquettes, Biomass and Bioenergy, Vol 26, pg 251 – 253. 13. Demirba A. (1997). Evaluation of biomass residue briquetting waste paper and wheat straw mixtures, Fuel Processing Technology Vol 55, pg 175–183. 14. Parikh Jigisha, Channiwala S.A. and Ghosal G.K. (2005). A correlation for calculating HHV from proximate analysis of solid fuels, Fuel, Vol 84 (2005), pg 487–494. 15. Bamgboye I. and Bolufawi S.J. (2009). Physical Characteristics of Briquettes from Guinea Corn (sorghum bi-color) Residue, Agricultural Engineering International: the CIGR Ejournal. 16. Husain Z., Zainac Z. and Abdullah Z. (2002). Briquetting of palm Fiber and shell from the processing of palm nuts to palm oil, Biomass and Bioenergy, Vol 22, pg 505 – 509. 46

Recent Advances in Bioenergy Research Vol. III 2014 17. Kumar N., Patel K., Kumar R.N. and Bhoi1 R.K. (2009). An assessment of Indian fuel wood with regards to properties and environmental impact, Asian Journal on Energy and Environment, Vol 10(02), pg 99-107 18. Saikia M. and Baruah D. (2013). Analysis of Physical Properties of Biomass Briquettes Prepared by Wet Briquetting Method, International Journal of Engineering Research and Development Volume 6, Issue 5 (March 2013), PP. 12-14. 19. Bikash B. Bhowmik R. and Saikia M. (2013). Challenges of Wet Briquetting from Locally Available Biomass with Special Reference to Assam, International Journal of Computational Engineering Research , Vol, 03, Issue, 7.

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Recent Advances in Bioenergy Research Vol. III 2014

Part II Thermochemical Conversion

48

Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 6 THERMAL AND CATALYTIC CRACKING OF NON-EDIBLE OIL SEEDS TO LIQUID FUEL Krushna Prasad Shadangi and Kaustubha Mohanty

Abstract

Verities of edible and non-edible oil containing seeds are available in nature. Out of these, the seeds containing edible oil habitually fulfill our food requirements. Hence edible seeds should not be use as a feedstock for production of fuel as it will directly affect the food chain. The seeds that are less edible or totally non-edible can be a considered as a feed stock for pyrolysis for the production of bio-oil. The quality and yield of pyrolytic product depends on biomass composition which include cellulose, hemicelluloses and lignin, oil and extractives. Higher amount of cellulose and extractive content enhances the yield of oil whereas the presence of lignin results in char during pyrolysis. The product yield and its quality is a function of reactor type, physical and chemical properties of biomass, final pyrolysis temperature, gas residence time in the reactor, pressure, ambient gas composition and catalyst types. The high viscosity, acid and water content of the thermal pyrolytic oil as well as low stability retards its use as an alternative fuel which can be overcome by cracking in presence of a suitable catalyst. Literatures revealed that catalytic cracking of non-edible oil seeds such as mahua, karanja, castor in presence of zeolite, Al2O3, CaO, Kaolin, Criterion-424 and BP 3189 enhanced the yield of pyrolytic oil and the fuel quality by altering the pH, reducing the water content and viscosity. It has been observed that the catalytic activity varies with the types of feedstock and its composition. Moreover non-edible seeds yield more oil compared to other feedstocks and has closer fuel properties to that of diesel which indicates their suitability as an alternative fuel for diesel engine. Keywords: Non-edible seeds; Thermal pyrolysis; Catalytic pyrolysis; Fuel properties 6.1

Introduction The depletion of the fossil fuel, increasing prices along with environmental

degradation is a major global problem. The utilization of energy also increased due to the rapid industrialization and growth of population. Present sources of energy are not enough to 49

Recent Advances in Bioenergy Research Vol. III 2014 prevail over the increasing needs. Since 1973 the energy resources have been doubled in developed countries but the requirement is still higher. The major demand of energy is fulfilled by the conventional energy resources such as coal, petroleum and natural gas. It was estimated that these oil sources might be depleted by 2050. The process of generating energy from these sources causes atmospheric pollution and creates problems such as global warming, acid rain etc. In view of increase in the energy demand as well as inherent problems associated with environmental pollution the attention of energy researchers has shifted to nonconventional energy sources such as wind, solar, tidal and biomass etc. Biomass is available in abundance which is renewable, cheap and can be converted it to energy in the form of liquid, solid and gaseous fuel. Natural biomass and its derivatives are the major sources of biomass which include wood, wood wastes, agricultural crops, crop wastes and residues, agricultural processing wastes, mill wood wastes and urban organic wastes. Energy crops are another and important resources which include short rotation woody crops, herbaceous woody crops, grasses, starch crops (corn, wheat and barley), sugar crops (cane and beet) and oil seed crops (such as soyabean, sunflower, safflower, mahua, karanja, neem, linseed, niger seed, guava, castor and other edible and non-edible seeds). One of the suitable and efficient methods for production of energy such as oil, gas and char is pyrolysis. Pyrolysis of wood byproducts has been a standard method for the production of energy for decades. Improvements and modifications have resulted in a more efficient and profitable biomass pyrolysis process that yields solid (bio-char), liquid (bio-oil and fine chemicals) and gaseous products (syn gas) (Goyal et al., 2006). Pyrolytic liquid is usually separated in to two phases, one is organic rich phase (the oil phase) and the other contains water and water soluble compounds (the aqueous phase). Pyrolytic liquid is being looked for as an alternate sustainable liquid fuel substitute for diesel and jet engines (the oil phase) as well as a source for commodity and fine chemicals (the aqueous phase). The oil phase is typically known as pyrolytic oil or bio-oil. The solid product known as bio-char is traditionally used as a soil enhancer that can hold carbon, boost food security, and increase soil biodiversity, and discourage deforestation. Bio-char also improves water quality and quantity by increasing the retention of nutrients and agrochemicals in soil for plant and crop utilization (Goyal et al., 2006). The product yield and quality of pyrolytic oil varies with type of feedstocks, their composition. Literatures revealed that less lignin and more extractive content in the biomass resulted in higher yield as well as better quality pyrolytic oil. Among the plant derivatives, seeds contain less lignin and more extractives in the form of oil. Several edible and non-edible seeds exist in the world. Since the edible seeds come under the food chain, hence should not be used for the production of fuel. 50

Recent Advances in Bioenergy Research Vol. III 2014 Therefore, it is always better to focus on non-edible oil seeds as feedstock to produce liquid fuel as they are outside the food chain. In this work, the use of non-edible seeds for the production of liquid fuel following thermal and catalytic pyrolysis is reported. Various process parameters that affect the yield as well as fuel properties of pyrolytic oil are discussed here. The role of catalyst during pyrolysis on the yield and quality of pyrolytic oil along with some drawbacks of thermal pyrolytic oil are also reported. 6.2

Pyrolysis and its Types Biomass pyrolysis simply means the heating of selected biomass at the elevated

temperature in absence of oxygen. During pyrolysis of biomass, the biomass undergoes various phases such as dehydration and depolymerization. Initially, within 100-200 oC most of the moisture and water gets eliminated and at the elevated temperature depolymerization of such chemical bonds associated with extractives hemicelluloses, cellulose and lignin takes place respectively. At the lower temperature the weaker chemical bonds and at higher temperature stronger chemical bonds breaks and the long chain chemical bond breaks to form short chain compounds (Singh and Shadangi, 2011). Pyrolysis can be carried out thermally where the operating parameter is only temperature and known as thermal pyrolysis. Pyrolysis in presence of catalyst is termed as catalytic pyrolysis. To overcome the drawbacks of thermal process, pyrolysis is carried out in presence of several catalysts which is discussed latter. 6.3

Process parameters that affect the yield

6.3.1

Effect of temperature on products yields One of the most important parameters that affect the yield of pyrolytic products is

temperature. The yield of pyrolytic liquid, gas and char varies with final pyrolysis temperature. During non-edible seed pyrolysis the foremost aim is to produce high yield of pyrolytic liquid/oil other than that of char and non-condensable gas. However, char and noncondensable gases are the other by-products of biomass pyrolysis. The temperature at which the yield of liquid is highest is considered as the optimum temperature. The thermal pyrolysis of Pistacia khinjuk seed (Onay, 2007 a), rapeseed (Onay and Kockar, 2004), safflower seed (Beis et al., 2002, Onay, 2007 b), pomegranate seed (Ucar and Karagoz, 2009), cherry seed (Dumanet et al., 2010), rape seed (Sensoz et al., 2000), jatropha seed (Figueiredo et al., 2011), tamarind Seed (Kader et al., 2011), mahogany seed (Kader et al., 2012), castor seed (Singh and Shadangi, 2011), neem seed (Nayan et al., 2013), mahua (Shadangi and Mohanty, 2014 a) 51

Recent Advances in Bioenergy Research Vol. III 2014 and karanja seed (Shadangi and Mohanty, 2014 b) was carried out and the effect of temperature parameter on yield is reported in Table 1. It was found that non-edible oil seeds can be used as a suitable feed stock for the production of alternative fuel by pyrolysis. Table 1 also provides the optimum temperature for maximum yield of pyrolytic liquid of several nonedible oil seeds. It was observed that the degradation temperature and the optimum temperature for maximum liquid yield varied with the types of feedstock. The yield of pyrolytic liquid increased with increasing temperature, whereas the yield decreased after a certain temperature. The temperature beyond which the yield of liquid starts decreasing is termed as optimum temperature for maximum liquid yield. Several researchers reported similar findings for the decrease in the liquid products. At higher temperatures, the secondary decomposition of char may also produce non-condensable gaseous products which would also contribute to increase in gas yield with increase in pyrolysis temperature. 6.3.2

Effect of heating rate on yield During pyrolysis the feed biomass is heated from room temperature to the

depolymerization temperature. Hence the required rate of heating plays an important role for maximum liquid yield. Lower the heating rate, the yield of char is more and liquid is less during pyrolysis. Higher heating rate breaks the heat and mass transfer limitation during pyrolysis and resulted in maximum yield of oil. Onay (2007 a) reported that the yield of pistacia khinjuk seed pyrolytic oil increased by 25 % when the rate of heating raised from 5 o

C min-1 to 300 oC min-1. Similar results were also obtained by Onay (2007 b) during

pyrolysis of safflower seed, rape seed (Onay and Kockar, 2004) and safflower seed (Beis et al., 2002). 6.3.3

Effect of sweeping gas flow rate on yield Biomass pyrolysis may be carried out in an inert atmosphere or without flowing of any

sweep gas. The yield of pyrolytic liquid does increase with supply of any sweep gas. In general nitrogen is used as a sweep gas to maintain inert atmosphere during the process. The important role of flow of sweep gas during pyrolysis is that it helps in reducing the formation of char, which is controlled by mass transfer of tar molecules into the light gas species. Onay and Kockar (2004) pyrolyzed rape seed in a fixed bed reactor with and without sweeping gas atmosphere and observed that the oil yield increased from 41.4 % – 51.7%. Onay (2007 a) and Onay and Kockar (2004) has reported that the effect of nitrogen flow rate on the liquid yield was negligible if nitrogen flow rate exceed more than 100 cm3 min-1. Similar conclusions

52

Recent Advances in Bioenergy Research Vol. III 2014 about the use of sweep gas flow rate are also reported by Kader et al. (2011) during pyrolysis of mahogany seed and during pyrolysis of pistacia khinjuk seed (Onay, 2007 a).

100

Total liquid yield, % Char yield, % 6.3.4

300

5

40

10

No

Fixed-bed

Pomegrana te

Karanja

Mahua

Neem

Castor

Mahogany

Tamarind

Jatropha

Rapeseed

Cherry

Safflower

25

Fixed-bed

Heating rate, o C min-1 Temp., oC

200

Thermal pyrolysis No 500 600 500

Continuous

Process N2 flow rate, cm3 min-1 Reactor type

Safflower

Pistacia khinjuk

Seeds

Rapeseed

Table 1. Thermal pyrolysis of non-edible seeds

30

Semi-batch reactor

At final temp.

25

5

600

550

600

500

500

500

420

400

550

550

47 5

525

550

600

57.6

68

54

44

44

23

45

49

64.4

38

54.2

20. 5

16. 5

-

40

35

20.9 3

30

57.7 5 21.5 5

55.17

13.5 17. 2

46. 1 20. 44

19.81

29.28

15.2

Effect of particle size on yield The yield of pyrolytic liquid during pyrolysis is also affected by the particle size of the

feedstock. Larger feed size decreased the heat transfer rate and increased the pyrolysis time which is liable for the formation of more char. In general, lower particle size of feed materials enhances the pyrolytic yield of liquid. Kader et al. (2011) experimented pyrolysis on tamarind seed and reported that liquid yield first increased up to a maximum value for feed size of 1.07 cm3 and subsequently decreased for larger feed size above 1.07 cm3. Pyrolysis experiments performed by Onay and Kockar (2004) on rapeseed, Kader et al. (2012) on mahogany seed and Onay et al. (2007b) on safflower seed suggested that particle size more than 1mm decreased the yield of pyrolytic oil by producing more char. 6.4

Fuel properties of Seed Pyrolytic Oil

6.4.1

Calorific Value

53

Recent Advances in Bioenergy Research Vol. III 2014 The calorific values of several non-edible seeds are presented in Table 2. This confirmed that non-edible seed pyrolytic oils have higher calorific value and closer to diesel. The calorific value of pyrolytic oil is a function of feedstock, operating conditions and the process used. Zhang et al. (2007) reported that non-edible seed pyrolytic oil having more heating value in comparison with wood pyrolytic oil. Table 2. Fuel properties of seed pyrolytic oil Seeds Calorific value, MJ kg-1 Viscosity, cSt References Pistacia khinjuk 39.84 -Onay, 2007 a Rapeseed 38.4 43 (50 oC) Onay and Kockar, 2004 Safflower 41 33 (50 oC) Onay, 2007 b Cherry 38.4 -Duman et al., 2011 Tamarind 19.1 6.51 (30 oC) Kader et al., 2011 o Mahogany 32.4 3.81 (26 C) Kader et al., 2012 Castor 35.64 83.19 (25 oC) Singh and Shadangi, 2011 Neem 32.49 22.6 (40 oC) Nayan et al., 2013 Mahua 41 33.97(25 oC) Shadangi and Mohanty, 2014 a Karanja 37.65 51.67(25 oC) Shadangi and Mohanty, 2014 b Diesel 45-47 3.68 (40 oC) -o 4.57 (25 C) 6.4.2

Viscosity High viscosity is one of the principal drawbacks of seed pyrolytic oils. Viscosity of

the pyrolytic oil is concerned with water content and water insoluble compounds. The higher water content and less water insoluble components in the pyrolytic oil reduce the viscosity of the pyrolytic oil. Table 2 shows the viscosities of various non-edible seed thermal pyrolytic oils. It is concluded that viscosity of pyrolytic oil varies with biomass types and the process type. The pyrolytic oil viscosity is much more higher compared to conventional diesel. Thus, the direct use of thermal pyrolytic oil in combustion engine is not acceptable and it needs upgradation to match the fuel properties. 6.4.3

pH of pyrolytic oil Pyrolytic oil is acidic in nature having high pH value ranging from 3-5. The high acid

content is due to the presence of carboxylic acid, acetic acid and formic acid in the pyrolytic oils. These acids are formed by the depolymerization of cellulose and hemicelluloses. High cellulose content in the biomass produces more acid in the pyrolytic oil during pyrolysis. The presence of various acids in the pyrolytic oil also reduces the thermal stability during storage. The direct use of seed pyrolytic oil as a transportation fuel may damage the engine. Table 3

54

Recent Advances in Bioenergy Research Vol. III 2014 confirmed that the non-edible oil seed pyrolytic oils are highly acidic in nature and also varies with the feedstocks. 6.4.4

Water content The presence of water content in pyrolytic oil reduces the heating value and the flame

temperature. Less water content decreases the viscosity which enhances the atomization towards complete combustion and reduces the harmful emissions such as SOx and NOx. It is confirmed from the literature that non-edible seed pyrolytic oil contains very less water in it which can be observed from Table 3. It was reported that the water content of pyrolytic oil varies from 0.33 % to 35 %. Since water is insoluble with organic pyrolytic oil most of the produced water and the water soluble chemicals collected separately as aqueous pyrolytic liquid (Shadangi and Mohanty, 2014 a, b). This confirmed that the non-edible seed pyrolytic oil can be accepted as a future alternative fuel. 6.4.5

Pour point Higher pour point is one of the disadvantages of pyrolytic oil. The pour point of seed

pyrolytic oil varies from 5-27 oC. Table 3 shows the pour point of several non-edible seed pyrolytic oils. High pour point of pyrolytic oil reduces the flow ability in winter especially in low temperature regions due to the formation of crystals which clogs the filter and reduces the efficiency of the combustion engines. Table 3. pH, water and pour point of some non-edible seed pyrolytic oil Pyrolytic oil pH Water content, % Pour point, oC References Neem 3.9 30-35 11 Nayan et al., 2013 Mahua 4.86 0.33 26.63 Shadangi and Mohanty, 2014 a Karanja 4.05 1.33 12.05 Shadangi and Mohanty, 2014 b Castor 3.7 -5 Singh and Shadangi, 2011 6.5

Catalytic Pyrolysis of Non-edible Seeds

6.5.1

Effect of Catalyst on Yield Theoretically catalyst has an effect on the rate of reaction. The catalytic effect

enhances the rate of reaction which increases the conversion during pyrolysis. It might have positive or negative impact. The influence of various catalysts on the pyrolytic yield of nonedible oil seeds were studied by different researchers. Catalytic pyrolysis of Pistacia khinjuk seed was carried out by Onay (2007 a) using BP3189 and Criterion-424 as catalyst and a liquid yield of 66.5% and 69.2% was found for the two catalysts respectively while it was

55

Recent Advances in Bioenergy Research Vol. III 2014 only 57.6% without catalyst. Shadangi and Mohanty (2014 a,b) studied the catalytic pyrolysis of Mahua and Karanja seed using CaO, Al2O3 and Kaolin and observed little increase in the yield of pyrolytic oil (4-6%) over thermal pyrolysis. 6.5.2

Effect of catalyst on fuel properties of pyrolytic oil Literature revealed that the use of catalyst altered the physical properties of non-edible

seed pyrolytic oils. The catalyst pyrolysis decreases the viscosity, increases the calorific value and decrease the acidity of pyrolytic oil. Shadangi and Mohanty (2014 a) reported that the use of CaO catalyst increased the calorific value from 41 to 43.15 MJ kg-1, reduced the viscosity from 0.033 to 0.018 Pa s and alter the pH acidic to basic (4.86 to 8.58) for Mahua pyrolytic oil. The effect of catalyst Al2O3, CaO and Kaolin on pyrolysis of karanja seed was studied by Shadangi and Mohanty (2014 b) and it was reported that CaO at 8:1 ratio produced less viscous (0.019629 Pa-s) pyrolytic oil compared to other catalysts. The calorific value increased for all the three catalytic pyrolysis with CaO (40.42 MJ kg-1), Al2O3 (41.21 MJ kg-1) and Kaolin (39.04 MJ kg-1) compared to thermal pyrolysis (37.65 MJ kg-1). 6.6

Conclusion The study of thermal and catalytic pyrolysis of non-edible seed confirmed that the

pyrolytic oil can be used as an alternative fuel. Non-edible seed produced more organic liquid in comparison with other feed stocks due to its high extractive content. The thermal pyrolytic oil is highly acidic in nature and very viscous and hence is not suitable for direct use in diesel engine. Catalytic pyrolysis is one of the suitable processes which altered the pH, reduces the viscosity and enhances the calorific value. Hence, catalytic pyrolytic oil from seeds will be suitable as an alternative fuel. However, more emphasis should be given on the effect of various catalysts to found out the most suitable catalyst which will enhance the fuel properties and stability of pyrolytic oils.

References 1. Beis S.H., Onay O., Kockar O.M. (2002) Fixed-bed pyrolysis of safflower seed: Influence of pyrolysis parameters on product yields compositions. Renewable Energy, 26: 21–32. 2. Duman G., Okutucu C., Ucar S., Stahl R., Yanik J. (2011) The slow and fast pyrolysis of cherry seed. Bioresource Technology, 102: 1869-1878.

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Recent Advances in Bioenergy Research Vol. III 2014 3. Figueiredo M.K.K., Romeiro G.A., Silva R.V.S., Pinto P.A., Damasceno R.N., D’Avila L.A., Amanda P. (2011) Pyrolysis Oil from the Fruit and Cake of Jatropha curcas: Produced Using a Low Temperature Conversion (LTC) Process: Analysis of a Pyrolysis Oil-Diesel Blend. EPE., 3: 332-338. 4. Kader Md A., Islam M.R., Joardder M.U.H. (2011) Fast Pyrolysis for Better Utilization of Tamarind Seed from Renewable Energy Point of View. Proceedings of the International Conference on Mechanical Engineering and Renewable Energy. Chittagong, Bangladesh., 22- 24. 5. Kader M.A., Joardder M.U.H., Islam M.R., Das B.K., Hasan M. (2012) Production of Liquid Fuel and Activated Carbon from Mahogany Seed by Using Pyrolysis Technology. In Green Chemistry for Sustainable Development. Bangladesh, Jessore: July 14. Id code: 53696. 6. Nayan N.K., Kumar S., Singh R.K. (2013) Production of the liquid fuel by thermal pyrolysis of neem seed. Fuel, 103: 437–443. 7. Onay O. and Kockar O.M. (2004) Fixed-bed pyrolysis of rapeseed (Brassica napus L.). Biomass and Bioenergy, 26: 289–299. 8. Onay O. (2007a) Fast and catalytic pyrolysis of Pistacia khinjuk seed in a well-swept fixed bed reactor. Fuel, 86: 1452–1460. 9. Onay O. (2007b) Influence of pyrolysis temperature and heating rate on the production of bio-oil and char from safflower seed by pyrolysis, using a well-swept fixed-bed reactor. Fuel Process Technology, 88: 523–531. 10. Shadangi K.P., and Mohanty K. (2014a) Thermal and catalytic pyrolysis of Karanja seed to produce liquid fuel. Fuel, 115: 434–442. 11. Shadangi K.P., and Mohanty K.. (2014b) Comparison of yield and fuel properties of thermal and catalytic Mahua seed pyrolytic oil. Fuel, 117 (30): 372–380. 12. Singh R.K., and Shadangi K.P. (2011) Liquid fuel from castor seeds by pyrolysis. Fuel, 90: 2538–2544. 13. Sensoz S., Angn D., Yorgun S. (2000) Influence of particle size on the pyrolysis of rapeseed (Brassica napus L.): fuel properties of bio-oil. Biomass and Bioenergy, 19: 271-279. 14. Uçar S. and Karagöz S. (2009) The slow pyrolysis of pomegranate seeds: The effect of temperature on the product yields and bio-oil properties, J. Analytical Applied Pyrolysis, 84: 151-156.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 7 EVALUATION OF MICRO GASIFIER COOKSTOVE PERFORMANCE WITH HANDMADE BIOMASS PELLETS USING REGION-SPECIFIC FUELS AND ASSESSMENT OF DEPLOYMENT POTENTIAL Debkumar Mandal, Vikas Dohare, Vijay H. Honkalaskar, Anurag Garg, Upendra V. Bhandarkar and Virendra Sethi

Abstract In rural areas of developing countries, large populations depend on traditional biomass such as wood, crop residues, and cattle-dung for cooking. Emissions from these activities have been reported to cause regional environmental impacts and global climate change. While engineered cook stove designs could be considered as a solution for reducing emissions, it is not independent of the region and season specific availability of fuels. A design solution based on gasification using engineered solid fuel from agricultural residues/coal powder is a relatively new development, and accounts for both the design of the cookstove and the physical property of the fuel. Work is needed to fine tune these gasifier cookstoves, quantify emissions and fuel consumption with emphasis on the utilisation of region-specific fuels. Micro-gasifier type cookstove have become more popular among small scale commercial operations where replacement of LPG with biomass pellets is economically profitable. However, in rural areas, where wood/agricultural residue is “freely” available as a natural resource, purchase of commercial pellet-based fuel is not affordable. The purpose of the study was to determine how locally available biomass may be used to take advantage of the micro-gasifier cookstove design. Further, it is also important to understand the behavioural and cultural obstacles for deployment of these stoves, and the suitability for ensuring reduction in indoor kitchen exposure. A field campaign was carried out in Gawandwadi village in Maharashtra, to understand deployability based on cooking practices and fuel availability. Experiments were conducted using local biomass residues (wheat husk, rice husk and saw dust) with locally available binding materials (cow dung, wheat flour and rice flour) in different proportions for making handmade “pellets”. Spherical balls were made 58

Recent Advances in Bioenergy Research Vol. III 2014 by pressing mixture (biomass residues with binding material) by hand. A simple “sewai” making home machine was modified and also used for extrusion for making disk shaped “pellets”. Standard Water Boiling Test was conducted to evaluate the performance of the handmade “pellets” in micro-gasifier stove. Besides, locally available fuel woods in small pieces were also used and the results were compared with the handmade “pellets”. The evaluation was done on the basis of Input power (kW), fuel required per output power (kg/kWh) and Efficiency (%). The efficiency and input power of the handmade “pellets” ranged between 33% to 57% and 2.4 kW to 3.1 kW respectively. Keywords: Micro-gasifier coookstove, Biomass, Handmade pellets 7.1.

Introduction Three billion people across the world use biomass for cooking and heating purposes.In

India, 82% of the population use solid fuels for cooking, of which rural and urban population accounts for 88 % and 24.6% respectively (URL 1).The global use of biomass fuels from traditional stoves (three stone, open fire) has been linked to adverse health effects, climate change, and deforestation. In India, forest area grew by ~2% annually, but afforestation quality is poor and deforestation persists due to urbanization across many parts in the country. Encouraging use of non-solid fuel in improved cookstoves (such as, micro-gasifier cookstove) can play a crucial role in preventing land degradation and deforestation, particularly in areas with negligible or negative afforestation rates.Improved cooking stoves have been shown to reduce the amount of fuel used to cook food and the air pollution produced in kitchens.58% of Indian population relies on fuel wood and 11% uses cow-dung for cooking purposes. On the other hand only 0.4% uses other fuels which include agricultural residues for cooking (URL 1). The traditional cookstoves used in the rural areas lead to the emission of unburnt gases such as CO,a toxic air pollutant in indoor air in rural as well as urban areas. The CO emission factors ranged widely from 3.0×10-2 g/Kg for coal gas/traditional stoves to 2.8× 102 g/Kg for the charcoal/ Angethi stoves (Zhang et al., 1999). Besides this, at a temperature of around 800-1000°C particulate matter (PM) is dominated by soot (carbon aggregate) in conventional cookstoves (Bolling et al., 2009). The CO standards for residential and agricultural areas are 2 mg m-3 for an 8 hour average, or 16 h-mg m-3 exposure equivalent according to the WHO guideline. These could be easily exceeded by CO exposure caused by traditional cookstoves using biomass (Zhang et al., 1999).

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Recent Advances in Bioenergy Research Vol. III 2014 To reduce these emission of CO and PM, improvements have been made in the combustion devices by studying the combustion pattern of the raw biomass and its processed form (in form of briquettes and pellets) so that less amount of gases and PM are released. A gasifier stove is such an attempt in which the processed forms of biomass (i.e. briquettes or pellets) are used. While studying the combustion pattern of fuel in many stoves it has been found that if the ratio of fuel and air is maintained at certainlevel then clean combustion can be achieved (Mukunda, 2011). Many of the fundamental phenomena observed in biomass combustion, which are most relevant to cookstoves, have been extensively studied and mainly focus on understanding how wood burns. It includes modelling of air-flow rates both on the solid and gas phases and heat transfer (Burnham-Slipper, 2008 and Mukunda, 2011). The present work deals with the fan based gasifier stove, Oorja, built by BP, India. The main focus of the work is the issue of using different handmade pellets or briquettes (using powdery biomass) and to check their adaptability for the Oorjagasifier stove.The Oorja stove has two modes of air flow rates i.e. “low” and “high” modes. In the present study, the biomass handmade pellets (using commonly available biomass) have been tested mainly using the “high” mode. For most existing stoves, whether traditional stoves such as the three stone fire or other stoves developed over the last two decades, the efficiency (determined using water boiling tests) is between 15 to 35%. In contrast, the water boiling efficiency of the Oorja stove (used with pellets designed for Oorja) has been found to be up to 60% (Varunkumar, 2012). 7.2.

Materials and Methods Table 1 shows the equipment and materials used for this study. Several combinations

of biomass-binders were used to hand-make pellets and compacted “briquettes”. This section describes the method followed for making of the region specific fuel to adapt in and Oorja (gasification type) domestic scale stove. 7.2.1

Process for Making the Handmade Pellets The making of the pellet without any kind of sophisticated pellet making machine

using the locally available biomass and the binder was done in three steps, which are described in the following sub sections. a) Preparing the mixture of the biomass and the binder Preparing the mixture for the pellet has been the most important step in the process to ensure maximum energy density, by maximising the amount of biomass with the minimum 60

Recent Advances in Bioenergy Research Vol. III 2014 amount of binder. The local binder used in the preparation were cowdung, wheat flour and rice flour while the biomass used were saw dust, rice husk and wheat husk. Sophisticated pellet making machine (designed especially for the manufacturing of the pellets that are commercially available in the market) generally the binder is not required as the lignin present in the biomass, when compressed, at high temperature acts as the binder. For such cases high degree of the compaction is needed which cannot be achieved by the hand pressing of the mixture to make the pellets. Table 1 Equipment and materials used in the present study S.No

Materials Required Biomass fuel

Quantity

Remarks

4 types

Oorja pellets, Rice Husk, As per the requirement of the Wheat Husk and Saw Dust experiment Cow Dung, Rice Flour and Wheat Flour K- type Thermocouple to measure temperature up to 1200˚ C Thermometer of range up to 100˚C 3 L Aluminium vessel

1 Binders

3 types

2 3

Thermocouple

2

4

Thermometer

5

Cooking vessel

1 2

6

Hood

1

7

1

8

Weighing Balance Stove

9

Stirrer

10

Kerosene

11

Bomb Calorimeter

1

1 2l 1

References

As per the requirement of the experiment Varunkumar (2012)

Water Boiling Test version 4.1.2 (URL 2) Water Boiling Test version 4.1.2 (URL 2) Hood including exhaust Water Boiling Test version 4.1.2 (URL 2) Range 0-50 kg (accuracy in As per the requirement for the grams) experiment Oorja pellet gasifier Stove As per the requirement for the experiment For stirring the water for Water Boiling Test version constant mixing 4.1.2 and Varunkumar(2011) To ignite the stove As per the requirement for the experiment To determine the calorific As per the need for the value of the pellets experiment

During the making of the pellet, it was first determined as to what amount of minimum amount of binder could hold the maximum amount of biomass. Different binders were mixed in equal proportions with the biomass, and its binding capacity was checked. The biomass was then added continually until the binder could not hold more of the biomass. The composition, in which the binder cannot hold more of the biomass was determined, was then used as the mixture for the pellet making. Binding capacity of different binders for different biomass varied, in which the cow dung was found to have the minimum binding capacity and wheat flour with the maximum binding capacity. But, since the cow dung is available freely it 61

Recent Advances in Bioenergy Research Vol. III 2014 was preferred over the other binders, even though the binding property was low as compared with the wheat and the rice flour. Table 2 Different types of fuels and binders used for the pellet making S.No. 1 2 3

Biomass Saw dust Rice husk Wheat husk

Binders Cow dung Rice flour Wheat flour

b) Compaction of the mixture for the making of the pellets The compaction of the mixture was the second step in the making of the pellets making by hand pressing into small “balls” as shown in Fig 1(a). Besides the hand pressing technique a commonly available snack making machine called as “sevai machine” was used. This is a hollow cylinder fixed with a piston. The top of the piston is provided with a rotating shaft. When this shaft is rotated the piston moves downwards and presses the mixture filled in the cylinder. At a certain point shaft, cannot be rotated, at that particular point the piston is removed and then compacted form of the mixture is recovered in the form of the discs as shown in the Figure 1 (b). This compacted form of the mixture, which can also be called as pellets are then put for drying. c) Drying of the pellets These wet pellets are dried until the moisture content of the pellet reaches ~12 % or below (needed for the gasification of the pellets). The drying can either be done either in the sun for over a week or they can also be dried in the hot air oven for 48 to 72 hours at the temperature of 45°C. Figure 1 (a) and (b) shows the different pellets made by hand pressing and using household sewai machine. Table 3 Different types of the pellets and their composition (notation is indicated at the bottom of the table) S. No. 1 2 3 4

Pellets Composition CD+RH+SD (Balls) 400 g cow dung + 100 g rice husk + 100 g saw dust CD+SD (Balls) 500 g cow dung + 60 g saw dust SD+WF (Balls) 250 g Saw Dust + 270 g Wheat Flour + 375 g water CD+SD+WH (Briquette) 1120 g Cow Dung + 245 g Saw Dust + 180 g Wheat Husk CD+SD+WH+WF 650 g Cow Dung + 200 g Saw Dust + 150 g Wheat Husk + 5 (Briquette) 75 g Wheat Flour 6 WH+RF (Briquette) 340 g Wheat Husk + 110 g Rice Flour + 600 g Water CD = Cow Dung, SD =Saw Dust, RH = Rice Husk, WF = Wheat Flour, WH = Wheat Husk and RF= Rice Flour 62

Recent Advances in Bioenergy Research Vol. III 2014

Figure 1(a) Disk shaped pellets made using the sewai machine

Figure 1(b) Ball shaped pellets made using hand pressing (a) Cow dung + Rice husk + Saw dust (b) Cow dung + Saw dust, (c) Saw dust + Wheat flour and (d) Wheat husk + Wheat flour 7.3

Results and Discussion

Stove Performance with Different Types of Handmade Biomass Pellets with the Oorja Gasifier Stove To evaluate the performance of the different compositions of the handmade biomass pellets, the following parameters are discussed: 1. Determination of the calorific value 2. The input power for different types of pellets in Oorja stove 3. Specific fuel consumption for each fuel 4. Efficiency of the stove The calorific values and densities of the fuels are shown in the following Table 4.

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Recent Advances in Bioenergy Research Vol. III 2014 Table 4 Calorific values and the densities of the different biomass fuels Biomass fuels

Calorific Value (MJ/Kg)

Density (Kg/m3)

CD+RH+SD (Balls)

15.66

244.3

CD+SD (Balls)

16.13

270.5

SD+WF (Balls)

15.4

423.2

CD+SD+WH (Briquette)

16.04

307.9

CD+SD+WH+WF (Briquette)

15.34

275.4

WH+RF (Briquette)

17.02

566.0

Oorja pellet

16.04

___

Babool wood

17.3

___

Ain wood

16.8

___

To conduct the water boiling test, the standard 3 litrewater boiling test protocols were taken into accounts with slight modification to test the biomass handmade pellets on “high” mode in the domestic gasifier Oorja stove. Tests were conducted in a way only a batch process was considered.The operational procedure does not require refuelling. Table 4 and 5 shows the result of water boiling tests and the performance of the stove respectively, for biomass pellets. Figure 2 plots the temperature of the boiling water as a function of time for each fuel. From the plot, we can observe that Oorja pellets take only 15 minutes to boil the water and complete the test in 49 minutes for one batch process, which is comparatively faster than the other types of pellets. On the other hand, the small pieces of Babool wood takes only 10 min to boil the water which is comparatively faster than the other types of biomass fuels but lasts only 26 minutes for one batch process. For the other pellets, the water did not even reach the boiling temperature, except for the ball shaped pellets made from Saw Dust and Wheat Flour that took 50 minutes to reach the temperature of 99˚C.

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Recent Advances in Bioenergy Research Vol. III 2014 Table 5 Result of Water Boiling Test (3 L Water) in Oorja Stove Parameters

CD+SD (Balls)

CD+RH+ SD (Balls)

SD+WF (Balls)

CD+SD+WH (Briquette)

CD+SD+ WH+WF (Briquette)

WH+RF (Briquette)

Oorja Pellet (not handmade)

Ain wood

Small Babool pieces

Large Babool pieces

Room Temp 31 (°C) Initial water 31 Temp ( °C)

29

27

28

27

27

26

26

27

28

29

27

28

27

27

26

26

27

28

Final Water 58 Temp (°C)

69

96

63

89

78

99 (15 min, 89 Cold Start.) 99 (14 min, Hot Start.) 96 (20 min, Simmering)

100 (10 100 min, Cold Start.) 62 (16 min, Hot Start.)

Mass Pellet (g) Duration Burning (min.)

of 140

150

575

380

440

210

595

335

365

385

of 12

14

50

15

41

24

49

10

26

15

CD = Cow Dung, SD =Saw Dust, RH = Rice Husk, WF = Wheat Flour, WH = Wheat Husk and RF= Rice Flour

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Recent Advances in Bioenergy Research Vol. III 2014 Table 6 Stove performance with different biomass fuels on Oorja stove Input Power

Sl. No Pellets

(kW)

Fuel required per output power

Efficiency (%)

(Kg/kWhr)

1

CD+RH+SD (Balls)

3.1

0.44

54.1

2

CD+SD (Balls)

2.8

0.47

57.1

3

SD+WF (Balls)

2.9

0.73

42.9

4

CD+SD+WH (Briquette)

6.7

1.75

33.1

2.7

0.92

40.0

CD+SD+WH+WF

5

(Briquette)

6

WH+RF (Briquette)

2.5

0.62

48.8

7

Oorja Pellet

2.2

1.17

32.3

8

Babool small wood pieces

3.7

0.59

58.9

9

Babool large wood pieces

6.7

0.87

37.5

10

Ain wood

8.1

1.35

24.9

Temp (ºC)

WBT of Biomass Fuels in Oorja 120

CD+RH+SD(Balls)

100

SD+WF (Balls)

80

CD+SD+WH (Briquette) CD+SD+WH+WF (Briquette) WH+RF(Briquette)

60 40 20 0 0

20

40

60

Time (min)

Oorja (Karjat) small babool Pieces

Figure 2 Comparision of Temperature vs. Time profiles for different fuels in the Oorja stove during the Water Boiling Test

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Recent Advances in Bioenergy Research Vol. III 2014 7.4

Conclusions In this present work the analysis of gasification behaviour of the handmade pellets

made from different locally available biomass was carried out to address the issue of how region specific fuel can be used for the clean combustion process, i.e., the gasification in the already available Oorja pellet gasifier stove. It also focussed on what changes can be made in the existing stove and what could be the alternate fuel that can be used by the rural people without having to buy the commercially available pellets. During the study, it was found that if the pellets are more densified with some better technique, the locally available biomass could be pelletized and used in the forced draft gasifier stoves like Oorja.

References 1. Bolling, A.K., Pagels, J., Yttri, K. E., Barregard.,Sallesten, G., Schwarze, P.E., and Boman, C. (2009) Health Effect of Residential Wood Smoke particles: The Importance of Combustion Condition and Physiochemical Particle Properties, Particle and Fibre Technology, 6:29. 2. Burnham-Slipper, H., Clifford, M.J. and Pickering, S.J. (2007) A simplifiedwood combustion model for use in the simulation of cooking fires, in 5th International Conference on Heat Transfer, Fluid Mechanics and Thermodynamics. 3. Mukunda, H.S. (2011) Understanding Clean Energy and Fuels from Biomass, Wiley India. 4. Varunkumar, S (2012) PhD Thesis- Packed bed gasification-combustion in biomass domestic stove and combustion system, IISc, Bangalore. 5. Zhang, J., Smith, K.R., Uma, R., Ma, Y., Kishore, V.V.N., Lata, K., Khalil, M.A.K., Rasmussen, R.A. and Thorneloe, S.T. (1999) Carbon monoxidefromcookstoves in developing countries: 1. emission factors, Chemosphere: Global Change Science, 1(130: 353–366. Web References 6. http://www.cleancookstoves.org/ (Last accessed on 17th November, 2013). 7. www.aprovecho.org/lab/component/rubberdoc/doc/231/raw (Last accessed on 15th September, 2013).

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 8 PRODUCTION OF HYDROCARBON LIQUID BY PYROLYSIS OF CAMELLIA SINENSIS (TEA) SEED DEOILED CAKE AND CHARACTERIZATION OF PRODUCTS Nabajit Dev Choudhury, Priyanko Protim Gohai, Bichitra Bikash, Sashi Dhar Baruah and Rupam Kataki

Abstract Increasing demand of the liquid fuel can be partially fulfilled by utilization of the bio-wastes for biofuel production through pyrolysis. In this line, the present work aims to explore Camellia Sinesis de-oiled cake (CSDC) for bio-fuel production. CSDC was pyrolysed for obtaining liquid biofuel. The thermal pyrolysis of CSDC was carried out in a fixed-bed reactor made up of stainless steel at temperature range from 400-600oC and at heating rate 300Cmin-1. and at

150 mlmin-1 nitrogen gas flow rate to determine the yield and

characteristic of the liquid and solid product. The maximum bio-oil yield was 27.38% at 5000C. The chemical composition of the bio-oil was investigated using FTIR and GC/MS. Results showed that the bio-oil obtained from deoiled cake of CSDC is a valuable source of fuel and chemicals. Keywords: Pyrolysis, Deoiled cake, Bio-oil, FTIR, GC-MS. 8.1

Introduction Increasing energy consumption, depleting conventional fossil fuel and environmental

consideration intensified the research on renewable energy particularly on utilization of biomass for the production relatively clean fuel due to its easy availability, easy process ability and environment friendly nature in contrast to the fossil fuel. Biomass act as a sink for greenhouse gases as it absorbs CO2 during its cultivation. Biomass therefore can be considered as an alternative clean development mechanism (CDM) option for reducing greenhouse gas emission [Mulligan et al., 2010]. The different thermo-chemical and bio-

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Recent Advances in Bioenergy Research Vol. III 2014 chemical process such as pyrolysis, gasification, liquification and anaerobic digestion; are used to convert biomass to fuel. Among these processes pyrolysis is one of the suitable thermo chemical conversion processes to get maximum liquid product from biomass. Any type of biomass can give liquid fuel after pyrolysis which can be used as energy fuel and also for production of different chemicals [Bridgwater and Peacocke, 2004]. Pyrolysis is a conversion technique in which biomass is heated to a desired temperature in an oxygen or air free atmosphere to yield solid, liquid and gaseous products. There are many parameters that influence the yield and quality of the products obtained through pyrolysis. These parameters can be divided into processs parameters and non process parameters. The feedstocks properties such as particle size, fixed carbon, cellulose, hemicelluloses, lignin, ash and mineral content are non process parameters and heating rate, temperature, residence time, sweeping gas type and

flow rate, raction time and type of catalysts used are process

paremeters. The advantage of the pyrolysis process is that the process parameters can be controlled to maximize the production of either solid char, liquid or gaseous products. Ususally, fast pyrolysis with high temperature and longer residence time favour conversion of biomass into uncondensable gaseous product and moderate temperature with short residence time favour the production of bio-oil. Slow pyrolysis is preferred when solid char is desirable product. Deoiled cake of non-edible oil seeds can serve as potential feedstocks for pyrolysis to produce fuels including liquid and gas which can be used as substitute for petroleum or natural gas for internal combustion engines, power station and heat supplies. These biofuel are environment bening in contrast to fossil fuel as they contain low nitrogen and sulphur content. Many researchers have done pyrolysis experiment on different de-oiled cakes such as Jatropha [Raja et al., 2010], Soybean [Uzun et al., 2006], rapeseed cake [Ozcimen et al., 2004] etc. In present study, a new source of de-oiled cake, Camellia Sinesis de-oiled cake (CSDC) was taken and pyrolysis experiment was done. The objective of th present work is to determine the maximum bio-oil production condition in fixed bed reactor. The present work also reports on the characterization of the bio-oil by FTIR and GC-MS for chemical compositon.Thermogravimetric analysis (TGA) and was used to determine the thermal behavior of CSDC. The physical properties of the bio-oil such as kinematic viscosity, flash point, acid number, and pH were also determined.

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Recent Advances in Bioenergy Research Vol. III 2014 8.2

Materials and Methods

8.2.1

Materials In this study, the sample (Fig 1) was collected from Biomass Conversion &

Gasification Laboratory of Department of Energy, Tezpur University, India, after lipid extraction with mechanical oil expeller [Malnad type oil expeller (Indus)]. CSDC were ground using a Wiley mill to pass a 0.4 mm (40 mesh) screen (as per TAPPI T257 Om- 85 methods) to fine particles (420 micron) in order to eliminate heat transfer effects during pyrolysis and then samples were oven dried and kept in a desiccator. The proximate and ultimate analyses data for CSDC are given in Table 1.

Figure 1: Camellia Sinesis amd CSDC 8.2.2

Characterization of feed stocks and biochar The proximate analysis of tea seed deolied cake and biochar obtained after pyrolysis

were done by ASTM D 3173-75 and ultimate analysis was done using CHN analyzer (Perkin Elmer, 2400Series-II). The percentage of oxygen was determined by means of difference. Higher heating value (HHV) was determined by 5E-1AC/ML, Auto bomb calorimeter according to ASTM D2015. The thermal behavior of CSDC to 900oC at different heating rates of 20oC/min were studied non-isothermally using Pyris Diamond TG/DTA analyzer (PERKIN ELMER). A high purity N2 gas (99.99%) was used as a carrier gas at a flow rate of 100 ml min-1. 8.2.3

Pyrolysis set-up The schematic diagram of the experimental setup is shown in Fig 2. The pyrolysis

setup consists of fixed bed reactor made of stainless steel with a length of 48 cm and an internal diameter of 3 cm, equipped with an inert gas (nitrogen) connection. The reactor was heated externally by an electric furnace, with the temperature being controlled by a Ni–Cr–Ni

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Recent Advances in Bioenergy Research Vol. III 2014 thermocouple fitted inside the reactor. The thermocouple was connected to a PID controller for controlling the temperature and heating rate. Before the experiments, the reactor was purged by nitrogen gas for 10 min at a flow rate of 30 ml min-1 to remove the air inside. Then, nitrogen flow rate was set to the desired value. A 10g of deoiled cake were loaded for each run of experiment. The liquid portion was recovered with diethylether washing. The aqueous phase was separated from oil phase with a separating funnel. The bio-oil and solvent mixture was passed over dry sodium sulphate to make it water free and then the solvent was evaporated from bio-oil by rotary evaporator and the residual was weighed as bio-oil. The residual solid in the reactor was weighed as char. The gas yield was calculated from the material balance. The reactions were carried out at different temperatures ranging from 400 – 600oC. V1

V2

Thermocouple FC

C F

F Gas

R

Liquid product collector

N2 cylinder

F- Furnace R- Reactor C- Condenser V- Valve FC- Flow controller

Figure 2: Schematic diagram of experimental unit for pyrolysis 8.2.4

Characterization of bio-oil Fourier Transformer Infrared spectroscopy (FTIR) of the pyrolytic oil obtained at

maximum yielding condition was taken with a Nicolet Impact I-410 model Fourier Transform Infrared Spectrometer to chemical composition of the bio-oil. The components of the bio-oil were analyzed using Perkin Elmer Clarus680 GC/600C MS. A capillary column coated with a 0.25 µm film of DB-5 with length of 30 m and diameter 0.25 mm was used. The GC was equipped with a split injector at 2000C with a split ratio of 1:10. Helium gas of 99.995% purity was used as carrier gas at flow rate of 1.51 ml min-1. The oven initial temperature was set to 70oC for 2 min and then increased to 290oC at a rate of 10oCmin-1 and maintained for 7

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Recent Advances in Bioenergy Research Vol. III 2014 min. All the compounds were identified by means of the NIST library. Mass spectrometer was operated at an interface temperature of 200oC with ion source temperature of 180oC of range 40–1000 m/z. The physical properties such as density, kinematic viscosity , flash point and calorific value were determined using standard test methods. 8.3

Result and Discussion

8.3.1

Characterization of feedstock and obtained biochar The proximate and ultimate analysis of CSDC and biochar are shown in the Table 1.

The volatile matters and ash content of the sample in proximate analysis was found to be 80.7% and 5.34% respectively. Low ash content of deoiled cake indiacate that the sample is good candidate for thermochemical conversion process. The volatile matter of the sample was 80.7% which is reduced to 22.72% after pyrolysis. It indicates that a large portion of the sample was converted to condensable and incondensable gases. As a result of significance decrease in the volatilie matters the fixed carbon content of the solid material increased which indicates relatively less liberation of the fixed carbon during pyrolysis. Moisture content play an important role in selection of the conversion process. Sample with less moisture content are suitable for thermal conversion while those with high moisture content are more suitable for biochemical conversion process such as fermentation. In this regard, tea seed deoiled cake with moisture content 4.61 % is a potential candidate for thermal conversion. Ultimate analysis presented in the Table1 showed a significance increase in carbon content of biochar whereas its oxygen content decreased in comparison to the oxygen content of the raw material. 8.3.2

TGA and DTG analysis of CSDC Thermogravimetric analysis (TGA) technique is applied in determination of thermal

stability of the sample in various ranges of temperatures. The TGA plot of CSDC at heating rate of 200C min-1 under nitrogen atmosphere is shown in Fig 3(a). The TGA of the sample shows that the temperature range from 133- 363oC associates with maximum weight loss. In this region this zone of TGA can be referred to as active pyrolytic zone. The intial weight loss in the temperature range 43- 133oC represents the evaporation of moisture contents physically absorbed in the deoiled cake. There is also possibility of loss of some light volatile matters. During the final stage of decomposition starts at 363oC, the rate of weight loss is very slow. In initial stage 7.24%, in the active pyrolytic zone or the second stage of decomposition,

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Recent Advances in Bioenergy Research Vol. III 2014 52.76 % and in the final stage 40% weight loss was observed. During the Second stage, the intermolecular associations and weaker chemical bonds are destroyed [Jinno et al., 2004; Chan et al., 2009]. The side aliphatic chains may be broken and some small gaseous molecules are produced at the lower temperature [Biswal et al., 2013]. During the Third stage at higher temperature chemical bonds are broken and the parent molecular skeletons are destroyed. As a result, the larger molecule decomposes to form smaller molecules. DTG curve (Fig 3(b)) shows that there is only one major peak at 294oC which was present in active pyrolytic zone at temperature range 133-363oC. Table 1: Proximate and ultimate analysis of CSDC Properties Proximate analysis (wt%) Moisture Volatile matter Fixed Carbon Ash Ultimate analysis (wt%) Carbon Hydrogen Nitrogen Oxygen Sulphur H/C molar ratio (on ash free basis) Emperical formula Gross calorific value (MJ/kg) 8.3.3

Tea seed deoiled Biochar obtained Rapeseed char cake at 5000C (Ucar et al., 2008) 4.34 ± 0.27 80.22 ± 0.48 10.66 ± 0.41 4.78 ± 0.66

6.23 ± 0.34 22.45 ± 0.27 38.46 ± 0.02 9.78 ± 0.41

20.01 16.41 18.54

47.63 7.34 3.48 41.55 1.849

76.35 3.76 5.67 14.22 0.591

56.48 3.22 7.32 32.25 0.23 0.68

CH1.849N0.062O0.654 19.65

CH0.591N0.063O0.139 27.92

CH0.08N0.114O0.43S0.001 23.88

Effect of temperature on the product distribution The pyrolysis of the tea seed deoiled cake yielded three different products viz. liquid,

gas and solid residue (biochar). Results of mass balance of pyrolytic decomposition products were presented in Table 2. The bio-oil yield increased from 22.25% to 27.38% with increased in temperatures from 400-500oC but decreased from 27.38% to 23.74% in temperature range 500-600oC. The maximum char yield 37.43% was obtained at temperature 400oC. The yield of char decreased with increasing temperature. The decrease in char yield with increase in temperature may be due to the higher decomposition of the biomass sample in higher pyrolytic temperature or may be due secondary decomposition of char residue

[Horn and

William, 1996]. The yield of gas increased with increase in temperatures. This is may be due

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Recent Advances in Bioenergy Research Vol. III 2014 to the secondary decomposition of the char and secondary cracking of the pyrolysis may enrich the content of the gas product at higher temperature.

(a) 100

Weight remaining (%)

80

60

40

20

0 0

200

400

600

800

1000

0

Temperature ( C)

101.4

(b)

Derivative Y1 (mg/min)

101.2 101.0 100.8 100.6 100.4 100.2 100.0 100

200

300

400

500

600

700

800

900

0

Temperature ( C)

Figure 3: (a) TGA and (b) DTG plot of Camellia Sinesis deolied cake Table 2: Effect temperatures on product distribution of pyrolysis Temperature Biochar (oC) (wt%) 37.43 400 32.23 450 29.13 500 27.67 550 25.47 600 8.3.4

Bio-oil (wt%)

Aquous Phase (wt%)

22.25 25.46 27.38 24.51 23.74

18.87 17.81 17.36 16.54 15.23

Gas (wt%) 20.31 23.67 25.43 30.67 35.28

FTIR of the bio-oil sample obtained at 500oC Biomass pyrolytic oil is composed of wide range of complex organic compound.

FTIR analysis was performed to investigate the chemical structure of the bio-oil sample. Figure 4 shows the FTIR spectra of tea seed deoiled cake. The O-H stretching vibrations at

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Recent Advances in Bioenergy Research Vol. III 2014 frequency 3379 cm-1 indicates the presence of alcohol or phenols. The presence of alkanes is detected at peaks around 2851 cm-1 and 2930 cm-1 with C-H stretching vibrations. C=O stretching vibrations cause band at 1717 cm-1. The presence of alkenes was detected by C=C stretching vibrations at 1610 cm-1. The peaks in the range of 950–1300 cm-1 show the presence of C–O stretching vibrations present in alcohol or ester. the C-H bending vibrations frequency 812 cm-1 indicates the presence of phenyl ring substitution bands. The results were found consistent when compared with the results of GC-MS.

110 100

% Transmittance

90 80 70

C-O ,C-H

60 50 40

C=O,C=C

O -H

30

C-H 20 4000

3500

3000

2500

2000

1500

1000

500

-1

W avenum ber (cm )

Figure 4: FTIR of CSDC bio-oil 8.3.5 GC-MS of the bio-oil sample The GC-MS analysis (Fig.5) of the oil sample is summarized in Table 3. More than 40 peaks are displayed in the GC/MS chromatograph but because of the complex nature the perfect separation of all the peaks are not possible and also the depending on strength of MS library, 10 peaks are evaluated. Comparing the mass spectra fragmentation pattern with Perkin Elmer NITS library and published data, the highest likelihood of compounds identification were obtained. The carbon distribution of the identified compounds were in the range of C5-C29. I-(b)_15-5-

Scan TI 1.78e

10

23.9

% 14.6 8.5

9.9

21.2 21.9 22.5

16.4

10.1 11.3

24.4

Figure 5: GC-MS of CSDC bio-oil

0 10.0

12.0

14.0

16.0

18.0

20.0

75

22.0

24.0

Tim 26.0

28.0

30.0

Recent Advances in Bioenergy Research Vol. III 2014 Table 3: Compound identified by GC-MS of CSDC bio-oil S.No.

TR (min)

Tentative compounds

1 2 3 4 5 6 7 8 9 10

8.57 9.93 10.14 11.37 14.67 16.47 21.28 21.94 22.53 23.97

2-PYRIDINECARBOXYLIC ACID, METHYL ESTER DECANOIC ACID, 2-METHYL3-FURANMETHANOL FURAN, 2,4-DIMETHYLMEQUINOL PHENOL, 2-METHOXY-4-METHYLN-TETRACOSANOL-1 1-HEPTACOSANOL METHYL OCTACOSANOATE CYCLOTRISILOXANE, HEXAMETHYL-

8.3.6

Empirical formulae C7H7NO2 C11H22O2 C5H6O2 C6H8O C7H8O2 C8H10O2 C24H50O C27H56O C29H58O2 C6H18O3Si3

Physical properties of the bio-oil Table 4 shows the results of the elemental analysis of the CSDC bio-oil. The result

shows that the bio-oil has higher heating value of 32.25 KJ/kg which is higher than some bio-oils produced from different de-oiled cake such as Neem cake bio-oil (30 MJ/kg) [Volli et al., 2012], Mustard de-oiled cake (25.2 MJ/kg) [Volli et al., 2012]. Table 4: Ultimate analysis of bio-oil PARAMETERS C H N O H/C O/C EMPERICAL FORMULA Higher Heating Value(MJ/kg)

CSDC BIO- OIL 71.35 8.05 4.65 15.95 1.354 0.167 CH1.354 N0.055 O0.167 32.25

The comparison of the various necessary properties of the oil obtained from the mustard de-oiled cake and diesel is shown in Table 5. The viscosity of the bio-oil is comparatively higher than that of diesel which may lead to poor atomization and incomplete combustion. Therefore the bio-oil is not suitable for direct use as a engine fuel but can be use in moder diesel engine by blending with the diesel. The higher heating value of bio-oil is also lower than that of diesel engine.

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Recent Advances in Bioenergy Research Vol. III 2014 Table 5: Fuel properties of of CSDC pyrolyitic oil Properties

Standard test method

Pyrolytic oil

Kinematic viscosity, 400C Flash point PH Acid Number Higher heating value (MJ/kg) Appearance

ASTM D 445 ASTM D 93 PH meter ASTM D664 -

28 62 3.24 33.56 32.25 Dark brown

Chemical formula

Identified

C5-C29

8.4

Diesel [Tuttle et al. 2004] 2-5.5 53-80 42-45

C8-C25

Conclusion Pyrolysis of Camellia Sinesis de-oiled cake was carried out in a fixed-bed reactor

made up of stainless steel at temperature range from 400oC to 600oC and at a rate of 30oC min-1 to produce bio-fuel. The maximum yield of oil is 27.38% on wt. % basis for Camellia Sinesis de-oiled cake, was obtained at a temperature of 500oC. The fuel and chemical analysis of bio-oil reveals that these pyrolytic oils can be used as fuel and as a source of chemicals. The carbon distribution in the chemical identified in bio-oil is in range C5-C29.

References 1.

Biswal B., Kumar S. and Singh R.K.(2013).Production of hydrocarbon liquid by thermal pyrolysis

of

paper

cup

waste.

Journal

of

Waste

Management,

URL:

http://dx.doi.org/10.1155/2013/731858. 2.

Bridgwater A.V. and Peacocke G.V.C., (2004).Fast pyrolysis: Fast pyrolysis processes for biomass. Renewable and Sustainable Energy Reviews, 4:41-73.

3.

Chan P.W., Atrey A., Howard R.B. (2009). Determination of pyrolysis temperature for charring materials. Proceedings of the combustion Institute, 32: 2471-2479.

4.

Horne P.A., Williams P.T. (1996). Influence of temperature on the products from the flash pyrolysis of biomass. Fuel, 75: 1051-1059.

5.

Jinno D., Gupta A.K.(2004). Determination of chemical kinetic parameters of surrogate solid wastes. J.Eng. Gas. Turb. Power, 126: 126-685.

6.

Mulligan C.J., Strezov L., Strezov V., (2010). Thermal decomposition of wheat straw and mallee residue under pyrolysis condition. Energy and fuel, 24:46-52.

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Recent Advances in Bioenergy Research Vol. III 2014 7.

Ozcimen D, Karaosmanoglu F. (2004) Production and characterization of bio-oil and biochar from rapeseed cake. Renew Energy; 29:779–87.

8.

Raja S.A., Kennedy Z.R., Pillai B.C., Lee C.L.R.(2010). Flash pyrolysis of jatropha oil cake in electrically heated fluidized bed reactor. Energy; 35:2819–23.

9.

Tuttle J., Kuegelgen V., (2004). Biodiesel Handling and Use Guidelines, Third edition in National Renewable Energy Laboratory.

10. Ucar S., Ozkan A.R. (2008). Characterization of products from the pyrolysis of rapeseed oil cake. Bioresour Technol; 99:8771–6. 11. Uzun BB, Putun AE, Putun E. (2006) Fast pyrolysis of soybean cake: product yields and compositions. Bioresour Technol; 97:569–76. 12. Volli V., Singh R.K. (2012). Production of bio-oil from de-oiled cakes by thermal pyrolysis. Fuel, 96: 579-585.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 9 COMPARATIVE STUDY OF DIFFERENT BIOMASS COOKSTOVE MODEL: AN EXPERIMENTAL STUDY K Pal, A K Pandey, P Gera and S K Tyagi Abstract This article presents the comparative experimental study of five different types of improved biomass cookstoves models based on their thermal efficiency, power output rating and emission reduction potential. All the cookstoves models are design and fabricated in the laboratory based on the gasification principle except NIRE-02, which is the modified form of the traditional cookstove. The performance of each cookstoves were evaluated following water boiling test as per Bureau of Indian Standard (BIS) protocols, whereas the emission reduction was calculated based upon the clean development mechanism (CDM) of United Nation Framework Convention on Climate Change (UNFCCC). The overall performances of these models were found to be much better than that of the traditional cookstoves being used by the majority of population around the globe. The emission reductions potential from these models were found to be between 2.0-3.0 tons per household annually, which not only shows the good agreement with the experimental values available in the literature but also represent a high potential for disseminating these cookstoves through CDM in the rural and remote area of the country, especially, where woody biomass is the major consumption as the cooking fuel. Keywords: Energy efficiency, Improved biomass cookstove, Clean development mechanism. 9.1

Introduction The past evidences of fire have been found as old as four lakh years i.e. during the

first ice age (Bronowski, 1973). However at that time fire was used for roast the meat. Mastery of fire is considered to be an important step toward human development which took off only about 12,000 years ago (FOA, 1993). A three stone fire arrangement was first time was used by people of ancient time for cooking their food. The use of three stone fire arrangement has many health and environment problems like exposure to heat as well as fire hazard beside the problem of poor thermal efficiency. The requirement for a better cookstove

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Recent Advances in Bioenergy Research Vol. III 2014 arose which gave rise to U-shaped mud cookstove/traditional cookstove. The traditional cookstoves solved many of the technical problems such as, enhancement of thermal efficiency by many folds. However, the health, socio-economic and cultural problems were not solved completely. The development of efficient cookstoves at International level was started in the 1940s (Anhalt and Holanda, 2013). However, in India the efforts for making improved biomass cookstoves started during 1950s with the main objective to improve the design of biomass-fired stoves and the widespread R&D started in 1970s. Later on, improved cookstove programs (ICPs) were considered as a solution to the fuel wood crisis and to reduce deforestation. A model of improved multiport stove was introduced by Raju in 1953 which was one of the high-mass and shielded-fire type and had a chimney to remove smoke and adjustable metal dampers to regulate the fire. Singer (1961) in Indonesia conducted cookstove efficiency tests on high mass mud stove with the main objective to increase efficiency and save fuel, without considering the socio-economic and cultural aspects. However, in 1990s the focus shifted more on the issues involving the Indoor air pollution and its effect on human health (World Bank, 2011). In addition to above factors, the comfort of cooking, smoke free kitchens, convenience and safety of stove users were tested and considered to be the important aspects as compared to the fuel savings. Smith et al., (2000) stated that the burning of biomass fuels emits high levels of smoke, containing hazardous pollutants such as, respirable suspended particulate matter (RSPM), carbon dioxide (CO2), carbon monoxide (CO), nitrogen oxides (NOx), sulfur oxides (SOx) and some cancer causing organic compounds like Benzopyrene, benzene and 1,3-Butadiene. Fullerton et al., (2008) found that different types of health risks were associated with the indoor air pollution such as, respiratory infections like pneumonia, tuberculosis, chronic obstructive pulmonary disease, birth defects, cataracts, cardiovascular events which cause serious problems in the women and child. Kleeman et al., (2000) studied the adverse health impacts of the particle size distribution and result of deposition of those pollutants in different areas of lungs. Keeping the above facts in mind, Ministry of New and Renewable Energy, Government of India has initiated a National level program to develop next-generation cleaner cookstoves and deploy them to the households where traditional cookstoves being used for cooking and heating applications. The initiative has set itself the lofty aim of

80

Recent Advances in Bioenergy Research Vol. III 2014 providing energy service comparable to clean sources like LPG without changing the fuels. Based on National surveys, published literature and assessments, and measurements of cookstove performance around the globe, it was found that about 570,000 premature deaths in poor women and children and over 4% of India's estimated greenhouse emissions could be avoided if such an initiative were in place. Although, the current advanced biomass stoves show substantial emissions reductions over the traditional stoves but there is a lot to be done to reach the LPG-like emission levels. WHO (2011) has estimated that every year indoor air pollution (IAP) is responsible for the death of 1.8 million people around the globe which comes out to be one death every 20 seconds. It was further estimated that the exposure to smoke from simple act of cooking constitutes the fifth worst risk factor for disease in the developing countries and causes almost two million premature deaths per year, exceeding the deaths attributable to malaria or tuberculosis (WHO, 2006). Ramanathan and Carmichael, (2008) recently found that the black carbon is playing a major role in the global warming. The Global Alliance for Clean Cookstoves (GACC), a new public–private partnership led by the UN Foundation was established to create a thriving global market for clean and efficient household cooking solutions (Global Alliance for Clean Cookstoves, 2011). The newly cookstoves which are known as advanced biomass cookstoves are based on better design principles; they have the better combustion efficiency and thus, reduce the fuel consumption to a greater extent. These cookstoves can deal with both the emissions and health issues resulting from cooking with open fires or traditional biomass cookstoves. These cookstoves have the ability to get carbon credits (UNFCCC, 2013), not only because of their contribution to climate-change mitigation but also they yield major co-benefits in terms of energy access for the poor people. Although, the current advanced biomass stoves show substantial emissions reductions over the traditional stoves, yet more improvements are needed to reach the LPG-like emission levels. 9.2

Mathematical Modeling An improved biomass cooking stoves have the ability to reduce indoor air pollution,

deforestation, climate change, and improve quality of life of rural peoples on a global scale. The better design of these cookstoves can significantly impact their performance and emissions. Although these improved biomass stoves have been studied for a long time however, a theoretical understanding of their operating behavior and the development of

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Recent Advances in Bioenergy Research Vol. III 2014 engineering tools for an improved cookstove based on natural convection is still lacking. This section presents the mathematical modeling of improved biomass cookstove based on various performance parameters as below: 9.2.1

Side feeding wood-burning cookstove Agenbroad et al. (2011a) developed a simplified model for the understanding the

fundamental operating behaviour of these natural convection based biomass cookstoves. This model was developed utilize the dimensional form of two equation system. This has been further developed into a dimensionless form at a later stage (Agenbroad et al. 2011b). A simplified model of the fundamental stove has been developed for predicting bulk flow rate, temperature, and excess air ratio, based on stove geometry including chimney height, chimney area, viscous and heat release losses and the operating firepower. The stove operator decided the operating firepower of the stove by controlling the fuel feed rate, excess air ratio. The resulting bulk flow rate, temperature, and excess air ratio etc. are the fundamental inputs for stove performance. The processes are categorized into two basic and fundamental, (a) heat addition from combustion, and (b) kinetic energy addition due to the chimney effect, and the details are given as below: 9.2.2

Heat addition from combustion The heat of combustion increased the temperature and decreased the density of bulk

flow passed over there. If we assume that the heat addition is efficient and instantaneous and the system is isobaric with no mechanical work done on/by the system, having ideal gas behavior, with constant potential and kinetic energies. The heat addition from the combustion .

showed an enthalpy increase (hC to hH) distributed in the mass flow rate ( m A ). The increased .

bulk flow temperature is calculated for a given mass flow rate m A , heat release rate

Q in , .

using

the constant pressure specific heat of air (Cp), as below (Kumar et al., 2013): . Q in =

. m A ( hC − h H ) =

TH . m A ∫ C p ( T ) dT Tc

. = m A C p , avg ( T H − T C

)

(1)

where T is the bulk flow temperature and the subscripts H and C denote the hot and cold conditions, respectively. 9.2.3

Kinetic energy addition due to the chimney effect The air flow through the stove depends on the chimney effect resulting from the

buoyant force of the decreased density of air after heat addition of combustion. The decreased

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Recent Advances in Bioenergy Research Vol. III 2014 density of air in the chimney creates a lower pressure as compare to the ambient pressure. In a fluid, the relationship between pressure variation with depth can be determined using the hydrostatic equation and the net pressure difference (∆P) as given below (Kumar et al., 2013):

∆P = g ∫ ρ (h)dh

(2)

where g is the acceleration due to gravity, and ρ is the density of medium which is a function of chimney height (h). The pressure can be calculated as below (Kumar et al., 2013): 1

2

2

3

3

3

∆P1−2 = ( P3 + g ∫ ρ amb dh) − ( P3 + g ∫ ρ (h) dh) = g ρ amb h − g ∫ ρ ( h)dh

(3)

If chimney walls are assumed to be adiabatic, than the temperature and density, ρ of the flue gases in the chimney (TH and ρH) remain constant, so Eq. (3) can be simplifies which determined the chimney effect due to the pressure difference as (Kumar et al., 2013):

∆ P1− 2 = gh ( ρ

amb

−ρ

H

) (4)

The gain in kinetic energy from the stagnant ambient air can be calculated using the integral form of Bernoulli's equation for the compressible flow, as (Kumar et al., 2013): ∆ P1 − 2 = gh ( ρ amb − ρ H ) =

1 ρ H v 22 2

where ρ H is the density of hot gas and

(5) v2

is the velocity use for calculating the kinetic

energy. Again assuming ideal gas behavior of the flue gases, the density is related to the temperature by the ideal gas law. Using ideal gas equations and solving Eq. (5) for volume and mass flow rate gives (Kumar et al., 2013):

 .  1 V  gh( ρ amb − ρ H ) = ρ H   2  A   .

V = CA

2 gh

.

V = CA .

m

A

2 gh

 P = CA   Rs

2

ρ amb − ρ ρH

(6) H

(7)

T H − T amb T amb  1     ×  T H 

(8) 2 gh

T H − T amb T amb

83

(9)

Recent Advances in Bioenergy Research Vol. III 2014 where C is the loss coefficient introduce to account for uncertainties and inefficiencies in the chimney effect including viscous losses, chimney wall heat transfer, and the unrealistic ideal .

point heat addition at state point 2 and its range is 0≤ C ≤1. The mass flow rate of fuel ( m ) F

in the cookstove model can be calculated from the firepower ( Q. in ) and the heating value (HV) of the fuel as (Kumar et al., 2013):

. m

F

. Q = LHV

(10)

The air fuel ratio (AFR) and excess air ratio (EAR) can be determined as below (Kumar et al., 2013): .

AFR =

mA

(11)

.

mF

Φ=

AFRstoich AFR

%EAR =

(12)

(1 − Φ).100% Φ

(13)

where AFRstoich is the air furl ratio (AFR) for stoichiometric combustion. The lower heating value (LHV) is used because the latent heat of the water vapor is significant, and seldom recovered. 9.2.4

Dimensionless chimney effect equation The advantages of working in the dimensionless form include the scale similarity and

reducing the number of independent parameters for experimentation independent of stove geometry. The dimensionless temperature form can be determined by inspection and is given as below (Kumar et al., 2013): *

T



T

H

− T T

amb

(14)

amb

Using Eq. (14) and Eq. (9), the mass flow rate of air in the cookstove model can be expressed as given below (Kumar et al., 2013): .

m

A

 P = CA   R T amb 

 2 ghT * ×  (T * + 1) 

(15)

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Recent Advances in Bioenergy Research Vol. III 2014 Substituting P/RTamb as the ambient density ρamb in the above equation and rearranging it as (Kumar et al., 2013): .

mA CAρ amb gh

=

2T * T* +1

(16)

This dimensionless mass flow rate can also be defined as the ratio of the actual mass flow rate to the characteristic natural convection and from Eq. (16), the dimensionless mass flow rate of air for the given geometry given as below (Kumar et al., 2013):

. m A* ≡

. mA CAρamb gh

(17) .

Using the dimensionless mass flow rate m A

*

of Eq. (17), the final form of chimney

effect equation is given as below (Kumar et al., 2013): .

mA = *

2T * T * +1

9.2.5

(18)

Dimensionless heat addition equation The mass burn rate of fuel is used instead of the firepower. The relation between the

firepower, mass burn rate and heating value is given as below (Kumar et al., 2013): .

.

Q in = m F HV

(19)

Substituting Eq. (19) and Eq. (14) into Eq. (1) and rearranging it, a dimensionless heating value group (HV*) is formed as (Kumar et al., 2013): .  HV  .  = mA T* mF  C T   p amb 

HV * ≡

(20)

HV C pTamb

(21)

This group defined as the ratio of the combustion heating energy to the initial thermal energy of the flow. Substituting the dimensionless mass air flow rate as defined in Eq. (17), into Eq. (20) which gives the relation between dimensionless heating value, dimensionless mass flow rate of air and dimensionless temperature as (Kumar et al., 2013):

85

Recent Advances in Bioenergy Research Vol. III 2014 . m

. HV * = m * CAρamb ghT * F A

(22)

A dimensionless mass burn rate similar to the dimensionless air flow rate of Eq. (17) is given as below (Kumar et al., 2013): . .

mF ≡ *

mF CAρ amb gh

(23) .

*

Using the dimensionless mass burn rate m F , the final form of the dimensionless heat addition equation becomes (Kumar et al., 2013): .

.

m F HV * = m A T * *

9.2.6

*

(24)

Air/fuel ratio from dimensionless model The air/fuel ratio (AFR) is defined as the ratio of mass flow rate of air to the mass

burn rate of fuel and can be given by rearranging Eq. (24) as shown below (Kumar et al., 2013):

. m* * AFR = .A = HV T* m*F

(25)

The dimensionless heating value (HV*) is considered as remain constant throughout the stove operation. From the above equation a simple inverse linear relationship can be seen between dimensionless temperature (T*) and the air fuel ratio (AFR). 9.3

Materials and Methods A comparative study on modified traditional cookstove and three different improved

biomass cookstoves (NIRE-03, NIRE-05, NIRE-06) has been presented in this research article. These improved cookstove work on the principle of down-draft gasifier where pyrolysis, gasification and combustion of biomass are taking place simultaneously. These cookstoves were made of mild steel whereas for insulation clay and wheat straw mixture was used. The line diagram of NIRE-02, NIRE-03, NIRE-05 and NIRE-06 are shown in the figure 1-4. 9.3.1

Materials Use

The following instruments/equipments have been used during the experiments: (a) Platform balance

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Recent Advances in Bioenergy Research Vol. III 2014 (b) Glass cylinders for measuring water (c) Aluminium vessels with lids of proper volumes as per BIS standard (d) Kerosene oil to ignite the process (e) Match stick (f) Stopwatch (g) Thermometer/Thermo-couple (h) Bomb calorimeter (i) Wood fuel in proper size

260

260

35 190 Space for flame 35 40

Grate

Air

Platform 200 200

Fig. 1: Line diagram of the modified traditional Cookstove, (NIRE-02)

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Recent Advances in Bioenergy Research Vol. III 2014 253 25

150

Top view Pot support

25 Wick

50

60

Secondary air inlet 15 Handle Bottom view 410

23

Insulation

Grate

50

Handle for primary Air adjustment

50

Primary air inlet

Fig. 2: Line diagram of improved cookstove (NIRE-03) 253 Top view 25

125 Pot support

25 60

Wick

50

Secondary air inlet 15 Handle 410

23

Insulation

Grate

50

Handle for primary Air adjustment

50

Primary air inlet

Fig. 3: Line diagram of improved cookstove (NIRE-05)

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Bottom view

Recent Advances in Bioenergy Research Vol. III 2014 230 25

Top view

110 Pot support

25

Wick

50

60

15

Secondary air inlet Handle

410

45

Insulation

Bottom view

Air gap Grate

50

Handle for primary Air adjustment

50

Primary air inlet

Fig. 4: Line diagram of improved cookstove (NIRE-06) 9.3.2

Methods As per the Bureau of Indian Standards (BIS, 2013) water boiling test of all above

mentioned four cook stoves were performed for the measurement of thermal efficiency. According to the BIS protocol stepwise procedure was follow for cookstove testing. At the time of experimental run, the temperature of water, flame and cookstove body was measured with the help of digital temperature sensor whereas the temperature of pot, plate and ambient was measured with mercury-glass thermometer. The measured value of thermal efficiency of each cookstove is compared with the open three stone fire cookstove which generally have the thermal efficiency 8-10%. The stepwise procedure of fuel preparation, burning capacity rate and water boiling test is as follow: a) Fuel Preparation The fuel wood cut from the same log into pieces of 3x3 cm square cross-section and length of half the diameter/length of combustion chamber so as to be housed inside the combustion chamber. The fuel pieces shall be oven dried by the following method (BIS, 2013): a) Weigh total quantity of wood (say 'M' kg.). b) Pick up one piece and mark 'X' by engraving and take its mass (say 'm' g.).

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Recent Advances in Bioenergy Research Vol. III 2014 c) Raise the temperature of oven up to 105 ºC. d) Stack the wood pieces in a honey comb fashion inside the oven. e) Maintain the oven temperature at 105 ºC. f) After 6 hours, remove the marked 'X' piece, weigh it and note reduction in mass from 'm' g, if any. If reduction is observed put the marked piece in the oven again and repeat the weighing of 'X' marked piece after every subsequent 6 hours period till the mass is constant and no further reduction in mass is observed. g) At this stage, weigh the total quantity of wood and note loss of mass from 'M' kg. h) Determine the calorific value of the prepared wood with the help of bomb calorimeter. b) Burning Capacity Rate Fuel burn per hour is known as the burning capacity rate of a cookstove, which can be calculated as: Stack the combustion chamber with test fuel in honey comb fashion up to 3/4 of the height or in a pattern recommended by the manufacturer. Sprinkle 10 to 15 ml. of kerosene on the fuel from the top of chulha/fire box mouth. Weigh the chulha with fuel; let the mass be M1 kg. After half an hour of lighting weigh the chulha again and let the mass be M2 kg. If the calorific value (CV) of the test fuel in kcal/kg then calculate the burning capacity of the chulha as heat input per hour as follows (BIS, 2013): Heat input: per hour = 2 (M1 - M2) x CV kcal/h

(26)

c) Water Boiling Test a) Take the test fuel according to burning capacity rating for one hour. Let the mass be 'X' kg. b) Stack the first lot of test fuel in the combustion chamber in honey comb fashion or as indicated by the manufacturer. c) Select and weigh the vessel with the lid in accordance with the table above. A minimum of two such vessels in a set will be required. Put the recommended quantity of water at 23 + 2 ºC (T1). d) Sprinkle measured quantity 'X' ml. (say 10 - 15 ml.) of kerosene for easy lighting on the test fuel and light. Simultaneously start the stop watch. e) Feeding of fresh test fuel lot shall be done after every 15 minutes. f) The water in the vessel shall be allowed to warm steadily till it reaches a temperature of about 93 ºC, then stirring is commenced and continued until the temperature of water reaches 5 ºC below boiling point at a place. Note down time taken to heat the

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Recent Advances in Bioenergy Research Vol. III 2014 water up to final temperature (less than 5 ºC below the boiling point) T2 ºC. g) Remove the vessel of from the chulha and put the second vessel immediately on the chulha. Prepare first vessel for subsequent heating. h) Repeat the experiment by alternatively putting the two vessels taken till there is no visible flame in the combustion chamber of the chulha. Note down the temperature of the water in the last vessel. 9.4

Analysis of Cookstove Improved biomass cookstoves can reduce indoor air pollution, deforestation, climate

change, and therefore, the quality of life can be improved on a universal scale. The better design of these cookstoves can significantly impact their performance and emissions. Although these improved biomass cookstoves have been studied for a long time however, a theoretical understanding of their operating behaviour and the development of engineering tools for an improved cookstove based on natural convection is still missing. 9.4.1

Thermal efficiency Thermal efficiency of a cookstove may be defined as the ratio of heat utilized to the

heat produced by complete combustion of a given quantity of fuel based on the net calorific value of the fuel and this can be written as below [Kumar et al., 2013, Kishore and Ramana, 2002, Tyagi et al., 2013, BIS, 2013]. Thermal efficiency η =

Heat Utilized × 100 Heat Produced

(2)

Heat utilized= {(n - 1)(W × 0.896 + w × 4.186 8)(T2 - T1 )(W × 0.896 + w × 4.186 8)(T3 - T1 )} kJ Heat produced = 4.186 8 {(X × c1 ) + (V × ρ × c 2 /1000)} kJ

η=

{(n-1)(W× 0.896 + w× 4.1868)(T2 - T1 )(W× 0.896 + w× 4.1868)(T3 - T1 )} × 100 4.1868 {(X × c1 ) + (Vρc2 / 1000 )}

where w is the mass of water in vessel, W is the mass of vessel complete with lid and stirrer, X is the mass of fuel consumed, c1 is the calorific value of wood, V is the volume of kerosene consumed, c2 is the calorific value of kerosene, ρ is the density of kerosene, T1 is the initial temperature of water, T2 is the final temperature of water, T3 is the final temperature of water in last vessel at the completion of test, in °C; and n is the total number of vessels used.

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Recent Advances in Bioenergy Research Vol. III 2014 9.4.2

Exergy efficiency Exergy efficiency may be defined as the ratio of output exergy to the input exergy and

given by [Dincer, 2007, Tyagi et al., 2013]:

E xo Exergy output × 100 = × 100 Exergy input E xin

ψ=

T T    m w C p (Tfw − Tiw )(1 − a ) + m pot C pA1 (Tfp − Tip )(1 − a )  Tfw Tfp   =  × 100 Ta m wd c1 (1 − ) × η+ x× d× c 2   Tfuel  

(3)

where mw is the mass water in the pot, Cp is the specific heat of water, Tfw is the final temperature of water, Tiw is the initial temperature of water, mpot is the mass of pot, CpA1 is the specific heat of Aluminium pot, Tfp is the final temperature of pot, Tip is the initial temperature of pot, mwd is the mass of wood, c1 is the calorific value of wood, Ta is the ambient temperature, Tfuel is the flame temperature, η theoretical efficiency, x is the volume of kerosene, d is the density of kerosene and c2 is the calorific value of kerosene. 9.4.3

Power Output Rating The power output rating of a cookstove is a measure of total useful energy produced

during one hour burning of fuel wood. It shall be calculated as follows (BIS, 2013): Power output rating =

m × CV× η , kW 3600 × 100

(4)

where m is the quantity of fuel wood burnt per hour, CV is the calorific value of fuel wood and η is the thermal efficiency of the cookstove, as calculated above. 9.4.4

Emission reduction calculations The emission reduction from each cookstove is based upon fraction of biomass that

can be saved during the project year and the calorific value of the biomass used and this can be calculated according to the formula given below (Global Alliance for Clean Cookstoves, 2011):

ER

y

= B y , savings × f NRB

,y

× NCV

biomass

× EF

projected

_ fossilfuel

(5)

where ERy is emission reductions during the year y in tCO2 , B y , savings is quantity of woody biomass that is saved in tonnes, f NRB , y is the fraction of woody biomass saved by the project

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Recent Advances in Bioenergy Research Vol. III 2014 activity in year y that can be established as non-renewable biomass, NCV biomass is the net calorific value of the non-renewable woody biomass that is wood fuel, 0.015 Tj/tonne), EF projected

_ fossilfuel

substituted (IPCC default for

is emission factor for the substitution of non-

renewable woody biomass by similar consumers, use of value of 81.6 tCO2/TJ and B y , savings can be calculated using the following formula:

 η  B y , savings = Bold .1 − old   η new 

(6)

Where Bold is Quantity of woody biomass used in the absence of the project activity in tonnes, Ƞold is the efficiency of the baseline system being replaced, measured using representative Sampling methods or based on referenced literature values (fraction), use weighted Average values if more than one type of system is being replaced; a default value of 0.10 may be optionally used if the replaced system is the three stone fire or a Conventional system with no improved combustion air supply or flue gas Ventilation system i.e., without a grate or a chimney; for other types of systems a Default value of 0.2 may be optionally used and Ƞnew is the efficiency of the system being deployed as part of the project activity (fraction), as Determined using the water boiling test (WBT) protocol. Use weighted average Values if more than one type of system is being introduced by the project activity. 9.5

Results and Discussion Based on the biomass characteristics, the performance of different cookstoves has

been carried out using burning capacity rate, power output, thermal efficiency and carbon emission reduction analysis at a typical location in India, while the discussion of results is given as below: Figure 5 shows the burning capacity rate and power output rating of the different model of cookstoves. As shown from the figure the burning rate and power output of NIRE02 are 3.6 kg/hr and 4.80 kW respectively. The burning rate and power output of NIRE-03 are 2.42 kg/hr and 3.45 kW respectively. NIRE-05 has the burning rate 3.22 kg/hr with 5.23 kW power output rating and NIRE-06 has the value of these quantities 2.32 kg/hr and 3.22 kW respectively. The maximum power output is found with NIRE-05 with 3.22 kg/hr burning rate which is the highest among all the other improved biomass cookstoves (NIRE03 and NIRE-06) and also from the modified traditional cookstove. These results are shows that the combustion bf biomass in NIRE-05 is better than that of the other cookstove models.

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Recent Advances in Bioenergy Research Vol. III 2014 Burning Rate(kg/hr)

Power Output(kW) 5.23

4.80 3.6

3.45

3.22

2.42

NIRE-02

3.22 2.32

NIRE-03 NIRE-05 Cookstove Model

NIRE-06

Fig-5: Burning rate and power output of different cookstove model. On the basis of results obtained from the water boiling test the thermal efficiency, exergy efficiency and emission reduction of different cookstove models was calculated using the equation (2)-(5) and the results obtained are presented in Fig 6. The thermal efficiency, exergy efficiency and emission reduction potential of modified traditional cookstove (NIRE02) was found to be 25.31%, 1.86% and 2.08 tCO2/year respectively. The performance of this modified model (NIRE-02) is two to three folds higher that of the traditional three stone fire. This cookstove model also has the good agreement with the carbon emission reduction as shown in the figure. However the thermal efficiency of improved cookstove models NIRE03, NIRE-05 and NIRE-06 are 35.19%, 37.47%, and 32.26 percent respectively, exergy efficiency 6.17%, 6.23% and 5.45% respectively and emission reduction potential from different model was 2.46, 2.52, and 2.37 tCO2/year respectively. Among all the improved cookstove models, NIRE-05 has the maximum value of thermal efficiency, exergy efficiency and as well as the CO2 emission reduction potential. This is because of the fact that the burning of the fuel wood is very good in this cookstove with higher power output and minimum heat loss into the environment. Based on the results obtained from different cookstove models it is found that the NIRE-05 cookstove is a best designed model which is steadied in this present study.

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Recent Advances in Bioenergy Research Vol. III 2014 Thermal Efficiency,η (%)

Exergy Effficiency ,ψ(%)

Emission Reduction (tCO2/year)

37.47

35.19

32.26

25.31

6.23

6.17 1.86 2.08 NIRE-02

2.52

2.46 NIRE-03

NIRE-05

5.45

2.37

NIRE-06

Cookstove Model Fig-6: Thermal efficiency, Exergy efficiency and emission reduction of different cookstove at burning capacity rate. 9. 6

Conclusions Based upon the analysis of experimental data following conclusions are drawn: •

Based on the experimental observations the energy and exergy efficiency and as well as the emission reduction potential for NIRE-02 model was found to be best when both grate and top space was provided.



The thermal efficiency 25.31% of modified traditional cookstove NIRE-02 was found which is around two-three times higher than that of the open three stone fire.



The performance and the CO2 emission reduction potential of NIRE-05 model is found to be higher than that of all other models for all set of operating parameters.



NIRE-05 model performs best and gives a large amount of emission reduction value i.e. 2.52 tonnes of CO2 reduced per household per year which means if approximately 10,000 NIRE-5 cookstove are provided to the consumers it will save approximately 25,200 tonnes of CO2 in one year.



Also energy efficiency was found to be always higher than that of exergy efficiency which is due to the energy gained by the hot water at that particular temperature.

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Recent Advances in Bioenergy Research Vol. III 2014 Acknowledgment Authors gratefully acknowledge the financial assessment provided by Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala and Ministry of New and Renewable Energy, New Delhi.

References 1.

Anhalt J., and Holanda S. (2013) Policy for subsidizing efficient stoves. Project No. 10604030. Accessed from internet on 25.07.13, 10604030_2.pdf

2.

Agenbroad J, DeFoort M, Kirkpatrick A, Kreutzer C, A simplified model for understanding natural convection driven biomass cooking stoves—Part 1: Setup and baseline validation, Energy for Sustainable Development 2011;15: 160–168.

3.

Agenbroad J, DeFoort M, Kirkpatrick A, Kreutzer C, A simplified model for understanding natural convection driven biomass cooking stoves-Part 2: With cook piece operation and the dimensionless form, Energy for Sustainable Development 2011; 15: 169–175.

4.

Bronowski J. (1973) The Ascent of Man, Little, Brow and Co., Boston.

5.

Bureau of Indian Standards. www.bis.org.in. Accessed 2013.

6.

Fullerton D.G., Brucec N. and Gordona S.B. (2008) Indoor air pollution from biomass fuel smoke is a major health concern in the developing world, Transactions of the Royal Society of Tropical Medicine and Hygiene; 102: 843-851.

7.

FAO, (1993) Improved solid biomass burning cookstoves: A development manual, Regional Wood Energy Development Program in Asia, file document 44; accessed from internet from wgbis.ces.iisc.ernet.in/energy/HC270799/RWEDP/acrobat/fd44. pdf on 4 September, 2013.

8.

Global Alliance for Clean Cookstoves (2011) (http://www.cleancookstoves.org)

9.

Household cookstove, Environment, health and climate change. The world bank, 2011 Accessed from internet on 13.09.13 climatechange.worldbank.org/…/ Household%20Cookstoves-web.pdf

10. http://cdm.unfccc.int/ accessed from internet on 30-08-13. 11. Ibrahim D. and Marc A. Rosen (2007) Exergy, energy, environment and sustainable development (Elsevier).

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Recent Advances in Bioenergy Research Vol. III 2014 12. Kleeman M.J., Schauer J.J. and Cass G.R. (2000) Size and composition distribution of fine particulate matter emitted from motor vehicles. Environmental Science & Technology; 34 (7):1132-1142. 13. Kumar M., Kumar S. and Tyagi S.K. (2013) Design, development and technological advancement in the biomass cookstoves: A review, Renewable and Sustainable Energy Reviews, 26: 265-285. 14. Raju S.P. (1957) Smokeless Kitchens for the millions, Rev. edn., The Christian Literature Society, Madras, India. 15. Ramanathan V, and Carmichael G (2008) Global and regional climate changes due to black carbon, Nature Geo-science, 1:221–227. 16. Singer H. (1961) Improvement of fuel wood cooking stoves and economy in fuel wood consumption, Report to the Government of Indonesia, Report no. 1315, Expanded Technical Assistance Program, FAO, Rome. 17. Tyagi S.K., Pandey A.K., Sahu S., Bajala V. and Rajput J.P.S. (2013) “Experimental study and performance evaluation of various cookstove models based on energy and exergy analysis,” Journal of Thermal Analysis and Calorimetry, 111 (3): 1791-1799. 18. Smith KR, Uma R, Kishnore VVN, Zhang J, Joshi V and Khalil MAK (2000) Greenhouse implications of household stoves: an analysis for India. Annual Reviews of Energy and the Environment, 25:741–763. 19. Kishore V.V.N. and Ramana P.V. (2002) Improved cookstoves in rural India: how improved are they?: a critique of the perceived benefits from the National Programme on Improved Chulhas (NPIC). Energy, 27: 47–63. 20. WHO (2006) Fuel for Life. Household Energy and Health, Geneva.

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Recent Advances in Bioenergy Research Vol. III 2014

Part III Biochemical Conversion

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 10 BIOPROSPECTING HALOTOLERANT CELLULASE FROM SALINE ENVIRONMENT OF BHITARKANIKA NATIONAL PARK, ODISHA Dash Indira, Sahoo Moumita, Dethose Ajay, C. S. Kar, R. Jayabalan

Abstract Research interest on biofuels gained much attention with depleting fossil fuel reserves and growing concern on environment. Bioethanol and biodiesel from biomass will fulfil the future energy needs. Lignocellulosic and algal biomass are sustainable and cost-effective resources for bioethanol production. However, algal biomass is preferred over lignocellulosic biomass due to its high cost of pre-treatment and production of fermentation inhibitors. Systems, which use nonpotable water for biofuel production, are considered due to the future threats on availability of potable water. Halotolerant enzymes are one of the promising candidates to be used in seawater based systems. Halotolerant enzymes can also be used for saccharifying the biomass, which are pre-treated with ionic liquids. Odisha being a coastal state with a coastline of 450 Km and with a mangrove ecosystem has potential to evidence halotolerant microorganisms, which can produce salt tolerant enzymes. In the present study five isolates were confirmed for cellulase production using CMC agar plates and Congo red assay. Activity of FP-endoglucanase produced by the isolates was assayed by determining the amount of reducing sugar formed from Cellulose by methods recommended by IUPAC. Endoglucanase produced by halotolerant microorganisms, named as BHK1, BHK2, BHK3, BHK4 and BHK5 which were isolated from soil and water samples were having optimal pH 5.0, 4.0, 5.0, 5.0 and 6.0 and optimal temperature of action at 65°C, 45°C, 65°C, 55°C and 65°C respectively. Endoglucanase activity was found to be enhanced several folds by use of 5 mM MnSO4 as cofactor. Identification of microorganisms and further characterization of the endoglucanase are in progress. Keywords: Cellulase, halotolerant, optimal temperature, optimal pH, cofactor.

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Recent Advances in Bioenergy Research Vol. III 2014 10.1

Introduction In 2008, 88% of global energy consumption was dependent on fossil fuels (Brennan and

Owende, 2010). Fossil fuel however, now accepted as unsustainable resources due to its depletion and increasing environmental concern due to accumulation of green house gases (Schenk et al., 2008). To overcome the dependence on fossil fuels, maintenances of sustainable economy and for global environmental concern, it is necessary to focus over and promote renewable resources of energy (Brennan and Owende, 2010; Prasad et al., 2001 a,b; Singh et al., 2010 a,b). Biomass is one of the most promising renewable resources for the production of biofuels, such as bioethanol (John et al., 2011) and biodiesel (Ho et al., 2010). Most of the bioethanol production across the globe comes from sugar and starch crops and from lignocellulosic biomass. However, with increasing demand of food, decreasing water availability and cost involved in pre-treatment of lignocellulosic biomass, agriculture crops are losing importance. By overcoming the drawbacks of lignocellulosic and sugar rich biomass, microalgae has gained the title of third generation feedstock for biofuel production (Nigam and Singh, 2011). Microalgal biomass rich in carbohydrate forms an excellent substrate for bioethanol production. It grows faster and fixes carbon dioxide at higher rate than terrestrial plants (Ho et al., 2012), also, rich in starch and cellulose and lacks lignin, which makes monosaccharide conversion easier (Ho et al., 2012, John et al., 2011). The carbohydrates in the form of starch or cellulose or complex polysaccharides are not fermentable and therefore are hydrolyzed to fermentable sugar prior to fermentation (Hagerdal-Hahn et al., 2007). Acid or alkali hydrolysis are common chemical methods used as they are faster, cheaper and easier however, they lead to release of components that acts as fermentation inhibitors and as a result hampers the fermentation process (Harun et al., 2010; Girio et al., 2010; Moxley and Zhang, 2007). Enzymatic hydrolysis process is amiable to environment and gives higher glucose yield without production of fermentation inhibitors. Cellulases are the key enzymes of saccaharification of both lignocellulosic and algal biomass. However, it is interestingly observed that in saline system, which is independent of fresh water, cellulase and other glucoamylases are completely inhibited thereby making them unsuitable for industrial usage (Matsumoto et al., 2003). There is expanding knowledge on halophilic enzymes and organisms capable of effectively treating marine biomass. A recent review on industrial and environmental applications of halophilic microorganisms states that

100

Recent Advances in Bioenergy Research Vol. III 2014 demand of salt tolerant enzymes and microorganisms is still limited (Oren, 2010), nevertheless, continuous efforts in this field and favourable results may change the scenario. Potential salt tolerant enzymes have been isolated from hypersaline microorganisms, which basically includes the halophilic α-amylase from Haloarcula hispanica (Vasisht et al., 2005), glucoamylases from marine yeast Aureobasidium pullulans N13d (Oren et al., 2006), thermophilic and halophilic amyloglucosidase from Halobacterium sodomense with optimal temperature between 66°C to 76°C and optimal activity between 8% to 22% of NaCl (Duan et al., 2006). An alkalihalotolerant cellulase from Bacillus flexus NT, isolated from Ulva lactuca with residual activity of about 70% at 15% NaCl concentration (Trivedi et al., 2010). Reports suggests that commercially available accellerase-1500 (cocktail of different glycosidases) performs depolymerisation of cellulose and avicel in reaction media containing 1X, 2X and 4X concentration of seawater (Grande et al., 2012) . Regarding the fermentation of marine algal biomass, minimal progress reported in the past 30 years after the reports of Clostridium pasteurianum fermenting Dunaliella species to produce butanol, 1, 3-propanediol and ethanol in presence of 10% NaCl. To overcome the salinity issues micro algae is either washed in deionised water or grown in a less saline medium however, the salinity problem during fermentation can be encountered by using halotolerant yeast. Citeromyces siamensis is novel halotolerant yeast isolated from dry salted squid and fermented soybeans in Thailand (Nagatsuka et al., 2002) which is tolerant to higher concentration of cations (3M NaCl and 0.8M LiCl) and osmotic pressure (60% glucose). Pichia sorbitophila is also reported halotolerant yeast, which can grow in 4M NaCl concentration when glucose and glycerol are sole carbon source (Lages and Lucas, 1995). The present work aimed to determine the effect of temperature, pH and metal ions on the cellulase produced by salt tolerant cellulolytic bacteria isolated from Bhitarkanika National Park, Odisha. 10.2

Materials and methods

10.2.1 Isolation of cellulase producing bacteria Soil and water samples were collected from Bhitarkanika National Park, Odisha. Samples were serially diluted and inoculated in Trypticase soy agar (TSA) media (15 g Tryptone, 5 g Soytone, 5 g Sodium Chloride, 15 g agar/ 1 L Distilled water) and incubated at 37°C for 24 hours. Individual colonies were obtained by streaking the culture on same media. Confirmation 101

Recent Advances in Bioenergy Research Vol. III 2014 of cellulose-degrading ability of bacterial isolates was performed by streaking on the CMC agar media (5 g CMC, 1 g NaNO3, 1 g K2HPO4, 1 g KCl, 0.5 g MgSO4, 0.5 g yeast extract, 1 g glucose, 17 g agar/ 1L Distilled water). The use of Congo red (1 mg/ml) as an indicator for cellulose degradation in an agar medium and 1 M NaCl as destaining solution provides the basis for a rapid and sensitive screening test for cellulolytic bacteria (CB). Colonies showing discoloration of Congo red was taken as positive cellulose-degrading bacterial colonies (Lu et al., 2004) and only these were taken for further study. 10.2.2 Cellulase enzyme production The selected cellulolytic bacterial isolates were cultured at 37°C at 150 rpm in 100 ml of enzyme production media composed of 0.1 g NaNO3, 0.1 g KH2PO4, 0.0.1 g KCl, 0.5 MgSO4, 0.5 g yeast extract , 0.1 g glucose, 0.5 g filter paper at pH 6.8–7.2. Broth culture after three days of incubation period was subjected to centrifugation at 5000 rpm for 15 min at 4°C. Supernatant was collected and stored as crude enzyme preparation at 4°C for further enzyme assays (Tailliez et al., 1989). 10.2.3 Cellulase assay Filter paper cellulase activity was assayed by measuring the amount of reducing sugar formed from soluble form of cellulose, CMC. Determination of enzyme activity is measured using methods suggested by International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987). In these tests, 0.5 ml of crude enzyme extract is incubated with 0.5 ml of 2% CMC in 10 mM citrate buffer at different temperatures and pH and the amount of reducing sugars formed were estimated spectrophotometrically with 3,5-dinitrosalicylic acid using glucose as standards (Miller, 1959). Then enzymatic activities of total endoglucanase were defined in units. One unit of enzymatic activity is defined as the amount of enzyme that releases one micromole reducing sugars (measured as glucose) per minute. 10.2.3.1 Effect of temperature on cellulase activity The crude enzyme extract along with the substrate (2% CMC in 10 mM citrate buffer) was incubated for 15 min at different temperatures such as 30, 40, 50, 60 and 70°C to study their effect on cellulase activity and assayed spectrophotometrically for their effect of enzyme activity. 10.2.3.2 Effect of pH on cellulase activity 102

Recent Advances in Bioenergy Research Vol. III 2014 The pH of the test solution was adjusted to different pH range of 3.0, 4.0, 5.0, 6.0 and 7.0 with phosphate buffer (10 mM Na2HPO4.2H2O, 1.8 mM KH2PO4) at their respective optimal temperature for 15 minutes. The test samples were then assayed to study their effect on enzyme activity. 10.2.3.3 Effect of metals on cellulase activity After getting optimum temperature and pH value different metal ions such as MgCl2, ZnCl2, MnCl2, FeCl2, CoCl2, EDTA and PMSF were subjected to study their effect on enzyme activity. They all were taken in a concentration of (5 mM) and assayed. 10.3

Result and discussion

10.3.1 Isolation and screening of cellulase producing bacteria Five strains of cellulolytic bacteria were isolated from soil and water samples from Bhitarkanika National Park, Odisha, India. Three bacterial isolates from soil (BHK1, BHK2, BHK3) and two from water (BHK4 and BHK5) were confirmed for cellulase production on CMC agar plates on aerobic incubation of 48 hours at 37°C by production of clear zone which was identified using Congo red assay (Figure1). 10.3.2 Production of cellulase All the five isolates (BHK1, BHK2, BHK3, BHK4 and BHK5) were cultured on enzyme production medium containing filter paper as sole cellulosic substrate. All the isolates showed cellulase activity on filter paper ranging between 0.93 EU/ml to 0.98 EU/ml. Highest cellulolytic potential was exhibited by BHK1 with 0.98 EU/ml whereas lowest was recorded in BHK4 with 0.93 EU/ml (table 1). 10.3.3 Cellulase assay Cell free supernatant collected from culture of BHK1, BHK2, BHK3, BHK4 and BHK5 was used as crude enzyme extract for determination of effect of temperature, pH and metal ions. 10.3.3.1 Effect of temperature on cellulase activity Crude enzyme extract from all the five isolates (BHK1, BHK2, BHK3, BHK4 and BHK5) were assayed at temperature ranging between 30°C to 70°C for 15 minutes. Maximum enzyme activity was recorded by BHK1 at 65°C, BHK2 at 45°C, BHK3 at 65°C, BHK4 at 55°C 103

Recent Advances in Bioenergy Research Vol. III 2014 and BHK5 at 65°C (Figure3.a). The above result in correlation with the fact that most of the cellulases have optimal temperature of action at 55°C.

Figure1: Clear zone formed on CMC agar plates by BHK1, BHK2, BHK3, BHK4 and BHK5 after 48 hours of incubation and stained with Congo red confirming the presence of extracellular cellulase. Table 1: Production of cellulase by BHK1, BHK2, BHK3, BHK4 and BHK5 in enzyme production medium after 72 hours of incubation. Cellulolytic bacterial strains

BHK 1 BHK 2 BHK 3 BHK 4 BHK 5

Amount of cellulase produced after 72 hours incubation (EU/ml) 0.98 0.95 0.96 0.93 0.93

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Recent Advances in Bioenergy Research Vol. III 2014 10.3.3.2 Effect of pH on cellulase activity Crude enzyme extract from all the isolates (BHK1, BHK2, BHK3, BHK4 and BHK5) were assayed in pH ranging between 3.0 to 7.0. Optimal pH of action for all the five isolates recorded as BHK1 at pH 5.0, BHK2 at pH 4.0, BHK3 at pH 5.0, BHK4 at pH 5.0 and BHK5 at pH 6.0 (Figure3.b). Optimal pH of action ranging between 5.0 to 6.0 in most cases may be due to the fact that all the isolates were from estuarine environment and not from purely saline sources. 10.3.3.3 Effect of metal ions on cellulase activity With optimal temperature and pH value obtained from the above five cellulolytic isolates (BHK1, BHK2, BHK3, BHK4 and BHK5) were assayed for effect of metal ions (5 mM concentration each Of MgCl2, ZnCl2, MnCl2, FeCl2, CoCl2, EDTA and PMSF). In all cases, enzyme activity is recorded maximum in presence of Mn2+. Other ions (Mg2+, Zn2+, Fe 2+, Co2+, EDTA and PMSF) did not show any substantial effect on the enzyme activity except Co2+ in BHK1, which enhanced the enzyme activity but not to such extent as Mg2+ (Figure 3.c and d). It may be inferred that Mn2+ may act as cofactor or enhancer of enzyme activity in these filter paper cellulases isolated from cellulolytic bacteria isolated from Bhitarkanika National Park, Odisha.

(a)

(b)

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Recent Advances in Bioenergy Research Vol. III 2014

(b)

(d)

Figure 3. (a) Effect of Temperature on enzyme action, (b) Effect of pH on enzyme action, (c) Effect of metal ions on enzyme action and (d) Comparative effect of Mn2+ on different strains of cellulolytic bacteria. 10.4

Conclusion The present study focused on screening of cellulase producing organisms from saline

environment of Bhitarkanika National Park, Odisha. Five isolates tested positive for production of extracellular cellulase and utilized for production of cellulase using suitable enzyme production media with filter paper as raw source of cellulose. Crude enzyme so produced were analysed for effect of temperature, pH and metal ions on their activity. All the potential cellulaseproducing organisms had enzyme ranging between 0.93EU/ml to 0.98 EU/ml. Optimal temperature of enzyme action is between 45°C to 65°C suggests that the enzymes work at higher temperature and at thermotolerant in nature. Optimal pH of action in the range 5.0 to 6.0 suggests that strains from less saline environment. Endoglucanase activity was found to be enhanced several folds in presence of 5 mM Mn2+ suggesting its role as cofactor or enhancer molecule. Identification of microorganisms and further characterization of the endoglucanase are in progress. A further study also includes isolation of potential halotolerant cellulolytic bacteria from more saline environment for their utilization in seawater-based medium. Acknowledgement Authors are very much thankful to MHRD and National institute of Technology, Rourkela for the financial support for providing all research facilities and Office of the Principal

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Recent Advances in Bioenergy Research Vol. III 2014 CCF (Wildlife) & Chief Wildlife Warden, Odisha for authorization of permission for sample collection from Bhitarkanika National Park, Odisha.

References

1. Brennan L. and Owende P. (2010) Biofuels from microalgae- a review of technologies for production, processing and extractions of biofuels and co-products. Renew. Sustain. Energy Rev., 14: 557–577.

2. Schenk P. M., Thomas-Hall S. R., Stephens E., Marx U. C., Mussgnug J. H., Posten C., Kruse O., Hankamer B. (2008) Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenergy Res., 1: 20–43.

3. Prasad S., Singh A., Jain N., Joshi H. C.( 2007) Ethanol production from sweet sorghum syrup forutilization as automotive fuel in India. Energy Fuels., 21: 2415–2420.

4. Prasad S., Singh A., Joshi H. C. (2007) Ethanol as an alternative fuel from agricultural, industrial and urban residues. Resour Conserv Recy., 50: 1–39.

5. Singh A., Pant D., Korres N. E., Nizami A. S., Prasad S., Murphy J. D. (2010) Key issues in life cycle assessment of ethanol production from lignocellulosic biomass: challenges and perspectives. Bioresour. Technol., 101: 5003–5012.

6. Singh A., Smyth B. M., Murphy J. D. (2010) A biofuel strategy for Ireland with an emphasis on production of biomethane and minimization of land take. Renew. Sustain. Energy Rev., 14: 277–288.

7. John R.P., Anisha G.S., Nampoothiri K.M., Pandey A. (2011) Micro and macroalgal biomass: a renewable source for bioethanol. Bioresour. Technol., 102 (1): 186– 193.

8. Ho S.-H., Chen W.-M., Chang J.-S. (2010) Scenedesmus obliquus CNW-N as a potential candidate for CO 2 mitigation and biodiesel production. Bioresour. Technol., 101 (22): 8725–8730.

9. Nigam P.S. and Singh A. (2011) Production of liquid biofuels from renewable resources. Prog. Energ. Combust., 37 (1): 52–68.

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Recent Advances in Bioenergy Research Vol. III 2014 10. Ho S.-H., Chen C.-Y., Chang J.-S. (2012) Effect of light intensity and nitrogen starvation on CO 2 fixation and lipid/carbohydrate production of an indigenous microalga Scenedesmus obliquus CNW-N. Bioresour. Technol., 113: 244–252.

11. Hahn-Hagerdal B., Karhumaa K., Fonseca C., Spencer-Martins I., Gorwa-Grauslund M.F. (2007) Towards industrial pentose-fermenting yeast strains. Appl. Microbiol. Biotechnol., 74 (5): 937–953.

12. Harun R., Danquah M.K., Forde G.M. (2010) Microalgal biomass as a fermentation feedstock for bioethanolproduction. J Chem Technol Biotechnol.,85: 199–203.

13. Girio F.M., Fonseca C., Carvalheiro F., Duarte L.C., Marques S., Bogel-Lukasik R. (2010) Hemicelluloses for fuel ethanol: a review. Bioresour. Technol., 101 (13): 4775– 4800.

14. Moxley G. and Zhang Y.H.P. (2007) More accurate determination of acid-labile carbohydrates in lignocellulose by modified quantitative saccharification. Energy Fuel, 21 (6): 3684–3688.

15. Matsumoto M., Yokouchi H., Suzuki N., Ohata H., Matsunaga T. (2003) Saccharification of marine microalgae using marine bacteria for ethanol production. Appl Biochem Biotechnol., 105:247-254.

16. Oren A. (2010) Industrial and environmental applications of halophilic microorganisms. Environ Technol., 31:825e34.

17. Vasisht N., Bolhuis A., Hutcheon G.W. (2005). Characterisation of a highly stable aamylase from the halophilic archaeon Haloarcula hispanica. Extremophiles, 9:487-495.

18. Duan X., Sheng J., Wang L., Chi Z., Li H., Wu L. (2006) Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme. Process Biochem., 42:462-465.

19. Trivedi N., Gupta V., Kumar M., Kumari P., Reddy C.R.K., Jha B. (2010) An alkalihalotolerant cellulase from Bacillus flexus isolated from green seaweed Ulva lactuca. Carbohydrate Polymers.83(2)891-897.

20. Grande P.M., De Maria P.D. (2012) Enzymatic hydrolysis of microcrystalline cellulose in concentrated seawater. Bioresour.Technol., 104:799-802.

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Recent Advances in Bioenergy Research Vol. III 2014 21. Nagatsuka Y., Kawasaki H., Limtong S., Mikata K., Seki T. (2002) Citeromyces siamensis sp. nov., a novel halotolerant yeast isolated in Thailand. Int J Syst Evol Microbiol., 52(6):2315-2319.

22. Lages F., Lucas C. (1995) Characterization of a glycerol/H+ symport in the halotolerant yeast Pichia sorbitophila. Yeast., 11(2):111-9.

23. Lu W. J., Wang H. T., Nie Y. F. (2004) Effect of inoculating flower stalks and vegetable waste with ligno-cellulolytic microorganisms on the composting process. J Env. Sc. and Health, Part B, 39(5-6):871–887.

24. Tailliez P., Girard H., Millet J., Beguin P. (1989) Enhanced cellulose fermentation by an asprogenous and ethanol tolerant mutant of Clostridium thermocellum. Appl. Env. Microbiol. 55:207–211.

25. Ghose T. K. (1987) Measurement of cellulase activity. Pure and Applied Chemistry., 59:257–268.

26. Miller G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry. 31(3):426–428.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 11 ISOLATION AND MOLECULAR CHARACTERIZATION OF CELLULOLYTIC FUNGI USED FOR CONVERSION OF SUGARCANE BIOMASS FOR BIOETHANOL PRODUCTION Chetan, A. M., Harinikumar, K. M., Bhavani, P., Manoj Kumar, H. B., Madhu T., Ningaraj Dalawai

Abstract One of the major alternatives to fossil fuels that received major attention is bioethanol derived from biomass. The cellulosic material are potential sources of ethanol. Cellulolytic fungi are capable of degrading cellulose to smaller sugar components like glucose units. The aim of this research was to isolate and screen fungi capable of producing cellulases and to convert pretreated lignocellulosic material to fermentable sugars for the production of ethanol using Saccharomyces cerevisiae. The lignocellulosic material such as sugarcane bagasse and sugarcane trash were used as substrates for ethanol production. Fungi were isolated from soil and compost samples collected from various regions. The pure cultures were screened for the ability to degrade cellulose. The cellulolytic activity was determined by measuring the clearing zone created by the fungi. The cultures were further characterized using five random primers. The fungi capable to produce cellulases were identified as Aspergillus niger, Aspergillus fumigatus, Trichoderma viride, Trichoderma harzianum and Trichoderma reesei based on colony characters, microscopic observation and identification at molecular level based on DNA coding for 18S rRNA. The substrates were powdered and pretreated with fungal isolates using Mandels’ and Reese media. The substrates were used as a carbon source. Sugarcane bagasse and trash treated with Trichoderma reesei have shown highest concentration of reducing sugars of 45.95mg/g and 40.56mg/g respectively and ethanol yield of 11.56g/l and 10.92g/l respectively. From this study the fungal cultures having the potential to degrade cellulosic material were identified and they can be used for bioethanol production from lignocellulosic wastes.

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Recent Advances in Bioenergy Research Vol. III 2014 11.1

Introduction The growth of population and the associated demand for fuel and food coupled with more

restrictive environmental regulations have intensified the research and development of renewable energy feedstocks to substitute for and/or to complement fossil fuel sources (Pereira Jr. et al., 2008). The transfer of crude oil-based refinery to biomass-based biorefinery has attracted strong scientific interest which focuses on the development of cellulosic ethanol as an alternative transportation fuel to petroleum fuel (Zheng, et al., 2009). In India, ethanol is primarily produced from molasses which is a byproduct from sugar mills using Saccharomyces cerevisiae strains. Sugarcane bagasse and trash, the left-over residue of leaves and tops, can be converted to ethanol by enzymatic or acid catalyzed hydrolysis and fermentation of the released monosaccharides (Michael et al., 2006). Sugarcane, an important cash crop, is grown over an area of 4.2 million hectares in India with an average productivity of 70 tons ha–1. Sugarcane converts approximately 2% of solar energy into chemical bonds of carbohydrates where in two third of these carbohydrates are in the form of lignocelluloses. These lignocellulosic material are a potential source of bioethanol production. There are three major steps to be employed in the conversion of lignocellulosic to Bioethanol which are pretreatment for lignin breakdown, hydrolysis and fermentation for Bioethanol production. The most challenging part is the hydrolysis process in order to obtain the reducing sugars. Hydrolysis of lignocellulosic can be done in two ways, either by using enzymatic or chemical methods. However, enzymatic hydrolysis is more environmental friendly as compared to chemical hydrolysis. Although, the costs of using commercial enzymes are expensive, however this problem can be overcome by using cellulose degrading organisms (Zainan et al., 2011). This research work has been carried out in order to produce bio-ethanol from lignocellulolytic wastes by using cellulolytic fungi. 11.2

Material and Methods Present investigation was carried out to isolate and identify cellulolytic fungi for the

conversion of lignocellulolytic biomass into Bio-ethanol and to characterize them using ITS primers (Internal transcribed spacer) and RAPD primers. 11.2.1 Isolation

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Recent Advances in Bioenergy Research Vol. III 2014 The soil and compost samples were collected from various regions of Mandya. The samples were inoculated on potato dextrose agar (PDA) medium. For the isolation of fungi, dilution plate method was used. All the samples (i.e., three soil samples and two compost samples) were serially diluted and the dilutions 10-2, 10-3, 10-4 were plated using spread plate method. 11.2.2 Screening From the various isolates, screening for cellulolytic fungi was made using selective media (Mandels' and Reese agar medium) procedures to select the potential isolates that could saccharify lignocellulosic wastes. Fungi determined to be cellulolytic were then cultured in Mandels salt medium supplemented with cellulose. (Mandels & Reese, 1957). Cellulolytic fungi create a clearing zone around the colony on the agar. 11.2.3 Molecular characterization Molecular characterization was done using five random primers OPA-1; OPD-6; OPA-4; A-5 and AA-11. 11.2.4 Extraction of DNA DNA extraction protocol was followed according to Chakraborty et al., (2010). Isolation of fungal genomic DNA was done by growing the fungi for 4 days. The quality and quantity of DNA was analyzed both spectrophotometrically and in 0.8% agarose gel. The DNA from all isolates produced clear sharp bands, indicating good quality of DNA. 11.2.5 PCR Amplification of ITS Region of fungal Isolates: All fungal isolates were taken up for ITS-PCR amplification. Genomic DNA was amplified by mixing the template DNA (50 ng), with the polymerase reaction buffer, dNTP mix, primers and Taq polymerase. Polymerase Chain Reaction was performed in a total volume of 50 µl, containing Sample ~50ng of gDNA, 100ng of Forward Primer, 100ng of Reverse Primer, 2µl of 10mM dNTPs mix, 5µl of 10X Taq Pol. Buffer, 3U Taq Polymerase enzyme, PCR grade water to make the volume upto 50µl. PCR was programmed with an initial denaturing at 94°C for 5 min. followed by 30 cycles of denaturation at 94 °C for 30 sec, annealing at 52°C for 30 sec and extension at 72°C for 1 min and the final extension at 72 °C for 3 min in a Thermocycler. After

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Recent Advances in Bioenergy Research Vol. III 2014 PCR, all the amplified products were ran on 1.5% Agarose gel with 1X TAE buffer. Sequencing was done at Amnion biosciences using ABI3730xl sequencer. 11.2.6 RAPD of Trichoderma Isolates: For RAPD, six random primers i.e. OPA-1; OPD-6; OPA-4; A-5; AA-04 and AA- 11 were selected (Table-1). Polymerase Chain Reaction was performed in a total volume of 50 µl, containing Sample ~25ng of gDNA, 400ng RAPD Primer, 2µl of 10mM dNTPs mix, 5µl of 10X Taq Pol. Buffer, 3U Taq Polymerase enzyme, PCR grade water to make the volume upto 50µl. PCR was programmed with an initial denaturing at 94°C for 5 min. followed by 45cycles of denaturation at 94°C for 1 min, annealing at 38°C for 1 min and extension at 72°C for 2 min and the final extension at 72°C for 5 min in a Thermocycler. After PCR, all the amplified products were ran on 1.5% Agarose gel with 1X TAE buffer. 11.2.7 Scoring the data The image of the gel the ribosomal RNA genes (rDNA) possess electrophoresis was documented through gel documentation system and analysis software. All reproducible polymorphic bands were scored and analysed following UPGMA cluster analysis protocol. The RAPD patterns of each isolate was evaluated, assigning character state “1” to indicate the presence of band in the gel and “0” for its absence in the gel. The scored band data (Presence or absence) was subjected to cluster analysis-using STATISTICA. Production of Bio-ethanol from Pretreated Sugarcane Biomass Using Saccharomyces Cerevisiae The two substrates, sugarcane bagasse and sugarcane trash were powered and pretreated with the fungal cultures using Mandels' and Reese medium using the substrates as carbon source. Culture conditions: 10g /l of each residue was taken in conical flask containing 200ml of Mandle’s medium. The conical flasks were plugged with cotton and sterilized at 15lbs per sq.inch for 20 minutes. Each flask was inoculated with 4-5 discs of different fungi. These flasks were incubated at room temperature for 5days on an orbital shaker. After five days mycelium was separated by filtration through Whatman filter paper No.1. The filtrate was used for further studies (Kader et al., 1999).

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Recent Advances in Bioenergy Research Vol. III 2014 Determination of total carbohydrate: The carbohydrate content of untreated and pretreated raw material in the culture broth was measured by phenol sulphuric acid method with glucose as standard (Dubois et al., 1956; Krishnaveni et al., 1984). Determination of reducing sugars: Reducing sugars in untreated and pretreated raw material in the culture broth were determined by dinitrosalicylic acid (DNS) method with glucose as standard (Miller, 1972). Fermentation: Culture filtrate was further inoculated with Saccharomyces cereviseae strain and allowed for fermentation for seven days. After fermentation it was filtered and ethanol content was determined. Ethanol estimation: The ethanol was estimated by Colorimetric method as described by Caputi et al., (1968). 11.3

Result and discussion Nine fungal isolates were obtained using Potato Dextrose Agar medium from the soil and

compost samples collected. Among them five isolates were identified and pure cultures were maintained on PDA plates. The pure fungal isolates were screened for cellulolytic ability. Highest cellulolytic activity was detected in three isolates of Trichoderma sp. Zone of clearance was highest in Trichoderma reesei (2.10mm) followed by Trichoderma viride (2.00mm) and Trichoderma harzianum (1.60mm). The enzymatic activity was considerably low in other fungi such as Aspergillus sp the clearance zone was lesser in Aspergillus niger (1.50mm) and the least was Aspergillus fumigatus (0.80mm) . The screened fungal strains were used for further studies. The DNA isolated from the desired cellulose producing fungi tentatively identified as species of Apergillus and Trichoderma species. When checked for purity exhibited that the DNA isolated from the two sources was pure. The same DNA samples, when run on an agarose gel also, confirmed to be pure as the bands of DNA are single and distinct. Traces of contaminants were found when observed under the Gel doc and photographed. In the present study we focused on the ITS region of ribosomal genes for identifying fungal species. ITS region of rDNA was amplified using genes specific ITS-1 and ITS-4 Primers. Amplified products were of size in the range 100bp to 180bp. The results are in accordance with Chakroborthy et al., (2010) who

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Recent Advances in Bioenergy Research Vol. III 2014 studied the identification and genetic variability of fungal isolates. The ITS PCR has helped to detect polymorphism at ITS region of rDNA among fungal isolates. In this study, a set of 5 accessions were genotyped with 5 RAPD primers as these primers are considered as superior for assessing genetic diversity. The genotypes were grouped into two major clusters, cluster II was the largest with 3 genotypes i.e. Trichderma reesei, Trichoderma viride and Trichoderma harzianum indicating that the three species are genetically closely related followed by cluster I with 2 genotypes i.e. Aspergillus niger and Aspergillus fumigatus indicating that the two species are closely related. The results obtained in the present study are also in accordance with the cluster analysis results obtained by Chakraborthy et al., (2010). Total sugar, reducing sugar and nonreducing sugar content of fungal pretreated and untreated samples were estimated. The results obtained are presented in Table 1. Autoclaving for sterilization has affected and resulted in increase in sugar content. With fungal treatment still increase in the yield of sugars was observed. The treated samples were subjected to distillation and the ethanol thus obtained was estimated using potassium dichromate method and the concentration of ethanol obtained is presented in Table 2. The results obtained were analyzed statistically using completely randomized design. In Tables 1 and 2 mean values assigned with same superscript(s) do not differ significantly (P=0.05). Among the two substrates sugarcane bagasse pretretaed with Trichoderma reesei has given maximum reducing sugar content (45.95 mg/g) followed by sugarcane trash pretreated with the same culture (40.56 mg/g). The substrates pretreated with Trichoderma viride and Trichoderma harzianum have moderately increased the sugar content. Pretreatment with Aspergillus niger and Aspergillus fumigatus have shown comparatively lesser results. Among the two substrates sugarcane bagasse pretretaed with Trichoderma reesei has given maximum ethanol yield (11.56g/l) followed by sugarcane trash pretreated with the same culture 10.92g/l. The substrates pretreated with Trichoderma viride and Trichoderma harzianum have moderately increased the sugar content. Pretreatment with Aspergillus niger and Aspergillus fumigatus have shown comparatively lesser results.

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Recent Advances in Bioenergy Research Vol. III 2014

Fig (a)

Fig (b)

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Fig(c)

Recent Advances in Bioenergy Research Vol. III 2014

Fig (d)

Fig (e)

Fig (f)

Fig a: genomic DNA; Fig (b-f) Gel picture RAPD analysis of fungal isolates with OPA-1, OPD-6, OPA-4, A-5, AA-11. Lane TH: Trichoderma harzianum, Lane M: 100bp DNA ladder (100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp) TV: Trichoderma viride, TR: Trichoderma reesei, AF: Aspergillus fumigatus, AN: Aspergillus niger

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Recent Advances in Bioenergy Research Vol. III 2014 Table: 1: Concentration of reducing sugars, non reducing sugars and total sugars of the hydrolysates Sugarcane bagasse

Sl no.

Cultures

Reducing sugar (mg/g)

Sugarcane trash

Non reducing

Total sugar

sugar

(mg/g)

(mg/g)

Reducing sugar (mg/g)

Non reducing

Total sugar

sugar

(mg/g)

(mg/g)

Untreated

0.98f

1.27e

2.25f

0.88f

1.15e

2.030f

1.

Aspergillus niger

35.45d

23.55c

59.00d

31.12d

20.28c

51.40d

2.

Aspergillus fumigatus

33.32e

21.02d

54.34e

28.19e

18.82d

47.01e

3.

Trichoderma viride

39.22b

30.90a

70.12b

38.54b

28.89b

67.43b

4.

Trichoderma harzianum

37.98c

28.00b

65.98c

32.22c

29.32b

61.54c

5.

Trichoderma reesei

45.95a

30.05a

76.00a

40.56a

31.34a

71.90a

SEm±

0.212

0.220

0.107

0.171

0.189

0.082

CD at 1%

0.841

0.872

0.426

0.677

0.747

0.320

Control

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Recent Advances in Bioenergy Research Vol. III 2014 Tree Diagram for 5 Variables Unweighted pair-group average Squared Euclidean distances

AF AN TR TH TV

0

2

4

6

8

10

12

14

16

Linkage Distance

Table 2: Concentration of Ethanol obtained from hydrolysates of Sugarcane Biomass Ethanol (g/l) Sl no.

Cultures Sugarcane bagasse

Sugarcane trash

1.

Untreated

1.56f

1.20f

2.

Aspergillus niger

7.32d

6.58d

3.

Aspergillus fumigatus

6.10e

5.86e

4.

Trichoderma viride

10.15b

9.78b

5.

Trichoderma harzianum

9.01c

8.77c

6.

Trichoderma reesei

11.56a

10.92a

SEm±

0.112

0.066

CD at 1%

0.440

0.261

18

Recent Advances in Bioenergy Research Vol. III 2014 References 1. Caputi A, Ueda JM, Brown T. (1968) Spectrophotometric determination of chromic complex formed during oxidation of alcohol. AJEV. 19:160-165. 2. Chakraborty BN, Chakraborty U, Saha A, Dey PL, Sunar K. (2010) Molecular Characterization of Trichoderma viride and Trichoderma harzianum Isolated from Soils of North Bengal Based on rDNA Markers and Analysis of Their PCR-RAPD Profiles., Global J. Biotech. Biochem. 5(1): 55-61. 3. Dubois M., Gilles KA, Hamilton JK, Rebers PA, Smith F. (1956). Anal. Chem., 26:350351. 4. Kader AJ, Omar O, Feng LS. (1999) Isolation of cellulolytic fungi from the Barino Highlands, Sarawak. ARBEC. 5. Krishnaveni S, Balasubramanian T, Sadasivam S. (1984) Carbohydrates. Food Chem., 15:229. 6. Mandels

M, Reese ET. (1957) Induction of cellulase in fungi by cellobiose. J.

Bacteriology. 73:816- 826. 7. Michael S, Carlos M. (2006) Production of fuel ethanol from sugarcane bagasse and sugarcane trash. Congress on Sugar and Sugar Cane Derivatives, Havana, Cuba, 19 22. 8. Miller GL. (1972) Carbohydrates. Anal. Chem.31:426. 9. Pereira JR, Nei; Couto, Maria Antonieta PG, Santa Anna, Lidia Maria M. (2008)Series on biotechnology: Biomass of lignocellulosic composition for fuel ethanol production within the context of biorefinery. Rio de Janeiro, Amigadigital Press, 47 p. ISBN 97885-903967-3-4. 10. Zainan NH, Md. Alam Z, Ma’an Fahmi al-Khatib. (2011) Production of sugar by hydrolysis of empty fruit bunches using palm oil mill effluent (POME) based cellulases: Optimization study. Afr. J. Biotechnol. 10(81):18722-18727. 11. Zheng Y, Pan Z, Zhang R. (2009) Overview of biomass pretreatment for cellulosic ethanol production. Int. J.Agric and Biolo. Eng. 2(3):51-5.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 12 APPLICATION OF THERMOSTABLE CELLULASE IN BIOETHANOL PRODUCTION FROM LIGNOCELLULOSIC WASTE Neha Srivatsava, Rekha Rawat and Harinder Singh Oberoi

Abstract Lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis makes the overall process less economical than presently available fossil fuels. Cellulase is a group of enzymes, which can be classified into several types based on the reactions they catalyze, including endoglucanase (EG) or carboxymethyl cellulase (CMCase), exoglucanase or cellobiohydrolase (CBH),

cellobiase

or

β-glucosidase.

Auxilliary enzymes

like

β-xylosidase,

α-L-

arabinofuranosidase, and feruloyl esterase if present along with xylanases help in complete conversion of hemicellulose to sugars like xylose and arabinose. The synergistic action of these enzymes plays an important role in the hydrolysis of cellulosic and hemicellulosic fractions. It is therefore desirable to have a consortium of all the cellulase and xylanase components for effective hydrolysis of cellulosic biomass. The use of thermostable cellulases for hydrolysis of cellulosic biomass have significant advantages, such as (i) improved hydrolysis of cellulosic substrates because of the higher rate of reaction, (ii) higher masstransfer rates leading to improved substrate solubility, (iii) lowered risk of contamination, and (iv) increased flexibility with respect to process design, thereby improving the overall economics of the process.

Therefore, research on developing thermostable cellulase

consortium for hydrolysis of cellulosic biomass is gaining momentum. Thus, the current chapter covers sources and application of thermostable enzyme along with their characteristic features. Limitations and possible approaches are also discussed. Keywords:

Bioethanol, Lignocellulosic biomass, Cellulases, Thermostability, Protein

engineering, Hydrolysis

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Recent Advances in Bioenergy Research Vol. III 2014 12.1

Introduction The production and utilization of bio-ethanol is gaining attention worldwide because

of many advantages like reducing global warming and improving global energy crises (Chovau et al., 2013). Ethanol can be produced by fermentation of sugars from agro-industrial waste materials. Lignocellulosic biomass is known as the most abundant polymer and renewable source of energy which is finally converted into glucose and soluble sugars in ethanol production process (Reese and Mandels, 1984). Lignocellulosic agricultural biomass is used as substrate for the production of second generation biofuels. Lignocellulosic biomass is a complex which is composed of cellulose, hemicellulose and lignin and the conversion efficiency of lignocellulosic biomass into biofules depends upon the lignin content and degree of polymerization (DP) in cellulose and hemicellulose (Oberoi et al., 2012). Cellulose and hemicellulose are regarded as the potential sources of sugars for second generation biofuel production and cover around two-third of the lignocellulosic biomass (Hamelinck et al., 2005). Different pretreatment methods are applied to increase the accessibility of cellulosic substrate which helps to open the lignin sheath (Alvira et al., 2010). Lignocellulose-degrading enzymes, such as cellulases and hemicellulases are used to release fermentable sugars after pretreatment of biomass. Progressive research on cellulase enzymes started in the early 1950s due to their potential in conversion of lignocellulosic biomass, the most abundant polymer and renewable source of energy which is finally converted into glucose and soluble sugars in ethanol production process (Reese and Mandels, 1984). Continued research on cellulases and hemicellulases revealed their industrial potential in different sectors and industries, such as bioenergy, food, brewery and wine, animal feed, textile and laundry, pulp and paper, agriculture. Cellulases is a complex enzyme system which is divided into three major groups: endo-1,4-β-D-glucanases (EC 3.2.1.4) which cleaves β-linkages at random, commonly in the amorphous parts of cellulose; exo-1,4-β-D-glucanases (EC 3.2.1.91) (or cellobiohydrolase) which liberates cellobiose from the non-reducing or the reducing end, in general from the crystalline parts of cellulose; and β-glucosidases (EC3.2.1.21) which releases glucose from cellobiose and short-chain cello-oligosaccharides. Thermostable enzymes have number of commercial applications as the paper processing industries are always interested in such type of cellulases which can withstand higher temperatures. In addition, one of the most important applications of thermostable cellulase is in the bioconversion of cellulosic biomass into

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Recent Advances in Bioenergy Research Vol. III 2014 fermentable sugars for bioethanol production at elevated temperature. Generally, enzymatic hydrolysis reactions are carried out at 45oC-50oC which shows slow enzymatic hydrolysis rates, low yield of sugars, and incomplete hydrolysis, more amounts of enzyme requirement and is very sensitive to microbial contamination. These limitations could be resolved by using thermostable enzymes (Yeoman et al., 2010; Viikari et al., 2007). Figure.1 is showing possible advantages of thermostable cellulases in bioconversion of lignocellulosic waste. Therefore, the current chapter focuses on thermostable cellulase enzymes and their applications in biofuels production.

Complete hydrolysis

Fungi and bacteria are the best source of production of thermostable cellulases

High Sugars yield

High reaction rate

Pretreatment step can be avoided

Application of thermostable cellulases in biofuels production

Less risk contamination

of

Economically viable

Less time required for hydrolysis reaction

Figure-1 Application of thermostable cellulases in biofules production 12.2

Bioethanol production: Current production status and Challenges The production of biofules has reached 105 billion liters in 2010 and increased up to

17% from the year 2009. Biofuels contributed about 2.7% of world’s fuel road transportation 123

Recent Advances in Bioenergy Research Vol. III 2014 in which bioethanol and biodiesel are the most prominent. In 2010, global bioethanol production reached about 86 billion liters. United States and Brazil are the top producers of ethanol in the world accounting for 90% of the total global production (Kocar and Civas 2013). USA produces more fuel ethanol than any other country; Brazil is the second largest producer of ethanol in the world. The US and Brazil put together produced a little over 86% of the world’s fuel ethanol in 2010. Although production of bioethanol has improved using new technologies, there are still some challenges that need further investigations. For example, the cost of cellulase and ethanol distillation using lignocellulosic biomass as substrate, account for 30 to 50% and 20% of the total cost, respectively. To achieve the commercialization of cellulosic ethanol, a number of technological break throughs as well as cost reductions in all the process steps are required (Chen and Qiu, 2010). 12.3

Microorganism for thermostable cellulase production Thermophilic microorganisms are potential sources of highly active and thermostable

enzymes (Zambare et al., 2011; Liang et al., 2011; Yeoman et al., 2010). However, many mesophilic microorganisms are also known for significant production of thermostable cellulases (Gao et al., 2008; Lee et al., 2010). Numbers of bacteria and fungi have been reported to produce thermostable cellulases. Fungi are the most studied organisms with respect to degradation of cellulose and production of cellulolytic enzymes because bacteria degrade cellulosic biomass slowly due to lack of penetrating ability like fungi (Swaroopa et al., 2004). Thermophilic and mesophilic fungal genera belonging to Aspergillus, Rhizopus, Trichoderma, Sclerotium and Sporotrichum thermophile etc. (Barnard et al., 2010) are known for the production of cellulases. Synthesis of heat shock protein (HSPs) is a common phenomenon for production of thermostable enzymes. In presence of cycloheximide, the ability to produce thermostable enzyme and acquired thermo-tolerance is lost (Maheshwari et al., 2000). There are number of reports available showing HSPs synthesis in thermophillic microbes along with rapid breakdown of pulse label proteins (Trent et al., 1994, Maheshwari et al., 2000). Thermophilic microorganisms have specialised protein known as ‘chaperonins’ which help protein to refold and retain their native form and store their function even after denaturation. The cell membranes of thermophiles are made up of saturated fatty acids which create hydrophobic environment for cell and keeps it rigid so that it can survive at higher temperatures (Haki and Rakshit, 2000). The DNA of thermophiles has reverse gyrase which forms positive supercoils and hence enhances melting point. Besides this, thermophiles also

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Recent Advances in Bioenergy Research Vol. III 2014 have electrostatic, disulphide bridge and hydrophobic interactions in their cell membranes like thermotolerants which enhance the tolerant capacity of thermophiles at extremely higher temperatures (Kumar and Nussinov, 2001). Many thermophilic fungal species have been reported to produce cellobiose dehydrogenase and glycoside hydrolase 61 (GH61) family of proteins (Dimarogona et al., 2012), addition of which enhance cellulase performance in lignocellulose hydrolysis (Barakat et al., 2012; Harris et al., 2010). Thermophilic fungi produce multiple forms of the cellulase components like mesophilic fungi. However, two different strains of T. aurantiacus produced one form each of endoglucanase, exoglucanase, and β-glucosidase, but the forms from the two strains had somewhat different properties (Khandke et al., 1989). Several thermophilic bacteria, belonging to the genera Bacillus, Geobacillus,

Caldibacillus,

Acidothermus,

Caldocellum,

and

Clostridium

produce

thermostable cellulases. Hyperthermostable lignocellulolytic enzymes (optima above 80oC) have been isolated from Thermotoga anaerocellum (Evans et al., 2000). Most of the studies reported that hyperthermophilic microbes did not degrade crystalline cellulose, when temperatures increased beyond 75oC due to the lack of carbohydrate-binding modules (CBM) (Bhalla et al., 2013, Graham et al., 2011; Maki et al., 2009) while presence of a multi-domain hyperthermophilic cellulase in an archaeal enrichment, allowed maximum deconstruction of lignocellulosic biomass beyond 90oC (Bhalla et al., 2013, Graham et al., 2011). 12.4

Thermostable enzymes

12.4.1 Thermostable cellulases Cellulases are enzymes that catalyze the depolymerization of cellulose and work synergistically to efficiently hydrolyze substrate. Endoglucanases and exoglucanases are commonly referred to as cellulases (Blumer-Schuette et al., 2008). Endoglucanases having carbohydrate-binding modules (CBM) are considered as primary cellulases and are responsible for efficient utilization of crystalline cellulose. Trichoderma and Aspergillus sp. are referred to as model for significant cellulase production. Several fungi and bacteria have been reported earlier for the production of thermostable cellulases (Evans et al., 2000; Bok et al., 1998) (Table-1). Thermostable enzymes are stable and active at high temperatures which are higher than the optimum growth temperatures of the microorganisms. Endoglucanases (30 to 100 kDa) of thermophilic fungi are thermostable, with optimal activity between 55 and 80°C at pH 5.0 to 5.5 and with carbohydrate contents varying from 2 to 50%. Exoglucanases (40 to 70 kDa) are optimally active at 50 to 75°C and are thermostable (Maheshwari et al., 125

Recent Advances in Bioenergy Research Vol. III 2014 2000). Combination of thermostable enzyme with a wide pH range makes them suitable candidates for bioprocessing. Hydrolysis of substrate with thermostable enzyme increases the rate of reaction, decreases viscosity, increases diffusion coefficient, increases bioavailability of organic compounds and the solubility of the substrate, resulting in complete hydrolysis and less risk of contamination at elevated temperatures. Such types of enzymes can also be used for models studies for the understanding of temperature stability and activity, which is helpful for protein engineering (Haki and Rakhsit, 2003). Table- Thermostable cellulases from different microorganisms Organism Fusarium proliferatum Teheromyces lanuginosus (wild and mutant) Bacillus subtilis Thermotoga neapoltana (EndocellulaseA) Alicyclobacillus acidocaldarius ATCC27009 (Endoglucanases) E. coli expressing endoglucanase gene from Clostridium thermocellum E. coli expressing endoglucanase gene from Geobacillus sp. 70PC53 Geobacillus sp. WSUCF1 (Endoglucanases) E. coli expressing endoglucanase gene from Thermoanaerobacter tengcongensis MB4 Aspergillus fumigates SK1

Optimum temperature 55oC 70oC

Stability

References

50% 50%

(Badal, 2002) (Bakalova et al. 2002)

70oC 95oC

50% 50%

(Mawadza et al., 2000) (Bok et al., 1998)

80oC

60%

(Eckert and Schneider, 2003)

80oC

50%

(Zverlov et al., 2005)

65oC

80%

(Ng et al., 2009)

70oC

50%

(Rastogi et al., 2010)

80oC

50%

(Liang et al., 2011)

60oC

50%

(Ang et al., 2013)

12.4.2 Thermostable xylanases Xylan is the second most abundant polysaccharide in nature after cellulose. Complete degradation of xylan needs the combined action of a group of hydrolytic enzymes: the endoxylanases (EC 3.2.1.8), which cleaves β-1,4-linked xylose randomly (xylan backbone); the β-xylosidases (EC 3.2.1.37), which converts xylobiose to monomeric xylose units ; and the various side-branch splitting enzymes such as α-glucuronidase and α-arabinosidase, acetyl xylan esterase, and acetyl esterase, which liberate other sugars (glucuronic acid arabinose) that

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Recent Advances in Bioenergy Research Vol. III 2014 are attached as branches to the backbone (Bhalla et al., 2013, Biely., 1985). Thermostable endo-β-1,4 xylanases (referred to as endoxylanases) produced by thermophilic and hyperthermophilic bacteria such as Thermotaga, Acidothermus, Cellulomonas, Paenibacillus ,Thermoanaerobacterium, Actinomadura have been reported and are receiving considerable attention because of their application in bio- bleaching of pulp in the paper industry, wherein the enzymatic removal of xylan from lignin-carbohydrate complexes facilitates the leaching of lignin from the fibre cell wall, avoiding the need for chlorine for pulp bleaching in the brightening process. They also have applications in the pre-treatment of animal feed to improve its digestibility. Production of xylnase has also been reported from marine algae, protozoa, snails and insects but bacteria and fungi are the major producers of thermostable xylanases (Collins et al., 2005). According to Brienzo et al., (2009), xylanses obtained from bacteria are more diverse than fungi. Fungi including Laetiporus sulphureus (Lee et al., 2009), Talaromyces thermophiles (Maalej et al., 2009), Thermomyces lanuginosus (Singh et al., 2003), Rhizomucor miehei (Fawzi, 2011) produce thermostable xylanases. Thermostable and alkalistable xylanases have also been reported from many fungi and bacteria, such as thermoalkalophilic xylanase obtained from Enterobacter sp. MTCC 5112 retained its 90% of enzyme activity after 0.66 h at 80oC and pH 9, and 85% and 64% of its activity after 18 h at 60 and 70oC, respectively, showing its potential for industrial applications (Bhalla et al., 2013, Khandeparkar Bhosale, 2006). 12.4.3 Thermostable endoglucanses Endoglucanase is responsible for formation of cellobiose (oligosaccharides) from cellulose which is finally converted into glucose molecules by the action of β-glucosidase. Several studies have reported production of thermostable endoglucanase production from mesophillic and thermophillic microorganisms including fungi and bacteria both. Generally, endoglucanases show high thermal stability, for example endoglucanases from T. aurantiacus shows thermal stability at 70oC with half life of 98 h, (Gomes et al., 2000). In one of the study, Parry et al. (2002) discussed about an endoglucanase with a molecular weight of 34 kDa (based on SDS-PAGE) along with a pI of 3.7. Endoglucanases, broadly with molecular weight ranging from 30 to 100 kDa obtained from thermophilic fungi are thermostable, with optimal activity between 55 and 80°C at pH 5.0 to 5.5 and with carbohydrate contents varying from 2 to 50% (Maheshwari et al., 2000). Exoglucanases having molecular weight in the range of 40 to 70 kDa, are optimally active at 50 to 75°C and are thermostable; these enzymes fall into the category of glycoproteins (Brizeno et al., 2009). 127

Recent Advances in Bioenergy Research Vol. III 2014 12.4.4 Thermostable β-glucosidase Conversion of cellooligosacchrides into glucose is done by the help of enzyme βglucosidase (BGL). Several hyperthermophilic bacteria (Dion et al., 1999) such as archaeabacteria (Kim et al., 2009) as well as fungi (Parry et al., 2001) are known for the production of significant thermostable BGL which shows maximum optimal temperatures of 88, 90, and 80◦C, respectively (Handelsman J., 2005). In one of the study, Duex et al., (2012) reported thermostability of BGL at 66oC. Now days, with the help of metagenomics, complete microbial genomes may be screened and isolated directly from the natural environments (Feng et al., 2009; 2010, Jiang et al., 2011). Number of BGL genes have been isolated by employing metagenomic libraries of different samples, including rabbit cecum, marine samples (Jiang et al., 2009), soil (Jiang et al., 2009, 2011), and termite gut (Scharf et al., 2010, Matteotti et al., 2011). 12.5

Application of thermostable cellulases The significant industrial importance of cellulases lies mainly in the bioenergy

development, textile, and detergent and cellulosic pulp paper industries. In the present available industrial processes, cellulolytic enzymes are used in: (i) clarification of juices and wines; (ii) detergents causing colour brightening and softening; (iii) pretreatment of biomass to improve nutritional quality of forage; and (iv) pretreatment of industrial wastes (Bhalla et al., 2013, Brienzo et al., 2009, Haki and Rakshit, 2003, Bhat, 2000). Currently, lignocellulosic biomass conversion into fermentable sugar is one of the main thrust area for the production of biofuels. Some studies have shown that a long reaction time is required for complete hydrolysis when cellulases do not have this property (Sassner et al., 2008; Zhu et al., 2008; Borjesson et al., 2007). The use of thermostable enzymes like those produced from T. aurantiacus help in improving the hydrolysis process. In one of the study, Ang et al., (2013), reported optimum hydrolysis temperature of 60oC by using thermostable enzyme obtained from fungus Aspergillus fumigates SK1. It is therefore important to have a combination of all the forms of cellulases like exo-, endo-glucanases and β-glucosidases in a consortium along with the xylanases and accessory enzymes capable of hydrolysis of biomass at elevated temperatures, thereby giving a characteristic advantage to such a consortium. Development of this kind of consortium is the need of an hour in improving the sugar productivity and decreasing the enzyme cost in bioethanol production from lignocellulosic biomass. 128

Recent Advances in Bioenergy Research Vol. III 2014 12.6

Concluding remarks The remarkable progress has been done in the production and development of

thermostable cellulases and has attracted worldwide attention for further research. Although thermostable enzymes have many advantageous but for industrial scale processes, final selection depends on the many factors such as energy consumption for overall process, production cost, enzyme efficiency and environmental issues of the complete process of lignocelluloses biomass conversion. Cultivation of thermophiles on commercial-scale for the production of thermostable enzyme is still an economical challenge because of low cell yield and might even increase the overall production cost. Based on this, there is need of more research for development and optimization of thermophilic processes of lignocellulosic biomass conversion to gain an economical industrial biofuel production comparable to existing processes. Acknowledgements Authors thankfully acknowledge the financial assistance received from the NAIP sub-project (4183) funded by World Bank through Indian Council of Agricultural Research, Government of India for conducting a part of the study reported in this book chapter.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 13 ENDOGLUCANASES: CHARACTERIZATION AND ITS ROLE IN BIOCONVERSION OF CELLULOSIC BIOMASS Rekha Rawat, Neha Srivastava, Harinder Singh Oberoi

Abstract The growing global energy demand and negative environmental impacts created by growing greenhouse gas emissions from fossil fuels have forced the global scientific community to intensify research on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the production of bioethanol. Endoglucanases are the major component of cellulase enzyme involved in the initial stages of cellulose breakdown. These are classified into11 glycoside hydrolase (GH) families, including GH5, 6, 7, 8, 9, 12, 44, 45, 48, 51, and 74 on the basis of different sequences, specificity and tertiary structure. The application of this enzyme in biofuel industry requires identification of highly stable enzymes that are able to perform at extreme values of pH and temperature. Several bacteria and fungi are known to produce thermoacidophilic as well as thermoalkalophilic endoglucanases. The present chapter focuses on the importance of extremophilic endoglucanases in order to improve the existing biomass conversion processes. In addition to this, structural information, protein dynamics and models for thermostable endogucanases are also discussed. Key words: Endoglucanase, Structure, Classification, Catalytic Mechanism, Thermostability. 13.1

Introduction Biofuels are drawing increasing attention worldwide as substitutes to petroleum-

derived transportation fuels to help address energy cost, energy security and global warming concerns associated with liquid fossil fuels (Gray et al., 2006; Lee, 2011). It refers to energy obtained from biomass which is the biodegradable fraction of products, or waste and residues from agriculture, forestry and related industries, as well as the biodegradable fraction of industrial and municipal waste. Lignocellulosic biomass is the most abundant renewable organic material on earth which is composed of cellulose, hemicellulose and lignin. Cellulose, which is an unbranched linear homopolymer of glucose molecules with β (1-4) linkages,

Recent Advances in Bioenergy Research Vol. III 2014 generally accounts for 20- 45% of plant biomass. Cellulose is the main source of sugars for biofuel production (Hamelinck et al., 2005). Processing of lignocellulosic biomass into biofuel consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product recovery. A key step in the production of biofuel is the hydrolysis of biomass into fermentable sugars that is facilitated by cellulase enzyme. Cellulases comprise of three enzymes namely endo-1,4-β-glucanase (also referred to as carboxymethylcellulase or CMCase; EC 3.2.1.4), exo-1,4-β-glucanase or cellobiohydrolases (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) that synergistically convert cellulose into soluble sugars and glucose (Lynd et al., 2002). Among these, endoglucanases are cellulases that act synergistically in the initial attack on cellulose as they hydrolyze the β1,4 glycosidic bonds. The by-product is subsequently catalyzed by other enzymes, thereby making endoglucanases crucial for the bioprocessing of plant biomass (Bhat, 2000). Therefore, this chapter presents information about structure, mechanism of cellulose hydrolysis and applications of endoglucanases. In addition, importance of thermostable endoglucanases and factors affecting thermostability are also discussed. 13.2

Mechanism of cellulolysis The hydrolysis of insoluble cellulose requires the synergistic action of three types of

enzyme: endoglucanase (1,4-β-D-glucan-4-glucanohydrolase; EC 3.2.1.4), exoglucanase (1,4β-D-glucan cellobiohydrolase; cellobiohydrolase; EC 3.2.1.91) and β-glucosidase (βglucosideglucohydrolase; cellobiase; EC 3.2.1.21) as mentioned previously. Endoglucanases initiate cellulose hydrolysis by cleaving the cellulose polymer exposing both the reducing and non-reducing ends, while cellobiohydrolases acts upon these reducing and non-reducing ends to liberate cello-oligosaccharides and cellobiose units, and β-glucosidases cleave the cellobiose to liberate glucose, thereby completing the hydrolysis process (Bhat and Bhat 1997). Endoglucanases are classified as endo-acting cellulases because they are thought to cleave β-1,4-glycosidic bonds internally only and appear to have cleft-shaped open active sites. They are typically active on the more soluble amorphous region of the cellulose crystal, increase the concentration of chain ends and significantly decrease degree of polymerization by attacking interior portions of cellulose molecules (Tomas et al., 2009). On the other hand, cellobiohydrolases are classified as exo-acting cellulases as they cleave β-1,4-glycosidic bonds from chain ends and have a tunnel-shaped closed active site that retains a single glucan chain and prevents it from re-adhering to the cellulose crystal (Divne et al., 1998). They are usually 136

Recent Advances in Bioenergy Research Vol. III 2014 active on the crystalline regions of cellulose; shorten degree of polymerization incrementally by binding to the chain ends releasing mainly cellobiose units. Thus, endoglucanase activity is thought to be primarily responsible for chemical changes in solid-phase cellulose that occur over the course of hydrolysis, but plays a minor role in solubilization relative to exoglucanase, while exoglucanase activity is thought to be primarily responsible for solubilization but plays a minor role in changing the chemical properties of residual cellulose. 13.3

Classification of Endoglucanases Based on sequence and 3-dimensional structure, endoglucanases (EGs) are grouped

into 11 glycoside hydrolase (GH) families, including GH5, 6, 7, 8, 9, 12, 44, 45, 48, 51, and 74 (Cantarel et al., 2009). Glycoside hydrolases (EC 3.2.1.) are a widespread group of enzymes which hydrolyse the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. The International Union of Biochemistry and Molecular Biology (IUBMB) Enzyme nomenclature of glycoside hydrolases is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes. A classification of glycoside hydrolases in families based on amino acid sequence similarities has been proposed a few years ago. Because there is a direct relationship between sequence and folding similarities, such a classification: (i) Reflects the structural features of these enzymes better than their sole substrate specificity, (ii) Helps to reveal the evolutionary relationships between these enzymes, (iii) provides a convenient tool to derive mechanistic information (Henrissat, 1991; Henrissat and Bairoch, 1993). The details of each family are shown in the table 1. 13.4

Structure of endoglucanases Endoglucanase is made up of different domains. The main domain contains the large,

globular catalytic domain which expresses the active site. A loop of the protein chain forms a tunnel that encloses the active site. It is attached at the O-glycosylated B block hinge region of the catalytic domain to the smaller, globular cellulose binding domain (CBM) at its Cterminal A block by a linker peptide (Nimlos, et al., 2007).The overall shape of the complex looks like a tadpole, with the A and B blocks forming the extended tail and the catalytic domain forming the head (Pilz, et al., 1990). These structural domains contain three types of structure folds depending upon the endoglucanases (Yennamalli et al., 2011).

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Recent Advances in Bioenergy Research Vol. III 2014

Table 1: Classification of different endoglucanases and their properties Catalytic

Catalytic proton

Nucleophile/Base

donor

(α/β)8

Glu

Glu

Inverting

-

Asp

Asp

GH7

Retaining

β-jelly roll

Glu

Glu

GH8

Inverting

(α/α)6

Asp

Glu

GH9

Inverting

(α/α)6

Asp

Glu

GH12

Retaining

β-jelly roll

Glu

Glu

GH44

Retaining

(α/β)8

Glu

Glu

GH45

Inverting

-

Asp

Asp

GH48

Inverting

(α/α)6

-

Glu

GH51

Retaining

(α/β)8

Glu

Glu

GH74

Inverting

-

Asp

Asp

Family

Mechanism

Structure

GH5

Retaining

GH6

13.4.1 (α/β)8 fold This type of fold has an alternating pattern of eight α and β subunits in a single domain, such that the eight parallel β strands on the inside are protected by eight α helices on the outside. This very common fold has been reported to exhibit the highest diversity of enzymatic functions (Wierenga, 2001). 13.4.2 β-jelly roll fold This type of fold consists of 15 β-strands in two twisted antiparallel β-sheets, named A and B, that pack against each other. β-sheet A contains six antiparallel β-strands forming the back, convex surface while β-sheet B contains nine anti-parallel β-strands arranged to form the front, concave binding surface (Sandgren et al., 2005). Additionally two α-helices pack against the back side of β-sheet B. 13.4.3 (α/α)6 fold

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Recent Advances in Bioenergy Research Vol. III 2014 The substrate binding cleft in this type of fold has a tunnel shape that is formed at the N-termini of six central, parallel α-helices. These six helices are surrounded by six external αhelices (Alzari et al., 1996). 13.5

Mechanism of cellulose hydrolysis by endoglucanases Endoglucanases uses two types of catalytic mechanisms for hydrolysis of glycosidic

bonds of cellulose: 13.5.1 Retaining mechanism In this type of mechanism, the stereomeric configuration of the anomeric carbon is retained in the β configuration after hydrolysis. A pair of Glu amino acids, act as the catalytic residues: one as a nucleophile and the other as an acid-base donor. The first step in this double displacement mechanism is glycosylation, where one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme intermediate. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. The second step in this mechanism is deglycosylation step, where water molecule acts as a nucleophile and the first residue’s carboxylic group acts as a base. Once deprotonated, the water molecule is an activated nucleophile that then hydrolyzes the glycosyl-enzyme intermediate leading to a break in the polymer (Yennamalli et al., 2011). 13.5.2 Inverting mechanism In this type of mechanism, the configuration of the anomeric carbon is inverted; i.e., hydrolysis of β-glycosidic bond leads to α-configuration of carbon and vice versa. Contrary to retaining method, this method involves single displacement mechanism. The reaction typically occurs with general acid and general base assistance from two amino acid side chains, normally glutamic or aspartic acids (Guimaraes et al., 2002; Guerin et al., 2002) and a water molecule acts as a nucleophile. Utilizing the water molecule on the opposite side of the sugar ring to stabilize the transition, these residues catalyze the glycosylation or deglycosylation in one step. Unlike the retaining mechanism, this mechanism does not involve the glycosylenzyme intermediate. 13.6 Microbial sources of endoglucanase enzyme

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Recent Advances in Bioenergy Research Vol. III 2014 A variety of microorganisms including fungi, bacteria and actinomycetes produce endoglucanases for utilizing cellulose as a source of carbon and energy. Among them, most emphasis has been placed on fungi and bacteria because of their ability to produce plentiful amounts of endoglucanses. The list of microbes having potential for endoglucanase production is given in Table 2. 13.7 Application of endoglucanases Microbial endoglucanases can be used for the production of bioenergy and other value added products and chemicals with great potential. Table 2: Most extensively studied microbes employed for endoglucanase production Group

Organisms

Fungi

Agaricus bisporus, Aspergillus niger, A. fumigatus, A. oryzae, A. terreus, A. wentii,

A.aculeatus,

A.

awamori,

Chaetomium

cellulyticum;

C.

thermophilum, Daldinia eschscholzii, Fomitopsis sp., Fusarium solani, F. oxysporum, Humicola insolens, H. grisea, Macrophomina phaseolina, Melanocarpus albomyces, Mucor circinelloides, Paecilomyces inflatus, Penicillium pinophilum, P. chrysogenum, P. occitanis, P. purpurogenum, Phanerochaete chrysosporium, Phlebia gigantean, Pleurotus ostreatus, Pyrenochaeta lycopersci, Rhizopus stolonifer, R. oryzae, Schizophyllum commune, Trametes versicolor, Trichoderma reesei, T. atroviride, T.viridae, T. harzianum, Thermoascus aurantiacus Bacteria

Acetivibrio cellulolyticus, Acinetobacter junii, A. amitratus, Anoxybacillus sp., Bacillus subtilis, B. pumilus, B.amyloliquefaciens, B. licheniformis, B. circulan, B. flexus, Bacillus agaradhaerens, Bacteriodes cellulosolvens, Butyrivibrio fibrisolvens, Cellvibrio gilvus, Clostridium thermocellum; C. cellulolyticum; C. acetobutylium; C. cellulofermentans, C. cellulovorans, Eubacterium cellulolyticum, Geobacillus sp., Fibrobacter succinogenes, Microbispora

bispora,

Paenibacillus

campinasensis,

P.

polymyxa,

Pectobacterium chrysanthemi, Pseudomonas fluorescens, Rhodothermus marinus, Ruminococcus albus, R. succinogenes, Thermotoga maritime Actinomycetes

Cellulomonas fimi, C. flavigena, C. cellulans, C. uda, Streptomyces cellulyticus,

S.

aureofaciens,

140

Thermomonospora

fusca,

T.

curvata,

Recent Advances in Bioenergy Research Vol. III 2014 Thermobifida fusca, T. cellulolytica Kuhad et al., 2011 13.7.1 Biofuel industry Bioconversion of lignocellulosic biomass into the fermentable sugars is the most important application of endoglucanases. Bioconversion is carried out either enzymatically or chemically using sulfuric or other acids (Wyman, 1999). However, when sulfuric acid is used, it is necessary to remove the residual sulfuric acid from the hydrolyzing solution prior to yeast fermentation. Furthermore, it produces toxic compounds that inhibit fermentation. The advantages of enzymatic hydrolysis are the lower requirements for cooling water, gas, electricity and disposal costs and no corrosion issues for equipment together with lower environmental pollution (Sun and Cheng, 2002). 13.7.2 Textile and laundry Endocellulases play a key role in textile and laundry because of their ability to modify cellulosic fibres in order to improve the quality of fabrics. They especially help in biostoning and biopolishing of cotton and fabrics. In bio-stoning of denim fabrics, endoglucanases remove excess dye from denim fabrics and restore the softness of cotton fabrics without damaging the fibre. Similarly, in biopolishing, they facilitate the removal of excess microfibrils from the surface of cotton and non-denim fabrics. In addition to this, they improve the detergent performance and allow the removal of small, fuzzy fibrils from fabric surfaces and restore the appearance and color brightness (Galante et al., 1998a; Godfrey, 1996; Kumar et al., 1996). 13.7.3 Paper and Pulp industry Paper and pulp industry also require endoglucanases for substantial energy savings during mechanical pulping, improvements in hand-sheet strength properties and deinking of fibre surface for improving brightness and freeness of the pulp by partial hydrolysis of carbohydrate molecules (Akhtar, 1994; Leatham et al., 1990; Prasad et al., 1993). 13.7.4 Wine and Brewery Industry Endoglucanases are also vital for fermentation processes to produce alcoholic beverages including beers and wines. They improve color extraction, skin maceration, must clarification, filtration, and finally the wine quality and stability. These enzymes can improve both quality and yields of the fermented products (Singh et al., 2007; Galante et al., 1998b).

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Recent Advances in Bioenergy Research Vol. III 2014 13.7.5 Other applications Endoglucanases also find applications in clarification of fruit and vegetable juices, oil extraction, and in improving the nutritive quality of bakery products and animal feed (Bhat, 2000). The enzymes are used to improve cloud stability and texture and decrease viscosity of the nectars and purees from tropical fruits, thus clarify the juices (Singh et al., 2007). The enzyme causes degradation of β-glucan in feed which lowers the viscosity of the intestinal contents and thus, improves the quality of the feed (Bedford, 1995). 13.8. Significance of thermostable endoglucanases Currently, the bioconversion of lignocellulosic biomass into fermentable sugar is the major concern for the production of biofuel. Thermostable endoglucanases are economically important because they are able to contribute to the hydrolysis of cellulose at higher temperatures compared to their mesostable homologs, reducing the number of processing steps during biofuel conversion. Thus, lignocellulosic conversion using thermostable endoglucanses have attracted much attention because of their several potential advantages such as (i) higher reaction rates due to the increased solubility of substrates; (ii) higher productivity as hydrolysis time is shortened; (iii) lessen the amount of enzyme needed; (iv) decreased risk of contamination; (v) facilitated recovery of volatile products; (vi) decreased cost of energy for cooling as the thermostable enzymes can be used directly after the heating step without a pre-cooling step and (vii) loss of enzyme activity is low during processing, at higher temperature used during pre-treatments (Zhang et al., 2011; Viikari et al., 2007; Bhalla et al., 2013). 13.9 Factors responsible for thermal stability Thermostability is ability of an enzyme to maintain its active structural conformation at higher temperature for a prolonged incubation period (Bhalla et al., 2013).There are multiple factors that are responsible for increased thermostability of enzyme. 13.9.1 Amino acid composition Positively charged residues (Lys, Arg and Glu) on the solvent accessible surface are more significant in thermophiles than in mesophiles (Glyakina et al., 2007). Kumar et al., (2000) reported that Arg and Tyr are significantly higher whereas Cys and Ser are significantly lower in thermophilic proteins. It was reported that a decrease in the number of Gly residues in thermophilic proteins leads to greater stability at higher temperatures (Panasik

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Recent Advances in Bioenergy Research Vol. III 2014 et al., 2000). Yennamalli et al., (2011) observed that amino acids Arg and Met are statistically significant among thermophilic proteins, whereas Gln and Ser are statistically significant among mesophilic proteins. 13.9.2 Intramolecular interactions For the thermophiles, only ionic interactions were significant, whereas for mesophiles, no intramolecular interactions were significantly different from thermophiles (Yennamalli et al., 2011). Comparison of intramolecular interactions showed that cation-π interactions are highly significant in imparting thermophilicity (Chakravarty and Varadarajan, 2002). 13.9.3 Fold specificity: Recently, Yennamalli et al., (2011) conducted the study on thermostable endoglucanases and demonstrated fold specificity as a key factor for controlling thermostability in endoglucanases. For the (α/β)8 fold, Arg and Pro (significant in thermophiles) are replaced by polar amino acids whereas Leu is primarily replaced with aromatic amino acids in the mesophilic counterpart. The absence of arginine amino acids leads to a loss of ionic interactions in mesophiles, rendering them enzymatically inactive at higher temperatures. In the β-jelly roll fold, the amino acids Glu, Arg, and His are substituted with polar, hydrophobic amino acids. Substitution to Pro is higher for Arg indicating the potential for fewer salt-bridges in mesophiles whereas number of salt bridges is high in thermophiles. For the Ser and Thr positions (significant among mesophiles) the thermophilic protein has hydrophobic, acidic, and basic amino acids substituted. In the (α/α)6 fold Glu and Val are replaced with polar amino acids and to a lesser extent with other amino acid groups. Gln (significant in mesophiles) is substituted to a large extent by hydrophobic, acidic and to a lesser extent with basic amino acid groups in thermophiles indicating that in the thermophilic protein these substitutions contribute towards more intramolecular interactions and extend stability to proteins at higher temperatures. 13.9.4 Other factors These include a decrease in loop length and a concomitant increase in secondary structure, an increase in aromatic stacking, increased hydrophobic interactions, increased metal-binding capacity, and increased oligomerization (Yano and Poulas, 2003). Gromiha et al., (1999) found Gibbs free energy change of hydration, long-range non-bonded energy, βstrand tendency and average long-range contacts as factors responsible for imparting

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Recent Advances in Bioenergy Research Vol. III 2014 thermostability. Several studies implicated partial, reversible folding of the protein as a factor responsible for thermostability at high temperatures (Uversky, 2009). 13.10 Conclusion Endoglucanases, also called primary cellulases, are synergistically involved in the first stage of cellulose breakdown-a vital step in the bioprocessing of lignocellulosic biomass into biofuel and other industrial bioprocesses. Despite possessing several advantages, lignocellulosic biofuel has not yet been well implemented on a commercial scale because of high costs of cellulolytic enzymes and lack of robust cellulases that can function efficiently at high temperature. Thus, understanding the structure of enzyme, mechanism of reaction and basis for thermostability helps in engineering the protein for enhanced activity. It can lead to more cost effective processes for biofuels and other industrial applications by designing a more efficient endoglucanase enzyme. Acknowledgements Authors thankfully acknowledge the financial assistance received from the NAIP subproject funded by the World Bank through the Indian Council of Agricultural Research (ICAR) Government of India (PR/8488/PBD/26/68/2006) for conducting a part of the study reported in this book chapter.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 14 COMPARATIVE STUDY OF FERMENTATION EFFICIENCY FOR BIO-ETHANOL PRODUCTION BY ISOLATES Richa Arora, Shuvashish Behera, Sachin Kumar

Abstract Recent production of bio-ethanol through microbial fermentation as an alternative source has renewed its research interest because of the increase in the fuel price and environmental concern. Yeast strains are commonly associated with the ethanol production potential in sugar rich environments. In the present study, isolation of various yeast strains were carried out from different soil samples collected from dumping sites of sugar-mills. A total of four yeast strains were isolated with the ethanol producing ability, which were used for the further study. An attempt has been made to evaluate the pattern of sugar utilization and ethanol yield by the yeast strains using the salt medium. The results obtained in this study showed a range of ethanol production between 5.0± 0.2 and 22.0± 0.4 in all the strains. Two isolates NIRE K1 and NIRE K3 showed the highest ethanol yield of 0.49 and 0.41, respectively after 40 h of incubation at 45oC. This study revealed the characteristics of the isolate NIRE K1 allow it to ferment glucose efficiently to ethanol and have the potential to develop a bioprocess for bioethanol production. Key words: Bio-ethanol; Fermentation; Ethanol Yield; Thermotolerant. 14.1

Introduction The global rise in energy consumption, predicted increase in energy demands in the

near future, the depletion of low extraction cost fossil fuel reserves, and climate change are forcing the search for new and alternative energy sources (Agbor et al., 2011). A concern about energy security, the environmental impact of energy production has led to the implementation of policies designed to encourage the production and use of renewable bioenergy (Glithero et al., 2013).

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Recent Advances in Bioenergy Research Vol. III 2014 An alternative fuel must be technically feasible, economically competitive, environmentally acceptable, and readily available. Numerous potential alternative fuels have been proposed, including bioethanol, biodiesel, methanol, hydrogen, natural gas, liquefied petroleum gas (LPG), Fischer–Tropsch fuel, electricity, and solar fuels (Limayem and Ricke, 2012). Bioethanol has been considered in all over the world as an alternative renewable fuel with the largest potential to replace fossil derived fuels, responsible for a significant fraction of greenhouse gas emissions (Dias et al., 2013). Current bioethanol research focus on lignocellulosic feedstocks due to its abundance and renewability; especially in relation to reduce the cost and increase the efficiency of the key steps in the lignocelluloses-to-bioethanol process (e.g. lignocellulosic pre-treatment, enzymatic hydrolysis and fermentation) (Saratale and Oh, 2012; Mathew et al., 2013; Matsushita et al., 2013). The main advantage of the production of second-generation biofuels from lignocellulosic biomass is to reduce the limitation between food versus fuel competition associated with first generation biofuels (Nigam and Singh, 2011; Singh et al., 2010). Most of the potential ethanologens that are in industrial use belong to mesophillic group (28-37oC). However, the bioethanol production from lignocellulosic biomass by thermophillic/ thermotolerant species have some process advantages over mesophiles due to high growth temperatures, require less energy for mixing and product recovery, higher saccharification and fermentation rates, minimized contamination risk, lower costs of pumping and stirring and no aeration and cooling problems (Georgieva&Ahring, 2007; Oberoi et al., 2010; Frock & Kelly, 2012). Considering the above, this study was carried out to compare the performance of the thermotolerant yeast isolates for ethanol production. Further, the growth and fermentation parameters of the isolates during fermentation were compared. 14.2

Materials and methods

14.2.1 Microorganisms and culture conditions Microorganisms were isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mills at 45oC. Four yeast isolates were compared for high ethanol

production

rate

and

higher

sugar

consumption

rate.

For growing the isolated strains, salt medium (SM) was used in g l-1, di-sodium hydrogen ortho phosphate, 0.15; potassium di-hydrogen ortho phosphate, 0.15; ammonium sulphate,

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Recent Advances in Bioenergy Research Vol. III 2014 2.0; yeast extract, 1.0; glucose, 10.0 at pH 5.0. The cells were grown in 250-ml flasks in a shaker at 45oC and 150 rpm for 24 h. 14.2.2 Fermentation conditions The medium for fermentation was the same as that for the growth medium, except for glucose 50 g l-1. Fermentation was carried out in 250-ml capped flasks at 100 rpm at 45oC. 14.2.3Analytical methods At 4h intervals, fermented broths (in triplicate) were removed and the contents were analyzed for biomass, sugar and ethanol. Glucose was analyzed by high-performance liquid chromatography (HPLC) using Hi-Plex H column at 57oC with 1mM H2SO4 as the mobile carrier at a flow rate 0.7 ml min-1 and detected by refractive index detector. Ethanol was analyzed by gas chromatography using Nucon 5765 with a 2-m-long and 1/8-in. diameter Porapak-Q column with mesh range 80/100. The sample was injected at an inlet temperature 150oC, and flame ionization detector 250oC using nitrogen gas as a carrier. Dry cell weight (DCW) was determined in the broth by centrifuging 1 ml of broth in pre-dry weighted Eppendorf tube using Eppendorf centrifuge 5430 R at 10,000 rpm for 5 min, followed by washing twice with distilled water and drying in a vacuum oven at 80oC to a constant weight. 14.3

Result and Discussion The cultural characteristics of the isolates are shown in Table 1. In case of isolates

NIRE K1 and NIRE K3, the concentration of sugar dropped down in 24 h, with concomitant production of ethanol; thereafter, the decline was gradual. At the end of 40 h of incubation, the residual sugar concentration reached close to 5 g l-1with the ethanol concentration was 22 g l-1. In contrast to NIRE K1 and NIRE K3, there was a marginal difference in sugar consumption albeit, there was no ethanol produced in 20 h NIRE K4 and NIRE K5. The marginal decrease in sugar concentration might be due to their utilization for growth and metabolism by NIRE K4 and NIRE K5. After 24 h, there was a gradual increase in ethanol concentration over the incubation period with simultaneous decrease in sugar concentration. At the end of 40 h of incubation, the ethanol production from NIRE K4 and NIRE K5 reached

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Recent Advances in Bioenergy Research Vol. III 2014 8 and 5 g l-1, respectively. The pattern of sugar utilization and ethanol formation among the strains is shown in Fig. 1. The initial DCW of NIRE K1 and NIRE K3 was kept at 2.7 g l-1 and 2.8 g l-1, which was 2.6 g l-1and 2.8 g l-1 after fermentation, and shows almost constant DCW throughout the process, respectively. No significant change was observed during fermentation in the cellmass concentration, which means the ethanol formation is non-growth associated when using NIRE K1 and NIRE K3. Table 1 Cultural characteristic of four yeast isolates Isolate

Colour

Margin

Shape

Opacity

Elevation

NIRE-K1

White

Entire

Circular

Transparent

Flat

NIRE-K3

White

Crenate

Circular

Opaque

Raised

NIRE-K4

White

Crenate

Circular

Opaque

Flat

NIRE-K5

Creamish yellow

Undulate

Irregular

Opaque

Umbonate

Fig. 1Comparison of ethanol production between free cells of four yeast isolates at 45oC

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Recent Advances in Bioenergy Research Vol. III 2014 However, in case of isolates NIRE K4 and NIRE K5, the initial DCW was kept at 2.5 g l-1, which was 1.7 and 1.3 g l-1 after fermentation, respectively. The DCW declined slightly when ethanol concentration increased in broth. Table 2 shows the fermentation parameters evaluated among the isolates. The maximum ethanol concentration was 22± 0.4 g l-1 on initial glucose concentration of 50 g l-1 with 89% of sugar conversion and ethanol yield of 96% of theoretical yield in 40 h. Banat et al., 1992 reported the maximum ethanol concentration of 7.2% (w/v) with ethanol yield of 98% of theoretical yield and ethanol productivity 1.71 g l-1 h-1 on 140 g l-1 glucose by K. marxianusIMB2 at 45oC. Cazetta et al., 2007 achieved an ethanol concentration of 55.57 g l-1 with an ethanol yield of 63.03% of theoretical yield and productivity of 1.16 g l-1 h-1 on molasses containing 200g l-1 reducing sugar in 48 h at 30oC by using Zymomonasmobilis. Table 2 Growth and Fermentation kinetics of free cells of four yeast isolates at 45oC in Salt Medium Parameters evaluated Final ethanol (P, g l-1) Ethanol Yield (Yp/s, g g-1) Sugar utilisation (g l-1) Conversion rate (%) into ethanol Maximum specific growth rate, h-1

NIRE K1

NIRE K3

NIRE K4

NIRE K5

22.0 ± 0.4*

12.0 ± 0.4

8.0 ± 0.1

5.0 ± 0.2

0.49

0.41

0.21

0.17

44.90 ± 0.04

29.27 ± 0.05

38.10 ± 0.07

29.41 ± 0.03

89.80

58.54

76.19

58.82

0.51

0.43

0.16

0.14

*mean ± standard deviation The ethanol yield (Yp/s = 0.49) obtained with the free cells of NIRE K1 was more than that of free cells of NIRE K3 (Yp/s = 0.41) followed by NIRE K4 (Yp/s = 0.21) and NIRE K5 (0.17). Kumar et al., 2009 reported ethanol yield of 90% of theoretical yield on glucose by Kluyveromyces sp. IIPE453 at 50oC and concluded thatthe strain has the ability to convert hexose sugars to cell mass as well as ethanol during the growth phase. Similar effect was found in our studies where NIRE K1 and NIRE K4 follow the Crabtree rather than the Pasteur Effect. In this study, isolate NIRE K1 was found to be more efficient than the other 3 isolates in ethanol production.

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Recent Advances in Bioenergy Research Vol. III 2014 14.4

Conclusion The new isolated thermotolerant yeast strain NIRE K1 has shown the good

consumption of sugar for ethanol fermentation. The results showed that ethanol production by isolate NIRE K1 was the highest in comparison to other isolates. For all the isolates, the peak ethanol concentration was obtained after 40 h of fermentation. This study revealed the characteristics of the isolate NIRE K1 allow it to ferment glucose efficiently to ethanol. The yield could be further increased after optimization of the fermentation parameters andhave the potential to develop a bioprocess for bioethanol production. Acknowledgments The authors are thankful to Dr. Y. K. Yadav, Director, SSS-NIRE for all the possible support and encouragement. The authors also acknowledge MNRE, Govt. of India for providing financial assistanceship.

References 1. Amerine M.A. and Ough C.S. (1984) Wine and Must Analysis., Wiley, New York, USA. 2. Carvalho W., Silva S.S., Converti A and Vitolo M. (2002) Metabollic behaviour of immobilized Candida guillirmondii cells during batch xylitol production from sugarcane bagasse acid hydrolysate. Biotechnol. Bioeng., 79:165-169. 3. Hartmeier, W. (1988) Immobilized Biocatalysts Springer, Berlin. 4. Kierstan M. and Bucke C. (1977) The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Biotechnol. Bioeng. 19 :387–397. 5. Krajewska, B. (2004) Application of chitin- and chitosan-based materials for enzyme immobilizations. Enzyme Microb. Technol., 35: 126–139. 6. Lin Y. and Tanaka S. (2006) Ethanol fermentation from biomass resources: current state and prospects. Appl. Microbiol. Biotechnol., 69: 627–642. 7. Mohany S.K., Behera S., Swain M.R. and Ray, R.C. (2008) Bioethanol production from mahula (Madhuca latifolia L.) flowers by solid-state fermentation. Appli. Ener., 86:640644 .

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Recent Advances in Bioenergy Research Vol. III 2014 8. Narendranath N.V. and Power, R. (2005) Relationship between pH and medium dissolved solids in terms of growth and metabolism of lactobacilli and Saccharomyces cerevisiae during ethanol production. Appl. Environ. Microbiol., 71:2239-2243. 9. Sakaguchi K., Matsui M. and Mizukami F. (2005) Applications of zeolite inorganic composites in biotechnology: current state and perspectives. Appl. Microb. Biotechnol., 67 : 306–311. 10. Sakai Y., Tamiya Y. and Takahashi F. (1994) Enhancement of ethanol formation by immobilized yeast containing iron powder or Ba-ferrite due to eddy current or hysteresis. J. Ferment. Bioeng., 77: 169–172. 11. Swain M.R., Kar S., Sahoo A.K. and Ray, R.C.( 2007) Ethanol fermentation of mahula (Madhuca latifolia L.) flowers using free and immobilized yeast Saccharomyces cerevisiae. Microbiol. Res.,162:16293-16298 . 12. Tanaka T. and Kawamoto L. (1999) Manual of Industrial Microbiology and Biotechnology (2nd ed., Washington, D.C., American Society forMicrobiology Press, pp. 94-102. 13. Yadav P., Garg N. and Diwedi D.H. (2009) Effect of loca tion of cultivar, Fermentation temperature and additives in the physico-chemical and sensory qualities on mahua (Madhuca latifolia J.F. Gmel) wine Preparation. Natural Product radiance 8:406-408. 14. Yamashita Y., Kurosumi A., Sasaki C. and Nakamura Y.( 2008) Ethanol production from paper sludge by immobilized Zymomonas mobilis. Biochem. Eng., 42:314-319. 15. Yu, B., Zhang, F., Zheng, Y., Wang, P.U.( 1996) Alcohol fermentation from the mash of dried sweet potato with its drags using immobilized yeast. Process. Biochem., 31:1-6.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 15 SWEET SORGHUM - AN IDEAL FEEDSTOCK FOR BIOETHANOL PRODUCTION Reetika Sharma1, Gurvinder Singh Kocher1 and Harinder Singh Oberoi

Abstract Sweet sorghum can serve as a potential feedstock for ethanol production, largely because of its ability to grow under hot and dry conditions; short duration, so that two crops could be taken in a year in arid regions; low water requirement and high biomass yield. Since sweet sorghum juice cannot be crystallized as cane sugar, it could be converted to ethanol through fermentation. The sugar content in different varieties of sweet sorghum varies from 14-22 %. After juice extraction, the bagasse could also be used for ethanol production. Although, the ethanol production process from lignocellulosic biomass is cost and energy intensive, sweet sorghum bagasse (SSB) contains relatively higher cellulose content (40 % or higher) as compared to rice straw, sugarcane bagasse or wheat straw. This means that there is an opportunity to obtain higher glucose concentration, which is essentially required for ethanol production. In addition, relatively lower concentrations of lignin and ash in SSB in comparison to other agricultural residues makes it easily amenable to pretreatment and hydrolysis processes. Therefore, higher ethanol concentration obtained using both sweet sorghum juice and bagasse could reduce the cost and energy required during downstream processing, thereby making sweet sorghum as an ideal and cost-effective feedstock for bioethanol production for its use as biofuel. Key

words:

Lignocellulosic

biomass,

Sweet

sorghum,

Pretreatment,

Hydrolysis,

Fermentation, Ethanol. 15.1

Introduction Fossil fuel limitations and constraints on carbon dioxide emissions have a high impact

in the market of bioethanol, which is the most commonly used biofuel for petrol substitution in the world (Taherzadeh and Karimi, 2008). Ethanol can be produced from a variety of feed stocks such as saccharine materials, starchy materials and many types of lignocellulosic 156

Recent Advances in Bioenergy Research Vol. III 2014 wastes (Sanchez and Cardona, 2008). Lignocellulosic biomass is considered a future alternative as raw material for bioethanol production, because it is more abundantly available and is much less expensive than food crops, especially when waste streams are used (Hamelink et al., 2005; Prasad et al., 2007b). Furthermore, the use of lignocellulosic biomass is more attractive in terms of energy balances and green house gas emissions (Taherzadeh and Karimi, 2007). Sweet sorghum (Sorghum bicolor (L.) Moench) represents an analogous crop to sugarcane with similar accumulation of sucrose but with a higher agronomic stability to temperature fluctuations, less water requirement and better tolerance to salinity, alkalinity and drought (Prasad et al., 2007a; Almodares and Hadi, 2009; Goshadrou et al., 2011). It contains 43.6 - 58.2 % soluble sucrose, glucose and fructose in the stalk (Billa et al., 1997; Dolciotti et al., 1998; Amaducci et al., 2004; Antonopoulou et al., 2008) and 22.6 - 47.8 % insoluble cellulose and hemicellulose (Dolciotti et al., 1998; Rattunde et al., 2001; Antonopoulos et al., 2008). In addition, it is an annual crop with a typical growing season of 3-5 months instead of 9-12 months required by sugarcane. Additionally, the sweet sorghum bagasse has a comparatively higher nutritional value for ruminants because of its more favorable fiber composition and is a better alternative for further hydrolysis and fermentation (Almodares and Hadi, 2009). Because of its agronomic flexibility and productivity, sweet sorghum is viewed as a viable feedstock option for ethanol production in some regions of the world. Sweet sorghums have potential for specific tropical, subtropical and arid regions of the US, Mexico, China, India, Southern Africa and other developing countries where the use of maize and other crops for ethanol production is not feasible due to either economic, agronomic or social considerations (Reddy et al., 2005; Chuck-Hernandez et al., 2009; Wu et al., 2010; Zhang et al., 2010). Sweet sorghum has a high a ratio of energy output to fossil energy input in comparison to sugarcane, sugar beet, maize and wheat and its fermentation efficiency has been reported higher than 90 % (Almodares and Hadi, 2009; Wu et al., 2010). Lastly, sorghum is known as one of the most variable crops in terms of genetic resources and germplasm that allows the breeding and development of new cultivars adapted to different regions around the globe (Zhang et al., 2010). For all these reasons, bioethanol produced from sweet sorghum presents a high environmental, economic and energetically sustainable biofuel which ascribes GHGs saving upto 70-71 % (Liu et al., 2008).

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Recent Advances in Bioenergy Research Vol. III 2014 Despite these advantages, utilization of sweet sorghum as a potential energy crop presents some major technical challenges which must be resolved before sweet sorghum is widely planted to provide feedstock for ethanol biorefineries. These include, its high concentration of soluble sugars, due to which it can be easily contaminated, limiting its storage stability. Secondly, the lignocellulosic stalk cannot be easily hydrolyzed enzymatically to fermentable sugars due to the presence of free sugars inhibiting the hydrolytic enzymes action; thus, the sugars in the stalk should be removed before the enzymatic hydrolysis of the cellulosic part of the plant (Taherzadeh and Karimi, 2007; Molaverdi et al., 2013). Short harvest periods also increase the capital cost involved in building a central processing plant that may be operated only seasonally. Finally, the common practice of utilizing sweet sorghum either involves a stage of sugars extraction and separate utilization of soluble sugars and fiber fraction, which exhibits some technical difficulties, or involves a solid state fermentation, which makes fermentation process and ethanol extraction more difficult, leading to increased cost (Mei et al., 2009; Kundiyana et al., 2010, Wu et al., 2010, Matsakas and Christakopoulos, 2013). Moreover, fermentation performance of sweet sorghum can be affected by the microorganism used, bioreactor configuration, free amino nitrogen, sugar content and composition of juices (Lui and Shen, 2008; Laopaiboon et al., 2009; Chohnan et al., 2011). For this reason, evaluation of different sweet sorghum cultivars for their bioethanol production potential is critically important (Zhao et al., 2009; Davilla-Gomez et al., 2011). 15.2

Origin and biology of sweet sorghum Cultivated sorghum (Sorghum bicolor spp. bicolor L. Moench) is in the sub-genus

Sorghum and originates from semi-arid regions of Africa. However, due to its adaptive capacity, now- a -days it is cultivated on a wide spectrum of climates in every continent (Saballos 2008). Sweet sorghum varieties belong to the grain, forage and broomcorn sorghums. These names describe well the diversity of phenotypes, as well as the aim and direction of selection. Sweet sorghum has been selected for accumulation of high amount of sucrose in the stem (Murray et al., 2009). Sweet sorghum belongs to C4 crop with high photosynthetic efficiency and high productivity. The plant grows to a height of about 14 feet. Seeds are produced by self-pollination from the panicle at the top of the plant and contains the male and female inflorescences. The plant lodging is more likely to occur in high population fields because stalks become smaller in diameter due to competition. The plant can also be blown down in strong winds due to its height (Nahar, 2011).

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Recent Advances in Bioenergy Research Vol. III 2014 15.3 Cultivation and harvesting of sweet sorghum Cultivation of sweet sorghum possesses the following advantages arising from the physiology and biochemistry of C4 plants that usually generate high biomass yield with minimal inputs (Rooney et al., 2007; Saballos, 2008; Byrt et al., 2011): 1. High conversion efficiency of light into biomass (biomass yields competing with switchgrass and miscanthus) resulting in high sugar, and thus ethanol yields; 2. High water use efficiency and thus low water requirement that is 25% of that needed for sugar cane and 50-66% needed for maize production (no irrigation); 3. Drought tolerance - even though drought leads to reduction in plant growth, enhanced accumulation of sucrose and starch was observed in drought stressed stems and thus resulting in equal sugar yields (Massacci et al., 1996); 4. Reduced demand for fertilizer due to the high leaf nitrogen use efficiency and large fibrous root system; 5. Modest demand for soil quality (that are not appropriate for corn or wheat) 6. High tolerance towards salinity and water-logging; 7. Pest and disease management is less complex, 8. Greater tolerance towards climate changes (e.g. temperature extremities, droughts). 9. Its cultivation is also possible on marginal lands and therefore, it can contribute to sustainable ways to produce bioethanol, for instance, avoiding the food versus fuel debate often related to bioethanol production (Prasad et al., 2009; Allen et al., 2011); 10. Its untapped genetic diversity as presented by its viability in almost every climate condition, carries enormous potential for further breeding (Rooney et al., 2007; Saballos, 2008). Cultivation Technology: The plant can be grown on soils ranging from heavy clay to light sand. Loam and sandy loam soils generally allow the best syrup production. Good surface drainage is preferred although sweet along sorghum can withstand long waterlogged condition; clay loam is preferred with soil acidity not lower than pH 6. Sowing: Sowing can be done on ridges or in furrows at a spacing of 60 cm between rows and 15 cm between plants. Three to four seeds are dibbled in each planting hole and the seedlings are to be eventually thinned to one per hole. Sweet sorghum is not suitable for high density; recommended density is about 7000 plants/ha. The plant is ideally shown during June to

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Recent Advances in Bioenergy Research Vol. III 2014 September, when soil can hold much water (deep).The crop does not prefer high rainfall as high soils moisture or continuous heavy rain after flowering may decrease sugar content in plants. Setting of Furrow: Two planting seasons are possible for sweet sorghum. During the wet season, furrows are set 100 cm apart while in the dry season planting are set about 75 cm apart. Fertilizer Application: The plant needs adequate nutrients to produce good yields. Quality of syrup is also affected by the fertilizer applications. The recommended dose of fertilizer for sweet sorghum is 80 kg of nitrogen, 60 kg of phosphorous and 40 kg of potassium per hectare. Half of N and whole of P and K are applied as basal dose. Remaining N is top-dressed during 25-35 days after germination, following weeding and inter-cultivation. Nitrogen fertilizer should not be applied in the field when sweet sorghum is grown immediately after a legume crop, as the soil contains nitrogen. Intercropping: Sweet sorghum is suitable to intercropping with early maturing crops for its characteristics in growth and development. Sweet sorghum seedlings develop slowly at their early stage. It can be intercropped with potato, maize, wheat etc. Adaptation and Yield: It is relatively inexpensive to grow high yield sweet sorghum plants and can be used to produce a range of high value added products like ethanol, energy and dried grains. It can produce approximately 30 tons/ ha per year of biomass on low quality soils with low inputs of fertilizer, half of that required by sugar beet and a third of the requirement for sugarcane or corn. Harvesting: The plant varieties mature between 115-125 days after plantation. To obtain highquality syrup and high yields, the crop should be harvested when the seed is in the soft dough stage. Stalks can be harvested either along with the grain, or 4-5 weeks after the grain harvest. The ear-head and peduncle (between the base of the seed head and the top node) should be removed before processing the stalks. Ear heads may be dried and threshed so the seeds can be used for the next year's crop. 15.4

Inherent advantages of sweet sorghum Sorghum is an annual crop with a lot of varieties (at present estimated at about 4,600

approximately), and some of these have a very high sugar content in the stems. Its bioenergy applications are numerous: sweet sorghum can be used to produce ethanol, but alternatively also biogas through anaerobic digestion, fiber sorghum (pelletized or not) can supply combined heat and power (CHP) plants, grain sorghum can be employed for food, feed and energy needs of small isolated communities. Grain sorghum is not so suitable for the ethanol

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Recent Advances in Bioenergy Research Vol. III 2014 production, because in these varieties, sugars are prevalently polymerized to starch, which then require hydrolysis before the alcoholic fermentation. Sweet sorghum is a sugar cane-like C4 plant, containing juice in the stem with large amount of sucrose (11-23 % brix, depending on variety and conditions) that can be effectively extracted by squeezing and thereafter, readily fermented to ethanol by yeast (Almodares and Hadi, 2009, Byrt et al., 2011). While it is not well suited to the production of refined sugar, sweet sorghum has multiple inherent advantages which have been represented in Table 1. Most important, sweet sorghum is a seedpropagated annual crop, i.e., a crop established through sowing seed and which matures and is harvested in a single season. These key characteristic impacts both its fit within current production cycles as well as the pace of scale-up and on-going improvements to the varieties themselves. In addition, sweet sorghum can be utilized in rotation with other annual crops, and potentially, with sugarcane itself, where sweet sorghum could be sown on fallow sugarcane land, hectares destined for rotation and land where sugarcane yields are limited due to marginal soils. This flexibility is due to the sorghum plant’s natural hardiness and rapid growth. Table 1: Inherent advantages of sweet sorghum over sugarcane Sugarcane

Sweet sorghum

Sugar quality

Sucrose

Mixed sugars

Establishment cost

Vegetative propagation

Seed propagation

Sugar yield (% fw)

13-15

8-13

Input requirements

Limited by water, nitrogen

50% water, 60% nitrogen

Scale-up time

Vegetative propagation

Seed propagation

Biomass yield (tons/ha)

70-90 tons/ha

60-100 tons/ha

Marginal land

Limited yield

Significant yield

Season extension

12-18 months

70-120 days

Product development

Perennial, 10-16 years

Annual, 3-5 years Source: Wu et al., 2008

Traditional uses of the juice include production of syrup, alcoholic beverages, crystalline sugar and in some regions stalks are also consumed fresh (Saballos, 2008). The

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Recent Advances in Bioenergy Research Vol. III 2014 leftover, built up of lignocellulose, is called bagasse. The main advantage of sweet sorghum bagasse over other cellulosic biomass is relatively higher cellulose and low ash content (Table 2). Low ash content facilitates the use of milder and low energy intensive pretreatment, while higher cellulose content creates a possibility of obtaining higher quantity of glucose (Gao et al., 2010). Table 2: Comparative compositional analysis of sweet sorghum bagasse with other agro residues Agro Residue

Kinnow pulp

Cellulose

Hemicellulose

Lignin

(%)

(%)

(%)

8.82±0.70

4.44±0.55

3.71±0.40

Ash (%)

5.93±0.34

Reference

Oberoi et al., 2010

Sunflower

32.56±1.65

20.73±0.66

14.36±0.56

6.03±2.46

Díaz et al., 2011

33.49±3.18

17.15±0.04

9.88±0.01

4.71±0.07

Brijwani et al.,

Stalks Soybean hulls

2011 Wheat bran

7.57±0.17

31.19±0.30

4.06±0.09

6.53±0.01

Brijwani et al., 2010

Sugarcane

35.22±0.91

24.52±0.63

22.28±0.14

3.71±0.31

bagasse Sweet

Rezende et al., 2011

44.6± 0.13

27.1±0.23

20.7±0.21

0.4±0.11

sorghum

Kim and Day, 2011

bagasse Sorghum

40.4±1.01

35.5±0.91

22.3±0.43

bagasse

0.2±0.01

Dogaris et al., 2009

Besides these uses, another possibility is gaining growing attention i.e. to cultivate it as energy crop for bioethanol production (Almodares and Hadi 2009; Byrt et al., 2011; Ratnavathi et al., 2011). Ethanol yields of 2100-8000 L/ha have been reported with one harvest annually that significantly exceed the ethanol yields from starchy materials, such as

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Recent Advances in Bioenergy Research Vol. III 2014 corn and wheat grains (Byrt et al., 2011; Balat and Balat, 2009). Furthermore, when comparing the energy performance of wheat and sweet sorghum monocultures, a significantly higher net energy gain for sweet sorghum was demonstrated in Northern Italy (Monti and Venturi, 2003). In Asia (China, India and the Philippines) and South America fermentation of sweet sorghum juice is carried out on a small to medium scale (Saballos, 2008). Contrarily, the conversion of sweet sorghum bagasse to ethanol is still in an experimental phase (Ballesteros et al., 2004; Sipos et al., 2009; Li et al., 2010; Yu et al., 2010; Shen et al., 2011; Ratnavathi et al., 2011). Economic evaluation of a sweet sorghum biorefinery for ethanol production from bagasse has been studied under North Chinese circumstances (Gnansounou et al., 2005), while the economy of juice processing has been investigated more deeply, for example, in Indian context (Prasad et al., 2007), upper Midwest of the USA (Bennett and Anex, 2009), Zimbabwe (Woods, 2001) and Inner Mongolia (Wang and Liu, 2009). 15.5

Technical hurdles Under moderate climate, the technological difficulty of sweet sorghum processing is

the short harvest period making the juice available only for 1-2 months annually. Due to this reason, the juice cannot be stored because the microbes including its natural microbial flora degrade the easily fermentable sugar content. Without any preservation, up to 12-30 % of the fermentable sugar content can be lost in 3 days and 40-50 % in a week at room temperature (Daeschel et al., 1981; Wu et al., 2010). Many methods have been proposed to elongate the availability of the juice, for instance: refrigerating (Wu et al., 2010), evaporation (Hodúr et al., 2008), ensiling the whole stalks (Bennett and Anex, 2009), proper harvest and processing method (Lingle et al., 2012) and lowering the pH with the addition of different acids (Hodúr et al., 2008). But these alternatives lead to elevated energy demands and/or chemicals needs, thereby, influencing the overall process economy. The reports of Bennet and Anex (2009) and Gnansounou et al. (2005) suggested that fermentation of sweet sorghum juice under moderate climate could only be a complementary process, for example, in a biorefinery concept. 15.6

Bioethanol production from sweet sorghum Sweet sorghum is being considered as a SMART crop as it offers triple benefits of

‘F’, i.e., food, fodder and fuel, without significant tradeoffs in any of these uses in the

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Recent Advances in Bioenergy Research Vol. III 2014 production cycle. It’s relatively short vegetation cycle allows sweet sorghum to be grown in double cropping systems based on water availability, which in turn can lead to greater agrobiodiversity and a reduced demand for fertilizers and pesticides (Nahar, 2011). Sorghum has the potential to be an excellent diversified biofuel crop able to fill the needs of multiple bioenergy conversion process across many environments with reduced energy requirements. Besides environmental advantages, sorghum is one of the more acquiescent plant to genetic modifications because it is highly variable in terms of genetic resources and germplasms. This facilitates plant breeding and development of new cultivars adapted to different regions around the globe (Ratnavathi et al., 2011). Overall scheme for production of bioethanol from sweet sorghum involving various steps which include pretreatment, enzymatic hydrolysis and various fermentation methods is presented in Figure 1. For the effective conversion of lignocellulosic material into ethanol, the following three major steps are involved: 1. A thermo-chemical pretreatment is a pre-processing step that improves the access of enzymes to cellulose and hemicellulose. 2. Enzymatic saccharification that breaks down cellulose and hemicellulose into simple sugars. 3. Fermentation of released sugars by specialized organisms. It is clear from the figure that sweet sorghum bagasse (SSB) can be used as an ideal substrate for production of cellulases which can further be used for saccharification of pretreated biomass. Pretreatment is an important step for cellulose conversion processes, and is one of the important factors affecting ethanol production from lignocellulosics. It is essential for causing alterations in the structure of cellulosic biomass in order to facilitate the enzymatic saccharification process. The goal here is to break the lignin seal and disrupt the crystalline structure of cellulose (Agbor et al., 2011; Zhang and Shahbazi, 2011). Once the cellulose and hemicellulose fractions are exposed after pretreatment and are accessible to the action of cellulases and xylanases, a mixture of hexose as well as pentose sugars are produced (Sun and Cheng, 2002). The sugars thus produced are fermented by the fermenting micro-organisms, such as Saccharomyces cerevisiae, Pichia kudriavzevii or a combination of hexose and pentose fermenting microbial strains to exploit the full potential of the sugars, thus produced. However, in absence of robust pentose fermenting micro-

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Recent Advances in Bioenergy Research Vol. III 2014 organisms, generally the alcohol yields from the mixed sugar streams are low. Table 3 represents the different microorganisms used in the fermentation for ethanol production from sweet sorghum along with their ethanol yields. However, the biological/biochemical route for ethanol production from lignocellulosic biomass is a preferred method, because it is less energy intensive as compared to the thermo-chemical conversion method and the infrastructural and capital requirements are relatively lower.

Figure 1: Flow chart for the bioethanol production from sweet sorghum (Ratnavathi et al., 2011) In one of our studies on production of ethanol from sweet sorghum bagasse, we found the ethanol concentration in the range of 35-40 g/l using crude cellulases and thermotolerant Pichia kudriavzevii strain (unpublished data). 15.7

Energy ratio and environmental sustainability The available data about the European Union (EU) model for the ethanol produced

from all components of sweet sorghum crop suggest an output/input ratio of 1.7 -7.3 (depending on the strategy chosen for the by-products exploitation), a high greenhouse gases (GHGs) saving in accordance with the RES Directive and a low water footprint. The main

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Recent Advances in Bioenergy Research Vol. III 2014 environmental advantage relative to the use of bioethanol in substitution of gasoline and/or the use as bio-ETBE (ethyl tert-butyl ether) instead of fossil antiknocks is the abatement of the transport sector contribution to the GHG emissions. For bioethanol produced using sweet sorghum, this assertion assumes an absolute validity because in this case, the saving calculated with the methodology indicated by EU is 71% which is one of the most virtuous values among the attainable ones. The use of sweet sorghum for bioethanol production conciliates the production of sustainable bioethanol applying already mature technologies with an involvement of the agricultural sector in the pathway. Table 3: Micro-organisms used in the fermentation for ethanol production from sweet sorghum Sr. No.

Micro-organisms used in the fermentation of:

Ethanol yield

Reference

Juice 1

Saccharomyces cerevisiae CFTR 01 and SG

0.39-0.48 (g/g)

Ratnavathi et al., 2010

2

Fermax yeast (Saccharomyces cerevisiae)

77.07-79.58(g/l)

Kundiyana et al., 2010

3

Super start yeast (Saccharomyces cerevisiae)

73.18-76.95(g/l)

Kundiyana et al., 2010

4

Saccharomyces cerevisiae TISTR 5048

0.42-0.48 (g/g)

Laopaiboon al.,2007

5

Saccharomyces Strains

29-87% (sugar Bulawayo et al., 1996 conversion effienciency)

6

Saccharomyces cerevisiae (Nanayang)

91.61 %

Liu et al., 2008

7

Saccharomyces cerevisiae

0.42-0.45 (g/g)

Balint-Sipos 2009

8

Saccharomyces cerevisiae

Ethanol conc. - Li et al., 2010 42.3 (g/l)

9

Saccharomyces cerevisiae

0.147 (g/g)

et

et

al.,

Bagasse

Ban et al., 2008

Source: Ratnavathi et al., 2011

15.8

Small-medium scale bioethanol production plant from sweet sorghum Sweet sorghum could favour the diffusion of the bioethanol pathway in the local

agricultural sector, because the creation of small-medium plants involves the plant building

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Recent Advances in Bioenergy Research Vol. III 2014 near the fields where the biomass is harvested (max 50 km approximately). This choice allows an increase of sustainability of the bioethanol production, because the impacts of the long distance transportation is reduced. The size of small-medium plants does not exceed 15,000 t/y of ethanol capacity. Table 4 represents the comparative theoretical ethanol production from sweet sorghum with other feedstocks. There are many criteria to select a suitable bioethanol production technology, such as plant scale, investment and operation cost, management operations, and conversion yield, which have a reflecting effect on the production cost. Another important criterion is the energy balance that shows the efficiency of processing energy used. Pilot studies in India have indicated that ethanol production from sweet sorghum is cost-effective (Dayakar et al., 2004). The International Crops Research Institute for the SemiArid Tropics (ICRISAT) located at Patancheru in Andhra Pradesh, India, has developed several improved sweet sorghum lines with high stalk sugar content and a few of these lines are being tested in pilot studies for sweet sorghum-based ethanol production in India, of which, SSV 84, SSV 74 and NSSH 104 have been released (Reddy et al., 2008). Further, sweet sorghum bagasse (SSB) has a higher biological value than the bagasse from sugarcane when used as feed for cattle, as it is rich in micronutrients and minerals and is as good as stover in terms of digestibility (Panwar et al., 2000). In addition to the above characteristic features, short duration of this crop and a yield in the range of 54-69 tons/ha renders SSB an important and readily available substrate for ethanol production (Almodares and Hadi, 2009). Thus, sweet sorghum represents a potential opportunity to improve the economic and environmental sustainability of the bioethanol pathway. Table 4: Theoretical ethanol production from multiple feedstocks (kg/ha, dry basis) (Adapted from Kim and Day, 2011) Component

Sugarcane

Energy cane

Sweet sorghum

Feed stock

70,000

100,000

60,000

Juicea

0

53,600

43,140

Fiber

9,450

26,700

7,800

0

5,253

5,091

4,368

12,846

3,865

2,695

7,221

2,402

Sugar Monomeric sugar from juice Glucose from celluloseb Xylose from hemicellulosec

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Recent Advances in Bioenergy Research Vol. III 2014 Ethanol Ethanol from juiced

0

2,684

2,601

Ethanol from cellulosed

2,232

6,564

1,975

Ethanol from hemicellulosed

1,377

3,690

1,227

3,609

12,938

5,804

Total ethanol a

Juice is wet kilograms

b

Glucose (kg)= Glucan (kg) × 1.11; 1.11 is a conversion factor considering water addition during hydrolysis

c

Xylose (kg) = Xylan (kg) × 1.14; 1.14 is a conversion factor considering water addition during hydrolysis

d

Ethanol (kg) = Glucose, fructose, sucrose or xylose (kg) × 0.511; 0.511 is a conversion factor for sugar to ethanol based on stoichiometric biochemistry of yeast.

15.9

Conclusions Sweet sorghum has distinct advantages over the other lignocellulosic biomass for

bioethanol production because of some of the inherent characteristics of this crop. Higher biomass yield, ability of the crop to grow under drought and stress conditions, ability to get two crops in a year makes sweet sorghum as an attractive contestant for ethanol production. The juice extracted from sweet sorghum could be used directly for bioethanol production through fermentation and the sweet sorghum bagasse could be converted to ethanol through a series of steps, like, size reduction, pretreatment, hydrolysis and fermentation. Therefore, combining the ethanol yields from both the juice as well as bagasse confers a distinct advantage to sweet sorghum over other lignocellulosic biomass, such as rice straw, cotton stalks, bagasse, etc. Acknowledgements Authors thankfully acknowledge the financial assistance received from the project funded

by

the

Department

of

Biotechnology,

Government

of

India

((PR/8488/PBD/26/68/2006) for conducting a part of the study reported in this book chapter.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 16 FERMENTATION OF GLUCOSE AND XYLOSE SUGAR FOR THE PRODUCTION OF ETHANOL AND XYLITOL BY THE NEWLY ISOLATED NIRE-GX1 YEAST Shuvashish Behera , Richa Arora , Nilesh Kumar Sharma and Sachin Kumar

Abstract Hemicellulose is the second most abundant polysaccharide after the cellulose available on the earth in the form of lignocelluloses. The hemicellulose must utilized for the cost-efficient production of ethanol. Xylan, the major component in the hemicellulose in plant biomass, yields mainly xylose as pentose sugars on hydrolysis. The progress in fermentation of pentose sugars has gone on slow pace as there are few microorganisms known, which are capable of pentose metabolism. Therefore, this study was carried out to isolate and screen the yeasts from soil samples collected from different dumping sites for the production of ethanol using glucose and xylose sugar. About 16 yeast strains showed positive results in ethanol production from glucose sugar. Four isolates designated NIRE-GX1, NIRE-GX2, NIRE-GX3, NIREGX4 showed positive result in ethanol production from both glucose and xylose sugars. Further study was carried out using the isolate NIRE-GX1 yeast, which showed more growth and fermentation efficiency at a temperature of 40oC on both the sugars. Anaerobic batch fermentations were carried out using the yeast strain from the individual glucose and xylose sugar separately and further mixture of both at 40oC temperature. The strain also showed xylitol production from xylose. The strain showed maximum ethanol concentration of 7.1 ± 0.6 g l-1 with complete utilization of glucose (20 g l-1) in 24 h. However, in case of xylose fermentation, the strain showed maximum ethanol concentration of 0.8 ± 0.08 g l-1 as well as xylitol concentration of 0.64 ± 0.3 g l-1 in 72 h on initial xylose concentration of 20 g l-1. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 14 g l-1 and xylose concentration of 6 g l-1, achieving maximum ethanol and xylitol concentration of 5.3 ± 0.5 g l-1 and 0.95 ± 0.32 g l-1, respectively in 72 h. Key Words: Bio-ethanol; Fermentation; Glucose; Xylose.

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Recent Advances in Bioenergy Research Vol. III 2014 16.1

Introduction The largest potential feedstock for ethanol is lignocellulosic biomass, which includes

materials such as agricultural residues (corn stover, crop straws, sugarcane bagasse), herbaceous crops, short rotation woody crops, forestry residues, waste paper and other plant wastes (Kim and Dale, 2005). It varies among plant species but generally consists of cellulose (40–50%), hemicelluloses (25–30%), and lignin (10–20%) (Wiselogel et al., 1996). Complete hydrolysis of cellulose and hemicellulose results in glucose and xylose respectively, which can be ferment to ethanol by several microorganisms (Alvira et al., 2010; Sims et al., 2010). Cellulose contains glucose and hemicellulose contains mainly pentoses, such as a xylose, which are not fermentable by the natural industrial strains and can add up to 25% of the constitution of lignocellulosic materials (Schell et al., 2004; Olofsson et al., 2008). Yeasts that produce ethanol from D-xylose have been isolated from various locations, including tree exudates (Ipsit et al., 2013), wood-boring insects (Suh et al., 2003), decaying wood (Cadete et al., 2009), rotten fruit and tree bark (Rao et al., 2008). There are also several yeast species such as Candida shehatae, Pachysolen tannophilus, Brettanomyces naardenensis, C. tenuis, Pichia segobiensis, C. lyxosophila, C. intermedia, C. jeffriesii, Spathaspora passalidarum, Spathaspora arborariae, C. prachuapensis, and Scheffersomyces stipitis which has been reported as xylose fermenting yeasts (Barnett et al., 2000; Nguyen et al., 2006; Cadete et al., 2009; Nitiyon et al., 2011). Candida and Pichia are the two naturally occurring best ethanol producing organism from pentose. However, those yeast strains are neither well adapted to ethanol production nor can tolerate to by-products of lignocellulosic hydrolysates (Jeffries, 2006; Hahn-Hägerdal et al., 2007). Although Saccharomyces cerevisiae combines several desired attributes such as high ethanol tolerance, general robustness and operation experience which favored for industrial scale of ethanol production; the success is restrained by the organism’s inability to naturally ferment pentose sugars (Helle et al., 2004; Hahn-Hägerdal et al., 2007). In a study, cocultivation of S. cerevisiae and S. stipitis strains to co-ferment glucose and xylose was unsatisfactory, due to their difference in fermenting condition and ethanol tolerance (Agbogbo et al., 2006). However, S. stipitis strain prefers to ferment glucose rather than xylose having lower ethanol tolerance than S. cerevisiae strain (Watanabe et al., 2007). Further, in order to assimilate xylose, some strains produce xylitol as the intermediate product with the production of ethanol. Despite the existence of these microorganisms, it is still

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Recent Advances in Bioenergy Research Vol. III 2014 challenging to reach high yields of ethanol from pentose sugars on a large scale (HahnHagerdal and Pamment, 2004) because no microorganisms that robustly convert pentose sugars into ethanol at high yields while withstanding fermentation inhibitors have been identified (Chandel et al., 2011). Therefore, this study was carried out for a new search of yeasts from soil samples collected from different dumping sites for the production of ethanol using glucose and xylose sugar. Finally fermentation of both glucose and xylose sugars were carried out using NIRE-GX1 yeast for the production of ethanol. 16.2

Materials and methods

16.2.1 Sample collection Samples for isolation of ethanol producing yeasts were obtained from the dumpyard at Jalandhar, Punjab and from Wahid Sandhar Sugars Ltd., Punjab. Exploration for collecting potential samples was carried out by walking-through within 60-90 min for each site. Soil samples were put into plastic or polythene bags and finally sample details were recorded. 16.2.2 Isolation of yeast To isolate yeast, 1g of each soil samples was suspended in 10 ml of sterile water by vortexing for 2 minute on maximum speed, followed by a 10x serial dilution. About 0.1 ml of each dilution in the series was spread onto the surface of yeast extract-peptone-dextrose (YPD) phytagel (yeast extract, 1%; peptone, 2%; dextrose, 2%; phytagel, 1.5%; ampicillin, 50 mg/ml; water, 1000ml; pH, 5.5) plates and incubated at 40o C for 24 hour. For isolation of xylose utilizing yeasts, xylose sugar (2%) was added in replacement of dextrose to the above medium. Various colonies were selected based on their morphology, size and color appear on the phytagel plates. The selected colonies were then subcultured on to separate phytagel plates to ensure their purity. 16.2.3 Microorganism and culture condition The yeast culture was maintained on yeast extract-peptone (YP) medium (g/l): yeast extract, 1%; peptone, 2%; phytagel, 1.5%; ampicillin, 50 mg/ml; water, 1000ml; pH, 5.5 and sugar (glucose and xylose) was added according to the use by the isolates. The culture was stored at 4±0.5oC for further use. 16.2.4 Preparation of inoculum

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Recent Advances in Bioenergy Research Vol. III 2014 The inoculum was prepared in 100 ml YP growth medium (as mentioned above but without phytagel) containing glucose and xylose as the sugar source, taken in sterilized (at 121oC for 20 min) 500 mL erlenmeyer flask and cotton plugged. The flask was inoculated with a loopful of the yeast culture and incubated for 24 h at 40o C at 120 rpm in an orbital shaker incubator (Remi Pvt, Ltd, Bombay, India). The cells grown after 24 h were acts as the inoculum for the fermentation medium. 16.2.5 Fermentation medium Fermentation medium was prepared using salt medium (SM) in gl-1: di-sodium hydrogen ortho phosphate, 0.15; potassium di-hydrogen ortho phosphate, 0.15; ammonium sulphate, 2.0; yeast extract, 1.0; glucose, 10.0 at pH 5.5. The cells from the YP medium was centrifuged at 7500 rpm for 10 min and added in the fermentation medium. The cells were grown in 250-ml capped flasks in a shaker at 40oC and 120 rpm for 72 h. 16.2.6 Analytical methods At 24 h interval, fermented broths (in triplicate flasks) were removed and the contents were analyzed for total sugar, ethanol and xylitol production. Glucose was analyzed by highperformance liquid chromatography (HPLC) using Hi-Plex H column at 57oC with 1mM H2SO4 as the mobile carrier at a flow rate 0.7 ml min-1 and detected by refractive index detector. The pH was measured by a pH meter (Systronics, Ahmadabad, India) using glass electrode. The fermentation kinetics was studied as per the formulae described below (Bailey and Ollis, 1986) 16.3

Results and discussion About 16 yeast strains showed positive results in ethanol production from glucose

sugar. Four isolates designated NIRE-GX1, NIRE-GX2, NIRE-GX3, NIRE-GX4 showed positive result in ethanol production from both glucose and xylose sugars. The isolated strains were carefully identified by morphological characteristics including color of the colony and growth pattern studies with the help of microscope. Strain selection was based on anaerobic growth on xylose as well as on high xylose uptake rate. In fermentation studies on defined media the resulting NIRE-GX1 yeast was shown to possess the ability to grow on xylose under anaerobic conditions and to ferment both xylose and glucose to ethanol in good yield and productivity.

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Recent Advances in Bioenergy Research Vol. III 2014 Therefore further study was carried out using the isolate NIRE-GX1 yeast, which showed more growth and fermentation efficiency at a temperature of 40oC on both the sugars. Anaerobic batch fermentations were carried out using the yeast strain from the individual glucose and xylose sugar separately and further mixture of both at 40oC temperature. The strain also showed xylitol production from xylose. The strain showed maximum ethanol concentration of 7.1 ± 0.6 g l-1 with complete utilization of glucose (20 g l-1) in 24 h. However, in case of xylose fermentation, the strain showed maximum ethanol concentration of 0.8 ± 0.08 g l-1 as well as xylitol concentration of 0.64 ± 0.3 g l-1 in 72 h on initial xylose concentration of 20 g l-1. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 14 g l-1 and xylose concentration of 6 g l-1, achieving maximum ethanol and xylitol concentration of 5.3 ± 0.5 g l-1 and 0.95 ± 0.32 g l-1, respectively in 72 h (Table 1). Nigam et. al. (1985) found maximum ethanol concentration of 8.8, 10.9 and 9.8 g l-1 with initial D-xylose sugar concentration of 40, 60 and 80 g l-1. Table 1. Ethanol production from glucose and xylose sugar using yeast isolate NIRE-GX1 Glucose Fermentation Time (h) pH Sugar Consumed Ethanol (g -1

(g l )

Xylose Fermentation pH

-1

l )

Sugar Consumed

Ethanol -1

(g l )

Fermentation of mixture of sugar Xylitol

pH Sugar Consumed Ethanol (g

-1

(g l )

-1

(g l )

-1

l )

Xylitol (g l-1)

-1

(g l ) 24

2.93

19.98±0.04

7.3±0.06

3.48

7.26±0.2 0.12±0.03 0.47±0.1 3.08

16.75±1.4

4.43±0.1 0.79±0.05

48

-

-

-

3.39

8.38±1.2

0.2±0.04 0.6±0.05 3.06

17.07±0.2

4.97±0.3 0.85±0.2

72

-

-

-

3.19

9.3±0.1

0.8±0.06 0.64±0.3 3.03

17.12±0.8

5.3±0.5 0.95±0.32

The growth and fermentation kinetics of free cells in presence of individual and mixed of sugars were also studied (Table 2). The ethanol concentration (P) obtained with glucose fermentation with NIRE-GX1 yeast (7.3±0.06 g l-1) was 89.04 % more than that of xylose fermentation (0.8±0.06 l-1), where as the volumetric substrate uptake (Qs) was found to be 84.34 % more in case of glucose fermentation (0.83 g l-1 h-1) over xylose fermentation (0.13 g l-1 h-1). The ethanol yield (Yp/s= 0.37 g g-1) and volumetric product productivity (Qp=0.3 g l-1 h-1) obtained with glucose fermentation was found to be 75.68 and 96.7 %, respectively higher than that of Yp/s (0.09 g g-1) and Qp (0.01 g l-1 h-1) of xylose fermentation. However, the final biomass (X) concentration in case of xylose fermentation (1.5 ± 0.2 g l-1) was considerably lower than glucose fermentation (1.2 ± 0.2 g l-1), which is useful during product separation and purification process (Diderich et al. 1999). Cadete1 et al. (2012) reported that the newly isolated S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and

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Recent Advances in Bioenergy Research Vol. III 2014 productivities (0.62 g l-1 h-1 to 0.75 g l-1 h-1). Also another strain Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g g-1 to 0.59 g g-1), with concentrations up to 25.2 g l-1. Table 2. Growth & Fermentation Kinetics of Ethanol Production Glucose fermentation

Xylose fermentation

Fermentation of mixed sugar

Final ethanol (P, g l-1)

7.3±0.06

0.8±0.06

5.3±0.5

Final biomass concentration (X, g

1.9±0.2

1.5±1.2

1.8±0.7

0.37

0.09

0.3

Volumetric substrate uptake (Qs, g 0.83

0.13

0.24

0.01

0.07

l-1) Ethanol yield (Yp/s, g g-1) l-1 h-1) Volumetric product productivity

0.3

(Qp, g l-1 h-1)

16.4

Conclusion Efficient ethanol production from xylose is crucial for Bioethanol production from

lignocellulosic biomass. We attempted to enhance the ethanol productivity of the xylosefermenting yeast NIRE-GX1. The strain showed utilization both hexose and pentose sugar for the production of bioethanol with a low amount of xylitol. Further studies like pentose sugar uptake, presence of sugar transporter and inhibitor tolerance are required to increase the uptake rate of pentose sugar. Acknowledgment We greatly acknowledge the Ministry of New and Renewable Energy, New Delhi, Govt. of India for providing funds to carry out research work.

References 1. Agbogbo F.K., Coward-Kelly G., Torry-Smith M. and Wenger K.S. (2006) Fermentation of glucose/xylose mixture using Pichia stipitis. Process Biochem., 41:2333–2336.

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Recent Advances in Bioenergy Research Vol. III 2014 2. Alvira P., Tomas-Pejo E., Ballesteros M. and Negro M.J. (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol., 101:4851–4861. 3. Bailey J.E. and Ollis D.F. (1986) Biochemical engineering fundamentals. McGraw-Hill., New York. 4. Barnett J.A., Payne R.W. and Yarrow D. (2000) Yeast Characteristics and Identification, 3rd edn, Cambridge Univ press, UK. 5. Cadete R.M., Santos R.O., Melo M.A., Mouro A. and Gonc¸alves D.L. (2009) Spathaspora arborariae sp. nov., a D-xylose-fermenting yeast species isolated from rotting wood in Brazil. FEMS Yeast Res., 9:1338–1342. 6. Cadete1 R.M., Melo1 M.A., Dussan K.J., Rodrigues R.C.L.B., Silva S.S., Zilli J.E., Vital M.J.S., Gomes F.C.O., Lachance M.A. and Rosa1 C.A. (2012) Diversity and physiological characterization of D-xylose-fermenting yeasts isolated from the Brazilian Amazonian forest. Plos One, 7:1-11. 7. Diderich J.A., Schepper M., Van H.P., Luttick M.A., Van D.J.P. and Pronk J.T. (1999) Glucose uptake kinetics and transcription of HXT genes in chemostat cultures of Saccharomyces cerevisiae. J. Biol. Chem., 274:15350-15359. 8. Hahn-Hägerdal B., Karhumaa K., Fonseca C., Spencer-Martins I. and GorwaGrauslund M.F. (2007) Towards industrial pentose-fermenting yeast strains. Appl. Microbiol. Biotechnol., 74:937–953. 9. Helle S.S., Murray A., Lam J., Cameron D.R. and Duff S.J.B. (2004) Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor. Bioresour. Technol., 92:163–171. 10. Ipsit H., Bidisha S., Anindita R. and Subhra K.M. (2012) Ethanol production from xylose and enzymatic hydrolysate of hemicelluloses by a newly isolated yeast strain. J. Microbiol. Biotechnol. Res., 08:54-58. 11. Jeffries T.W. (2006) Engineering yeast for xylose metabolism. Curr. Opin. Biotechnol., 17: 320–326. 12. Kim S. and Dale B.E. (2005) Life cycle assessment of various cropping systems utilized for producing biofuels: bioethanol and biodiesel. Biomass. Bioenerg., 29:426– 439.

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Recent Advances in Bioenergy Research Vol. III 2014 13. Nguyen N.H., Suh S.O., Marshall C.J. and Blackwell M. (2006) Morphological and ecological similarities: wood boring beetles associated with novel xylose-fermenting yeasts, Spathaspora passalidarum gen. sp. nov. and Candida jeffriesii sp. nov. Mycol. Res., 110:1232–1241. 14. Nigam J.N., Margaritis W.A. and Lachance M.A. (1985) Aerobic fermentation of Dxylose to ethanol by Clavispora sp. Appl. Environ. Microbiol., 50:763-766. 15. Nitiyon S., Boonmak C., Am-In S., Jindamorakot S., Kawasaki H., Yongmanitchai W. and Limtong S. (2011) Candida saraburiensis sp. nov. and Candidaprachuapensis sp. nov., xylose-utilizing yeast species isolated in Thailand. Int. J. Syst. Evol. Microbiol., 61:462–468. 16. Olofsson K., Bertilsson M. and Liden G. (2008) A short review on SSF–an interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnol Biofuels, 1:7. 17. Rao R.S., Bhadra B. and Shivaji S. (2008) Isolation and characterization of ethanol producing yeasts from fruits and tree barks. Lett. Appl. Microbiol., 47:19–24. 18. Schell D.J., Riley C.J., Dowe N., Farmer J., Ibsen K.N., Ruth M.F., Toon S.T. and Lumpkin R.E. (2004) A bioethanol process development unit: initial operating experiences and results with a corn fiber feedstock. Biores. Technol., 91:179–188. 19. Sims R.E.H., Mabee W., Saddler J.N. and Taylor M. (2010) An overview of second generation biofuel technologies. Bioresour. Technol., 101:1570–1580. 20. Suh S.O., Marshall C.J., Hugh J.V.M. and Blackwell M. (2003) Wood ingestion by passalid beetles in the presence of xylose-fermenting gut yeasts. Mol. Ecol., 12:3137– 3145. 21. Wiselogel A., Tyson J. and Johnsson D. (1996) Biomass feedstock resources and composition. In: Wyman, C.E. (Ed.), Handbook on Bioethanol: Production and Utilization. Taylor and Francis, Washington, DC, pp. 105–118. 22. Watanabe S., Saleh A.A., Pack S.P., Annaluru N., Kodaki T. and Makino K. (2007) Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADH-preferring xylose reductase from Pichia stipitis. Microbiology, 153:3044–3054.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 17 COMPARATIVE BIOETHANOL PRODUCTION BY S. cerevisiae AND Z. mobilis FROM SACCHARIFIED SWEET POTATO ROOT FLOUR (Ipomoea batata L) USING IMMOBILIZED α- AMYLASES AND GLUCOAMYLASE Preeti Krishna Dasha, Sonali Mohapatraa , Manas Ranjan Swainb, Hrudaya Nath Thatoi

Abstract Sweet potato (Ipomoea batatas L) represents an important biomass resource for fuel alcohol production, because of its chemical composition which has a dry matter content of 30-40% and high density of starch (70%), compared to other forms of biomass and thus promises as an alternative bioresource for the production of ethanol through fermentation. The starch present in sweet potato can be hydrolysed to simple sugars and can then be used by microorganisms in fermentation process. Fermentation, by using immobilized enzyme is an advanced technology which has several benefits as compared to free cell culture which include enhanced ethanol yield, easy to separate, reduced percentage of contamination, better operational stability and viability of enzyme actions for several cycles of fermentation. In this experiment batch fermentation is carried out in flask condition to compare the bioethanol production by Saccharomyces cerevisiae (S. cerevisiae) and Zymomonas mobilis (Z. mobilis) from saccharified sweet potato root flour (SPRF) using immobilized α-amylase and glucoamylase. The ethanol yields were 485.5 g/ kg and 448.7 g/ kg of SPRF for S. cerevisiae and Z. mobilis respectively after an incubation period of 96 hours maintained at pH 4.5 and 30oC temperature. The study demonstrated that S. cerevisiae can produce 10.6% higher bioethanol in comparison to Z. mobilis. The highest ethanol concentration was obtained after 96 hour of fermentation. Thus, it was evident from the present study that Z.mobilis does not appear to be an efficient microbial source for commercial bioethanol production, attributed to its lower tolerance to temperature, ethanol and utilization of substrate range compared to S. cerevisiae. Thus the use of S. cerevisiae has great scope for bioethanol production under enzyme immobilization condition having potential for commercial application.

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Recent Advances in Bioenergy Research Vol. III 2014 Keywords: Sweet potato, Saccharification, Immobilization, S. cerevisiae, Z. mobilis. 17.1

Introduction The uses of ethanol as an alternative motor fuel has been steadily increasing around

the world for a number of reasons such as it decreases dependence on foreign oil, reduces trade deficits, creates jobs in rural areas, reduces air pollution by replaceing aromatic and sulfur-content in gasoline, it also reduce nitrogen oxide (NOx) emission to improve air quality which can reduce urban smog and reduces global climate change carbon dioxide buildup (Wheals et al., 1999). The high oxygen content in bioethanol could reduce the generation of known hazardous volatile organic compounds (VOCs) and carbon monoxide in vehicle exhaust (Wyman, 1996; Yoon et al., 2009). Establishment of ethanol industry requires sufficient and cheap feedstock in order to reduce the costs of production that has been recognized as a critical point (Cardona and Sanchez, 2007). However, the transformation of some conventional raw materials (like corn, wheat and rice etc) is not feasible, due to food security issues (Lin and Tanaka, 2006). With high concentrations of starch, tuber crops are considered one of the most important sources for bioethanol. The tuber crops viz. potato, sweet potato, cassava are most promising feed stock due to their economic viability and availability. Sweet potato (Ipomoea batata L.) is one of the most important starch producing crops grown worldwide. The dry matter content in sweet potato ranges from 21 to 30% of which about 80% starch (Zhang and Oates, 1999). Starch is a complex carbohydrate which needs conversion into simpler sugars before being converted into ethanol. Starchy materials required to be converted to glucose monomers by saccharification process which can be achieved by enzyme treatments (by amylases and glucoamylases). After conversion to simpler monomers (glucose) it is fermented to produce ethanol with help of ethanol producing microorganisms (Lin and Tanaka, 2006). Saccharomyces cerevisiae and Zymomonas mobilis are usually the first choice for industrial ethanol production, because of their good fermentative capacity, high tolerance to ethanol and the capacity to grow rapidly under the anaerobic conditions that are characteristically established in large scale fermentation vessels (Agbogo and Kelly, 2008). The concept of immobilization provides a promising strategy for the use of enzymes in a bioreactor for easy scale up an industrial biomass conversion. Day by day number of applications of immobilized industrial important enzymes are increasing steadily.

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Recent Advances in Bioenergy Research Vol. III 2014 Immobilized enzymes could be employed in the bioethanol production with the aim of reducing the production cost by reusing the enzymes. Considering the above facts, the present study is carried out to compare the bioethanol production by S. cerevisiae and Z. mobilis from saccharified sweet potato root flour (SPRF) using immobilized α -amylase and glucoamylase. 17.2

Materials and methods

17.2.1 Sweet potato Freshly harvested and sweet potato (SP)

roots (var. ST-13) (starch, 178 g/kg; total

sugar, 25 g/kg dry weight basis) were collected from the experimental farm of Regional Centre of Central Tuber Crops Research Institute (CTCRI), Bhubaneswar during the month of November, 2011 and used within 24 h after harvest. The fresh SP roots were chipped manually, dried at 60 ºC to reduce the moisture level to ~ 12 % and finally grinded to flour by dry milling and consist particles with diameter 0.2- 1.7 mm (95 % or more particles pass through a 1.70 mm sieve). 17.2.2 Microorganisms and culture conditions The Z. mobilis MTTC 92 strain was maintained on ZS (Z. mobilis specific) medium [(g/L) yeast extract ,10; glucose, 20; MgCl2, 10; NH4SO4 ,10 ; KH2PO4, 10; agar, 15 and pH 6-6.5] and S. cerevisiae (CET) was maintained on malt-extract-yeast extract-glucose-peptone (MYGP) medium [(g/L): malt extract,3; yeast extract,5; peptone, 5; glucose, 20; agar, 15; pH 5.5 ]. Both the cultures were stored at 40C for further use. A loop of microbial culture (Z. mobilis and S. cerevisiae) was inoculated to a 250 ml Erlenmeyer flask which contained 100 ml sterile respective medium (as mentioned above) and were incubated at 30 0C in a rotary shaker (120 rpm) for 24 h. The liquid culture had initial cell density (3×109 colony forming unit (CFU)/ml). 17.2.3 Enzymatic saccharification of SPRF SPRF (Sweet Potato Root Flour) (10%) slurry was prepared in 250 ml Erlenmeyer flasks with a working volume of 100 ml by adding tap water in a ratio of 1:10 for experiment. In first step the slurry was dextrinized by addition of 32 µl of immobilized Palkolase- ®HT at pH 5.5 and 90°C for 1hour and then slurry was cooled down to room temperature. In second

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Recent Advances in Bioenergy Research Vol. III 2014 step, immobilized Palkodex® (329.7 µl) was added to the dextrinized slurry at pH 4.5 and incubated for 24 h at 60 °C for saccharification. 17.2.4 Immobilization of Enzymes The immobilized beads of α amylase were prepared with 0.3% sodium alginate, 0.5% glutaraldehyde, 0.3M CaCl2 solution and 0.2% starch concentration where as for glucoamylase 0.3% sodium alginate with 0.5% glutaraldehyde in 3M CaCl2 solution with starch concentration of 0.2% were required. 17.2.5 Ethanol fermentation from saccharified SPRF Ethanol fermentation was conducted by S. cerevisiae and Z. mobilis under anerobic condition in an Erlenmeyer flask sealed with rubber stopper equipped with opening for CO2 venting. The immobilized enzyme hydrolysed SPRF (100ml) was inoculated with freshly harvested Z. mobilis and S. cerevisiae starter cultures at [10 % v/v (3×109 CFU)/ml] aseptically. The fermentation medium containing flasks (n=3) were incubated in an incubatorcum shaker at 30±20C for 120 h with a constant shaking (100 rpm). The fermented broth was distilled to recover ethanol using alcohol distillation apparatus (Borosil Glass Works Ltd., Mumbai, India). 17.2.6 Study of Fermentation parameter (1) Incubation period: The enzyme enzyme hydrolyzed sweet potato slurry was inoculated with 10 % (v/v) starter culture with pH 4.5 and incubated for 24-120h. (2) Initial medium pH: The fermentation medium (saccharified 10% SPRF slurry) with pH (3- 5.5) was inoculated with 10 % (v/v) starter culture and incubated for 96 h at 30 0C. 17.2.7 Analytical techniques At appropriate time intervals, fermentation medium (in triplicates) were removed and contents were analysed for sugar and ethanol. The ethanol concentration was determined based on the density of the alcohol distillates obtained from the fermentation broth. Ethanol concentration of the fermentation liquid was determined by measuring the specific gravity of the distillate according to the procedure described by Amerine and Ough (1984). The pH was measured using a pH meter (Systronics, Ahmadabad, India) fitted with a glass electrode. The total sugars were assayed by Dinitrosalicylic acid and Anthrone’s method (1999). Fermentation kinetics was calculated using the formulae by Bailey and Ollis (1986).

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Recent Advances in Bioenergy Research Vol. III 2014 17.2.8 Population count Yeast and bacterial populations in the fermented mash were calculated by serially diluting the substrate (fermented SPRF slurry) in sterile distilled water and plating in suitable dilution (104-105) on Petri plates containing ZSM and MYGP medium for Z. mobilis (bacteria) and S. cerevisiae (yeast) respectively. 17.2.9 Calculations The maximum theoretical ethanol yield from sugar was calculated according to the stoichiometric relation represented by Eq. (1), i.e. 100 g of hexose produce 51.1 g of ethanol and 48.9 g of CO2. Ethanol yields over total initial sugar (Y1) and average ethanol productivity rate (Y2) were calculated according to Eqs. (2) and (3). C2H12O6 →2CH3CH2OH + 2CO2

(1)

Y1 = ethanol produced in fermentation/ ethanol produced in theoretical x 100%

(2)

Y2 = Final ethanol concentration/fermentation time 17.3

(3)

Results and discussion

17.3.1 Effect of incubation period In this study, the immobilized enzyme liquefied SPRF hydrolysate was used for subsequent fermentation (submerged) by yeast, S. cerevisiae and bacteria, Z. mobilis. A 10% inoculums was added and cultivated at 30 ºC for both the organisms and the comparison of sugar utilization and ethanol production profile of the two organisms are given in (Figure1 a and b) respectively. After 96 h S. cerevisiae and Z. mobilis produced 487.1 and 445.3g/kg of ethanol respectively. Both the organisms S. cerevisiae and Z. mobilis utilises 88.4 %, 84.7 % total sugar and 94.3%, 86.7% of reducing sugar respectively. Z. mobilis utilises less sugar content and produce less amount of ethanol with respect to Saccharomyces cerevisiae and produces 10.4% less bioethanol with comparison to S. cerevisiae. The ethanol productivity (1.95g/L/h, 1.77g/L/h), ethanol yield (0.614g/g, 0.573g/g) and final ethanol efficiency (98.7%, 94.4%) were obtained by S. cerevisiae and Z. mobilis respectively at optimum incubation period (96 h). In case of S. cerevisiae the concentration of total sugar decreased rapidly within 24 h (73.08% over initial content) of fermentation with concomitant production of ethanol (231.3 g/kg). Thereafter the decline of total sugar was gradual in between 24 and 96 h and then

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Recent Advances in Bioenergy Research Vol. III 2014 decreases. After the end of 96 h incubation period, the residual total and reducing sugar concentration was 100 g/kg and 47 g/kg respectively in culture broth. Similar ethanol production and sugar utilization were observed for bacterium (Z. mobilis) but after 24 h but the bioethanol production is 10.5% low in comparison to S. cerevisiae. The result shows that Z. mobilis was significantly less efficient than S. cerevisiae (7.3%) [Fishers LSD test, P0.2V in practice has been required for hydrogen production from acetate as a substrate (Cheng and Logan, 2007; Hu et al., 2008). This voltage has considerably lower than the water electrolysis process for hydrogen production (Lu et al., 2009). Recent years, the microbial electrolysis cell performance has been considerably improved. However, in practice, the high cost of the reactor and low production rates are main issues for its application (Cheng and Logan, 2007, Rozendal et al., 2007). Therefore, its recent advances in reactor design (Call and Logan, 2008, Tartakovsky et al., 2008 and Hu et al., 2008) and operation (Lalaurette et al., 2009 and Selembo et al., 2009) still need to be developed for realizing the applications of this technology. Also, the high cost of cathode is one of the most critical ones. Pt based cathodes have been widely used in MEC studies (Liu et al., 2005) owing to its effecient catalytic properties and popularity in microbial fuel cell (MFC) studies (Logan et al., 2008). The alternatives to Pt based cathode in MECs have been investigated recently. Selembo et al. (2009) studied the nickel oxide catalysts through cathodic electrodeposition of NiSO4 and (NH4)2SO4 on a metal sheet in MECs. Harnisch et al. (2009) reported that the electrocatalytic behavior of synthesized tungsten carbide powder in MECs by pasting the powder onto graphite disc with Nafion. However the main challenges associated in an MEC for hydrogen production including low volumetric efficiency and the use of an expensive catalyst (platinum). Hence, the researchers have led to examine non-precious metals and alternatives for Pt based cathodes.

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Recent Advances in Bioenergy Research Vol. III 2014 A single-chamber membrane less MEC with cost effective cathode has been attention recently. Lack of membrane in MECs reduce the lower the internal resistance, eliminate the pH gradient across the membrane, and enhance the hydrogen production rate (Hu et al., 2008, Lu et al., 2009). However, in membrane less MEC, hydrogen reoxidization by exoelectrogens and methanogens are main challenges. The present study attempts to the develop the efficient, stable and cost-effective stainless steel cathode for hydrogen production in MECs. This electrode performance was also examined for hydrogen production in membrane less singlechamber MECs. 38.2

Materials and methods

38.2.1 Reactor construction Single-cell MEC was constructed with graphite brush (1.9cm diameter x 3.2cm length) as anode. Stainless steel mesh (3.5 cm diameter X 4.8cm length) was used as cathode. The distance from the middle of brush anode to the cathode was 1.75 cm. Both anode and cathode chambers were connected to electrode and outside circuit. A power source was supplied at a steady voltage (0.2 –1.0 V) between anode and cathode electrode. A power source (DC-3002, Beetech, India) was connected to the circuit for providing voltage, and an external resistor (1000 Ω) was to calculate the current (Fig.1).

Fig.1. Photograph of MECs with stainless steel cathode 38.2.2 Start-up and operation MEC were first operated in microbial fuel cell (MFC), with the cathode exposed to air as mentioned previously (Lu et al., 2009). MFC was inoculated with sewage sludge, collected from the local wastewater treatment plant, in a nutrient buffer solution (NBS) (Na2HPO4, 4.58 g/l; NaH2PO4·H2O, 2.45 g/l; NH4Cl, 0.31 g/l; KCl, 0.13 g/l; trace mineral; 50:50 (v:v)wastewater and buffer) (Call and Logan, 2008) containing 1 g/l glucose. During each fed411

Recent Advances in Bioenergy Research Vol. III 2014 batch cycle, the solutions in the MFCs were replaced when the voltage was decreased to < 20mV (external resistance of Rex = 1000 Ω). Once a maximum voltage of >0.5V (Rex = 1000 Ω) was obtained the cathodes were sealed and the reactors were switched to MEC mode. The MEC was fed to a 200 mL of autoclaved medium containing (in 1 l of pH 7.0 phosphate buffer): NH4Cl, 310 mg; KCl, 130 mg; CaCl2, 10 mg; MgCl2.6H2O, 20 mg; NaCl, 2 mg; FeCl2, 5 mg; CoCl2.2H2O, 1mg; MnCl2.4H2O, 1mg; AlCl3, 0.5 mg; (NH4)6Mo7O24, 3 mg; H3BO3, 1mg; NiCl2.6H2O, 0.1mg;CuSO4.5H2O, 1mg; ZnCl2, 1 mg and 1.0 g/L glucose as a carbon source. pH was adjusted by 1N NaOH solution and monitored by pH meter. A fixed voltage (Eap) of 0.2–1.0 V was applied to the MEC circuit using a power supply (DC 3002, Beetech, India) by connecting the positive pole of the power supply to the anode, and the negative to the cathode. The voltage across a high-precision resistor (Rex =1000 Ω) in the circuit was measured using a voltmeter at 1 h intervals to calculate current. The volume of gas produced from MECs was measured by water displacement method. All experiments were conducted in fed-batch mode. Fresh medium was added to the reactors and sparged with ultra high purity nitrogen gas (99.999%) for 10min. All tests were conducted at room temperature. Applied voltage scans were obtained by varying the voltages stepwise from 0.2 to 1.0 V at 1 h intervals between each voltage (v) change. The current densities (cathode surface area) and the potential of electrodes data of every applied voltage step were averaged over each time interval. 38.2.3 Analytical methods The composition of biogas including H2, CO2 and CH4 was analyzed by Shimadzu gas chromatograph (GC-2014) (Shimadzu Co. Singapore) equipped with a thermal conductivity detector and a stainless column packed with Porapak Q (80/100 mesh). The operational temperatures at the injection port, column oven and detector were 40 °C, 40 °C and 80 °C, respectively. Nitrogen was used as carrier gas at a flow rate of 20 mL/min. The concentration of the volatile fatty acids (VFAs) was measured by Shimadzu gas chromatograph (GC-2014) (Shimadzu Co. Singapore) equipped with a flame ionization detector (FID) and stabilwax – DA capillary column. The injector, column oven and detector were operated at 180, 200 and 220°C, respectively. Nitrogen was used as carrier gas with a flow rate of 5 mL/min. The concentration of glucose was determined using phenol-sulfuric acid method. 38.2.4 Calculations

412

Recent Advances in Bioenergy Research Vol. III 2014 Hydrogen yields was calculated as YH2 = g H2/g glucose or mol H2/mol glucose and coulombic efficiency, CE (%), was calculated as CE = CT/CC, where CT is the total coulombs calculated by integrating the current over time, and CC is the total charge consumed. Cathodic hydrogen recoveries (rcat, e− to H2 in the cathode), overall hydrogen recovery (RH2 = CErcat), and maximum volumetric hydrogen production rates (mL/h) were calculated as previously described assuming standard biological conditions (T=298.15K, P = 1 bar, pH 7) (Logan et al., 2008). 38.3

Results and discussion

38.3.1 Biohydrogen production from glucose Hydrogen production rates were substantially increased from 1.42 (at 0.2 V) to 1.64 ml/h (0.6 V) using buffered fermentation effluent by increasing the applied voltage (Fig. 2). Further, the hydrogen production rate was decreased from 1.64 ml/h to 0.43 ml/h, when the Eap was varied from 0.6 to 1.0 V. The control experiment (without applied voltage) showed that the hydrogen production rate was 1.47 ml/H. On the other hand, hydrogen content was found to be same in Eap upto 0.5V and control experiment. The maximum hydrogen content of 41 % was obtained at 0.6 V. Furthermore, the hydrogen content was decreased from 41 to 31 % as the Eap was varied from 0.6 to 1.0V. The maximum cumulative hydrogen volume of 149 ml was observed at 0.6V. The hydrogen production remained decreased with the further increasing of applied voltage upto 1.0 V. These findings indicate that the hydrogen production was considerably enhanced at an optimum applied voltage (0.6V) in single-chamber membrane-less MEC using mixed culture when compared to the control experiment (110 mL) (Fig.3).

413

Recent Advances in Bioenergy Research Vol. III 2014 Fig.2. Hydrogen production rate, hydrogen content and cumulative hydrogen production with different applied voltage

Fig.3. Cumulative hydrogen production with different time interval of control and 0.6V experiment 38.3.2 Coulombic efficiency and current density As seen in the Figure 4, the Coulombic efficiencies (CE) were increased from 5.4 to 33.7 % by varying the applied voltage from 0.2 to 0.7V. Further it was decreased at above 0.7 V. At low applied voltages of 0.2 and 0.4 V, CE values were found to be low due to methanogenesis. Similar results were obtained by Tartakovsky et al., (2008). As the Eap was increased from 0.7 to 1.0 V, the anode potential was increased and the CE was decreased (Fig. 4). The decrease of CE at the highest applied voltage resulted in a reduction of hydrogen recovery (RH2). However, the current density was gradually increased from 0.04 to 2.32 mA/m2 when the applied voltage was varied from 0.2 to 1.0 V. According to literature, a higher resistor increased the output voltage of the power supply and shared a higher voltage on it (Hu et al., 2012). For the MEC, the hydrogen production rate was partially dependent on the resistor. In spite of this, a constant resistor (1000 Ω) was used in this study and the maximum output current density of the external power supply was reached to 2.32 mA/m2. Consequently, the high hydrogen production rate was achieved (Fig.2). Similar observation was obtained by Chae et al. (2008). 38.3.3 Hydrogen Recoveries

414

Recent Advances in Bioenergy Research Vol. III 2014 Factors affecting the hydrogen yield were observed to be current density (normalized to electrodes area), coulombic efficiency, and cathodic hydrogen recovery. From Figure 4, the cathodic hydrogen recoveries were increased from 37.5 to 79.3 % as the applied voltage was varied from 0.2 to 1.0 V. In addition, the overall hydrogen recovery was similar to the cathodic hydrogen recoveries. The overall hydrogen recoveries were remarkably increased remarkably from 34.7 to 75.9 % as the applied voltage was differed from 0.2 to 1.0V (Fig. 3). These results demonstrate that cathodic hydrogen recovery at below 0.5 V was very low. The high hydrogen recovery was achieved at above 0.5V of applied voltage. These results show that the low current density and coulombic efficiency reduced the hydrogen production rate. Further study requires on optimizing the architecture to lower the internal resistance for improving the hydrogen production.

Fig.4. Current density and Coulombic efficiency at different applied voltage

Fig.5. Hydrogen recoveries and glucose utilization at different applied voltage

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Recent Advances in Bioenergy Research Vol. III 2014 38.4

Conclusions The hydrogen recovery was improved in a low cost single-chamber membrane less

MEC with stainless steel cathode. In addition, small electrode spacing and lack of membrane used in this reactor contruction reduced the pH gradients and proton diffusion resistance which led to high hydrogen production rate. The maximum hydrogen production rate of 1.64 ml/h and overall hydrogen recovery of 69.7 % were achieved when the applied voltage was 0.6 V. In comparison, the control experiment (without applied voltage) indicated that the hydrogen production rate was 1.47 mL/h and hydrogen recovery was 31.5%. Further, optimizing the internal resistance and increasing the current density would improve the hydrogen production rate in this MEC. Acknowledgements This research was funded by the Department of Biotechnology (Ref. No. BT/PR12051/PBD/26/213/2009, dated 19th November 2010), New Delhi, India. Author Mohanraj gratefully thank Ministry of New and Renewable Energy, New Delhi, India for Senior Research Fellowship (NREF–SRF).

References 1. Call D. and Logan B.E. (2008) Hydrogen production in a single chamber microbial electrolysis cell lacking a membrane. Environ. Sci. Technol., 42:3401–6. 2. Chae K.J., Choi M., Ajayi F.F., Park W., Chang I.S., Kim I.S. (2008) Biohydrogen production via biocatalyzed electrolysis in acetate-fed bioelectrochemical cells and microbial community analysis. Int. J. Hydrogen Energy, 33:5184–92. 3. Cheng S. and Logan B.E. (2007) Sustainable and efficient biohydrogen production via electrohydrogenesis. Proc. Natl. Acad. Sci., 104:18871–3. 4. Harnisch F., Sievers G., Schroder U. (2009) Tungsten carbide as electrocatalyst for the hydrogen evolution reaction in pH neutral electrolyte solutions. Appl Catal B: Environ., 89; 455–458. 5. Hu H., Fan Y. and Liu H. (2008) Hydrogen production using single-chamber membrane free microbial electrolysis cells. Water Res., 42:4172–4178. 6. Liu H., Grot S. and Logan B.E. (2005) Electrochemically assisted microbial production of hydrogen from acetate. Environ. Sci. Technol, 39:4317–20.

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Recent Advances in Bioenergy Research Vol. III 2014 7. Lalaurette E., Thammannagowda S., Mohagheghi A., Maness P.C. and Logan B.E. (2009) Hydrogen production from cellulose in a two-stage process combining fermentation and electrohydrogenesis. Int. J. Hydrogen Energy, 34:6201–10. 8. Liu H., Hu H., Chignell J. and Fan Y. (2010) Microbial electrolysis: novel technology for hydrogen production from biomass. Biofuels., 1:129–142. 9. Logan B.E., Call D., Cheng S., Hamelers H.V.M., Sleutels T.H.J.A., Jeremiasse A.W. and Rozendal R.A. (2008) Microbial electrolysis cells for high yield hydrogen gas production from organic matter. Environ. Sci. Technol., 42:8630–8640. 10. Lu L., Rena N., Xing D. and Logan B.E. (2009) Hydrogen production with effluent from an ethanol–H2-coproducing fermentation reactor using a single-chamber microbial electrolysis cell. Biosen. Bioelect., 24:3055–3060. 11. Rozendal R.A., Hamelers H.V.M. and Buisman C.J.N. (2006) Effects of membrane cation transport on pH and microbial fuel cell performance. Environ. Sci. Technol., 40:5206–11. 12. Rozendal R.A., Hamelers H.V.M., Euverink G.J.W., Metz S.J. and Buisman C.J.N. (2006) Principle and perspectives of hydrogen production through biocatalyzed electrolysis. Int. J. Hydrogen Energy., 31:1632–40. 13. Rozendal R.A., Hamelers H.V.M., Molenkamp R.J. and Buisman C.J.N. (2007) Performance of single chamber biocatalyzed electrolysis with different types of ion exchange membranes. Water Res., 41:1984–94. 14. Selembo P.A., Merrill M.D. and Logan B.E. (2009) The use of stainless steel and nickel alloys as low-cost cathodes in microbial electrolysis cells. J. Power Sources., 190, 271– 278. 15. Tartakovsky B., Manuel M.F., Neburchilov V., Wang H. and Guiot S.R. (2008) Biocatalyzed hydrogen production in a continuous flow microbial fuel cell with a gas phase cathode. J. Power Sources., 182:291–7.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 39 FEASIBILITY OF INTERLINKING TWO TECHNOLOGIES FOR SIMULTANEOUSLY TWO BIOENERGIES GENERATION Prashant Pandey, Vikas Shinde, S.P. Kale, R.L. Deopurkar

Abstract Microbial fuel cell for bioelectricity generation rapidly extending interdisciplinary area, has attracted great research efforts to fulfill the demand of renewable energy. Here, we studied bioelectricity generation using food waste leachate obtained from aerobic predigestion tank of two-stage aerobic-anaerobic sequential biogas reactor (Nisargruna Biogas Technology). Volatile fatty acids content of food waste leachate was 8000 mgl-1; it inhibits the methanogens for methane production in anaerobic reactor. Hence, after electrochemical evaluation of MFC for food waste leachate, it was observed that maximum open circuit voltage (OCV) 0.830 V was obtained for the COD load of 1000 mgl-1. At optimum condition, MFC current density was reached up to 5.897 Am-2 and maximum power density 2.379 Wm-2 along with substrate removal more than 80% in terms of COD. This study infers that simultaneously bioelectricity and biogas production can be done using food waste by linking bioelectrochemical technology (Microbial fuel cell) to Nisargruna Biogas technology. Key words: Microbial fuel cell (MFC), Electricity, Biogas, Wastewater treatment 39.1

Introduction Three E’s (Energy, Environment and Economy) are the prime and inescapable concern

of modern society. Society is always ultimate beneficiary of scientific research, knowledge derived and its understanding. Biotechnology is employed for the betterment three E’s in the well being of human beings. The first E i.e. Energy resources scenario if considered, current global energies in use are coal, gas, oil, nuclear and renewable energies. There are limits of non-renewable resources. Oil reserves will not appreciably run out for at least 100 years or more, demand for 418

Recent Advances in Bioenergy Research Vol. III 2014 oil is expected to exceed production within 2015 to 2025 timeframe (Rifkin, 2002). Nuclear energy has its own limits having concern of attack on nuclear power stations, nuclear war, accidental discharge, thermal pollution, radioactive waste disposal problem etc. This vulnerability of conventional sources of energy bound world to think about reliable and sustainable energy sources. Hydrogen is considered the most viable energy carrier for the future. Hydrogen is high in energy content as it contains 120.7 KJg-1. This is the highest energy content per unit mass among known fuels (Krishna, 2013). Therefore, Fuel cell and Renewable energy resources are most promising technology. The second E i.e. environmental scenario if considered, presently, rapid population growth results in globalization, industrialization and rapid economic development that produces in addition enormous amount of waste in its daily activities. 1/3 food produced lost or wasted globally amounting to 1.3 billion tons per year. In India, Fruit and vegetables loss is more than the UK consumes and Grain loss is more than Australia produces (Gustavsson et al., 2011). 1,27,486 Tons per day municipal solid waste is generated in the India during 201112 (CPCB, MOEF, Govt. of India, 2013). Generally, food waste accounts about 27% of total MSW (Behera et al., 2010) which amounted to 34421.22 Tons per day. In India, Food waste is disposed by land filling. Alternatively, food waste is utilized in biogas production. Both utilization and disposal processes resulted in discharge of large amount of leachate, which is troublesome for handling, storage and transportation. Acidogenic food waste leachate has complex composition and heavy pollutant load. It causes ground and surface water contamination, vermin attraction, offensive odour emanation and produce putrefying gases (Han et al., 2004). For satisfying discharge quality standards of food waste, some easy treatment options are needed. For reducing global green house gas emission a method need to be developed that did not leak CO2 into the atmosphere at an average rate (globally!) of more than 1% over centuries (Lewis and Nocera, 2006). Thus, promising solution of these environmental concerns is MFC technology. If we consider scenario of third E i.e. Economics, need for energy in the world increases 1.6 % in average annually (Öztürk et al., 2013). Increasing world human population; declining reserves of cheaply extracted fossil fuels; increasing price of crude oil, coal and natural gas in international markets, affect economic stability in the world. Moreover, human population of the world will increase by 40 % by 2050 ( Holechek, 2013) if effective steps are not taken soon then the world will face economical consequences. Although, huge amount of 419

Recent Advances in Bioenergy Research Vol. III 2014 food waste is utilized as resource in two-stage aerobic-anaerobic sequential biogas reactor (Nisargruna Biogas Technology) but it produces acidogenic food waste leachate in anaerobic digestion or methane production process. High content of volatile fatty acids in acidogenic food waste leachate has adverse effect on growth of methanogens resulting in reduced amount of biogas production. Microbial fuel cell is promising sustainable technology for bioelectricity generation, wastewater treatment and resource recovery. Our aim is to evaluate the feasibility of interlinking of Nisargruna Biogas technology (anaerobic digester) to microbial fuel cell technology (anodic compartment) so that simultaneously both energies i.e.

Methane and Bioelectricity generation can be done.

Therefore, efficacy of acidogenic food waste leachate obtained through the Autothermal anaerobic digestion of Nisargruna Biogas plant predigestion tank’s food waste is examined as microbial fuel cell resource for bioelectricity generation. 39.2

Materials and Methods

39.2.1 Acidogenic food waste leachate : Production Food waste was collected from the predigester tank of Nisargruna plant based on kitchen waste from Symbiosis International University, S.B. road, Pune (India) developed by Bhabha Atomic Research Centre (BARC), Navi Mumbai, (India). Batch mode digestion was carried out in a simulated Autothermal thermophillic aerobic digester (ATAD). The total volume of the conical flask was 1000 ml with effective volume of 600 ml. The temperature of 45°C in the digester was maintained using water bath for 144 h. High amount of VFAs causes reduction in Biogas production (Wang et al., 2009) in Nisargruna Biogas plant. Therefore, Volatile fatty acids were used as indicators of process imbalance in anaerobic digesters as suggested by Ahring et al., 1995. After analysis of VFAs at regular intervals for 144 hrs, it was observed that with progression of time, the volume of leachate was increased with increased in COD. pH of leachate was depleted day by day due to accumulation of VFAs. hrs of digestion at 45°C gave maximum concentrations for all VFAs under observation. (Figure1). Then the Acidogenic food waste leachate digested at hrs at 45°C and was stored at 4 0C for use as substrate in anodic compartment. 39.2.2 Microbial innocula: Two types of inocula: 1) municipal wastewater 2) anaerobic sludge from a sequential two stage aerobic-anaerobic digester, were mixed in ratio of 3:7 (100 ml). First inocula were 420

Recent Advances in Bioenergy Research Vol. III 2014 collected from Sangam Bridge, Pune, Maharashtra, India. Second inocula was collected from sequential two stage aerobic-anaerobic digester (Nisargruna Biogas technology), Matheran, Maharashtra, India.

Figure 1: Variation in concentration of VFAs with respect to ATAD digestion time. 39.2.3 Preparation of Substrate: 3.36 g of acidogenic food waste leachate diluted to 300 ml (COD, 1000 mgl-1), then the 100 ml of mixed inocula were added, making the working volume of the reactor 400 ml. The prepared fuel is ultrasonically treated using Ultrasonic Processor (Piezo –U- Sonic brand) having constant supplied power of 100 W, frequency of 53 kHz for 5 minutes. This substrate was used in MFC’s anodic compartment. In Cathode, 400 ml of 100 mM potassium ferricyanide in phosphate buffer solution (pH 7.1) was inoculated.

39.2.4 Microbial fuel cell reactor configuration and operation Laboratory scale dual chamber MFC made of transparent Plexiglas material was used in this study. The basic concept of design was taken from Logan et al., 2006. The two chambers were designed with about 0.500 l capacities. The working volume of each chamber was 0.400 l. The anode compartment was maintained anaerobic condition. The anode and 421

Recent Advances in Bioenergy Research Vol. III 2014 cathode chamber had two openings connected through a pipe for inlet and outlet and for circulation of fuel. Two Pure carbon brush electrodes were used with 16*12.5*2 mm in dimensions in anodic compartment having 1.0 cm apart from each other. In cathode, a pure carbon brush electrode having dimensions of 16*12.5*2 mm was used in opposite side of the CEM in between anodic compartment electrodes. Electrodes were placed equal distance from membrane. The internal distance of each electrode was fixed i.e. 2.0 cm from membrane. The terminal of each electrodes were connected with ultrathin copper conceal wires. The effective surface area of both carbon electrodes in anode compartment was 0.00102 m2. Both the compartments were connected with CEM having 7 cm in diameter (Nafion T117; Dupont, Wilmington, Delaware ) by using rubber coupling arrangement. The two chambers with membrane coupling assembly were fixed with nut- bolts. The anode chamber was washed with nitrogen gas to remove traces of oxygen to maintain the anaerobic condition. The systematic diagram of dual chambered MFC is given in Figure 2 (A).

Figure 2: Systematic diagram (A) and Actual figure (B) of dual chambered MFC. MFC was operated in batch fed mode. For inoculation and start up of MFC, the synthetic wastewater was prepared with NH4Cl (0.31gl-1), KCl (0.13 gl-1), NaH2PO4.H2O (2.69 gl-1), Na2HPO4 (4.33 gl-1), Acetate (10mM), Trace element solution (12.5 ml), Vitamin solution (12.5 ml). At the starting condition, MFC was operated in open circuit voltage (OCV) mode. The pH of the solution influent was 4.70 in anode and 7.01 in cathode. The MFC was operated once with synthetic wastewater. After stable voltage generation, MFC was switched for different food waste leachate concentration to decide the optimum organic influent rate. The influent concentration was tested for 500 mg l-1, 1000 mg l-1 and 2000 mg l-1 of organic 422

Recent Advances in Bioenergy Research Vol. III 2014 matter as COD. The MFC was kept in incubator at 30°C. To prevent the sedimentation of fuel a magnetic bead was kept in anode chamber and placed at magnetic stirrer (REMI brand) at 170 rpm throughout the operation. The MFC operating condition is given in the figure 2 (B). 39.2.5 Data collection, Analysis and Calculation Voltage was verified directly by using digital multimeter (MASTECH brand Digital Multimeter 10 A DC) for every hour. Current (I) and Power (P = IV) were recorded. The power density was normalized to anode surface area and anode void volume. The polarization curve was prepared for measuring stable voltage for various external resistances (39-470 ohms). The curve then used to calculate the maximum power density. The current density (Id = V/RA) was calculated, where V (V) is voltage, R (Ω) is resistance and A (m2), the geometric anode surface area. The power density (Pd = V2/RA) was calculated. The columbic efficiency (CE) was calculated for batch fed mode system by using the formula (Logan et al., 2006) as:

where ∆ COD is the substrate concentration for batch fed mode system by the time ( tb ) , F is the faradays’ constant, ʋan liquid volume in anode chamber, I, is the current in ampere. pH and conductivity was measured by probes (HACH, USA). COD (including both soluble and particulate) was determined using a standard dichromate oxidation (open reflux) method and VS, SCOD was analyzed by the standard method (APHA, 2005). To quantify VFA 1ml filtrate sample of the supernatant was inserted into glass syringe and inserted into the Shimadzu GC -2010 with a flame ionization detector. Before the assay was started, the machine was calibrated and 1ml of standard VFA solution was added. The sample injection volume was 1ml. Nitrogen was the carrier gas. The injection port and detector were maintained at 180°C respectively. The determination result of VFA constituents were respectively expressed in the form of height of the peaks in the graph and their occupied area was used to calculate their respective concentration in mgml-1. Carbohydrate estimation was done by Anthrone’s method (Scott and Melvin, 1953). Protein content was measured by Biuret method (Layne, 1955). 39.3

Results

39.3.1 Acidogenic food waste leachate characterization

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Recent Advances in Bioenergy Research Vol. III 2014 Before anaerobic digestion raw material has pH of 6.1 that after anaerobic digestion gradually decreases with increase in VFA concentration. After 72 hrs of digestion the characterization of Acidogenic food waste leachate is given in the table 1. Table 1: Characterization of Acidogenic food waste leachate after 72 hrs digestion. Parameter

Unit

pH

-

Soluble COD Conductivity VS

4.40

mScm

12400

-1

19.39

gl

-1

78.3

-1

mgl

Temperature

°C

Protein

-1

mgO2 l

VFA Carbohydrate

Food waste leachate ( after 72 hrs digestion )

8010 45

mgml

-1

11.80

mgml

-1

7.61

39.3.2 Pretreatment of Anodic Substrate For the selective enrichment of specific group of bacteria, pre-treatment of parent inoculum was carried out to use in anode chamber of a MFC. Symbiotic relationship within population and composition of the substrates (wastewater) strongly affects the composition of the bacterial associations in the anode chamber. Inocula in present study are a mixture of different consortia of bacteria and therefore the susceptibility of this to ultrasonication depends on the resistance of the predominant bacterial groups. Resistant microorganisms to ultrasonication stress have more possibility to survive and become a dominant species in the mixed culture. Ultrasonication can suppress activity of Grampositive methanogens by retaining

Gram-negative

bacteria.

Major

electrogens

are

gram-negative

bacteria.

Methanogenic bacteria lacks protective spores forming capability in restrictive environment such as high temperature, extreme acidity and alkalinity, and also have a slower growth rate unlike other hydrogen producing and electrogenic bacteria (Zhu and Beland, 2006). Therefore, the application of moderate duration ultrasonic pre-treatment to inoculums might have enhanced the activity of electrogenic bacteria and somewhat change in leachate composition, which resulted in enhanced current production. The pretreatment of ultrasonic

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Recent Advances in Bioenergy Research Vol. III 2014 waves at 53 kHz at 100 W for 5 minutes ( Figure 4 ) causes reversible damage although 2, 10 and 15 minutes treatment produces poor results. Ultrasonication of fuel (acidogenic food waste leachate and mixed inocula) produced initial open circuit voltage of 0.130 V with respect to untreated 0.92 V only. Thus, with pretreatment initial OCV is increased by 41.30%.

Figure 4: Microscopic image of ultrasonically treated inocula before and after ultrasonic treatment (53 Htz, 100W for 5 minutes) at 1000X magnification. 39.3.3 MFC performance: 39.3.3.1 Determination of Organic Loading Rate (OLR) During start up, it was decided to operate MFC almost for 12 days to reach the stable OCV voltage of approx 0.536 V for 0.400 l of anode solution. It means that anode and cathode were considered fully enriched (Zhang et al., 2011). MFC was freshly re-inoculated with food waste leachate solution of varying concentration of 500, 1000 and 2000 mgl-1 of COD. It was observed that, at different COD input, MFC gave different OCV. For leachate concentration of 500, 1000, 2000 mgl-1 as COD, OCV was 0.631 V, 0.830 V, 0.638 V respectively. The MFC gave maximum voltage of 0.830 V with carbon brush electrode for 1000 mgl-1 COD. After that, voltage was reduced for further substance load. This was because of high COD concentration may affect the microbial activity of anode chamber. It also leads to increase in internal resistance of anode chamber and may increase the charge transfer

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Recent Advances in Bioenergy Research Vol. III 2014 resistance (Nam et al., 2010). Performance of MFC during in open circuit voltage mode to determine optimum organic loading rate is given in figure 5. Thus, the total substrate with concentration of 1000 mgl-1 was considered optimum to study the electrochemical performance of MFC for various external loads.

Figure 5: Open circuit voltage at various substrate concentration with time. 39.3.3.2 Polarization Behavior and Power density In closed circuit voltage mode with various external resistances (39 - 470 Ω) gave maximum closed circuit voltage output of 0.730 V at 220 Ω resistance i.e. good current, Afterwards sudden and continuous drop in voltage was observed.

Therefore, optimum

external resistance was considered 220 Ω for MFC operations. The Closed circuit Voltage (CCV) at a range of resistances is given in the figure 6. Maximum power density and internal resistance of MFCs were obtained by polarization curves. Polarization curves were plotted against potential and power density varying resistances. Current generation showed decreasing trend with increase in the resistance which is in concurrence with the literature reported earlier (Mohan et al., 2008). A low resistance allows more electrons flow in the fuel cell circuit and this result in the potential drop especially at lower resistances in spite of higher power density (Mohan et al., 2010). 426

Recent Advances in Bioenergy Research Vol. III 2014 According to Ohm’s law, at maximum power density, the internal and external resistances are equal. Thus, at Maximum Power density (2.379 Wm-2) MFC external resistance equal to its internal resistance i.e. 220 Ω at 3.258 Am-2 current density. MFC current density was reached up to 5.897 Am-2. The trend of Voltage and Power density as function of current density is given in the figure 7.

Figure 6: Closed circuit Voltage at a range of external resistances (39 to 470 Ω).

Figure 7: The polarization behavior of MFC and Power density. 39.3.3.3 Columbic efficiency and Substrate removal efficacy

427

Recent Advances in Bioenergy Research Vol. III 2014 Columbic efficiency (CE) is the ratio of the coulombs obtained in a MFC to the theoretical coulombs if all the substrate oxidized produces current. CE percentage was obtained 24 %. MFC demonstrated the high COD removal efficacy of 83%. 39.4

Discussion The application of low-frequency ultrasonication was reported to be very effective in

decreasing the amount of bacterial population in sewage sludge. As the frequency and time period increases, the bacterial population decreases (Kesari and Behari, 2008). However, with the prolonged exposure time, the flaw may expand and the structure of cell wall will be destroyed, which leads to the decrease of bacterial activity (Xie et al., 2009). Effect of pretreated anaerobic sludge was reported by More and Ghangrekar, 2009. They reported maximum 0.745 V OCV (40 kHz, 120 W for 5 minutes) with simple synthetic wastewater as substrate rather in present study high molecular wastewater is used and maximum OCV of 0.830 V (53 kHz,100 W for 5 minutes) is produced. The effects of three different inocula (domestic wastewater, activated sludge, and anaerobic sludge) on the treatment of acidic food waste leachate in MFCs were evaluated by Li et al., 2013. Using 1000 mgl-1COD food waste leachate (pH 4.76) as the substrate highest power (0.432 Wm-3) was obtained using anaerobic sludge inoculum. COD removal and CE% was obtained >87% and 20% respectively. In another experiment, using 5000 mgl-1 COD food waste leachate produced maximum power density 15.14 Wm-3 with COD removal efficacy of 90% and columbic efficacy of 66.40% (Rikame et al., 2012). Rather in present study, although the OCV is less than Rikame et al., 2012 but it is greater than Li et al., 2013. Volumetric power density is less than Rikame et al., 2012 and Li et al., 2013 because large volume to anode surface area ratio. If both power density is converted to Wm-2 (with respect to anode surface area) then there is no any literature available as best of my knowledge until date reporting such high power density with Acidogenic food waste leachate. Columbic efficacy and COD removal efficacy although is lesser than all of above because of lesser surface area of anode and respectively large anodic chamber working volume. 39.5

Conclusion This study infers the feasibility of interlinking of Nisargruna Biogas technology

(anaerobic digester) to microbial fuel cell technology (anodic compartment) is possible with 428

Recent Advances in Bioenergy Research Vol. III 2014 further research and improvement of Power density, Current density and Voltage, so that simultaneously both energies i.e. Biogas and Bioelectricity generation can be done.

References 1. Ahring B.K., Sandberg M. and Angelidaki I. (1955) Volatile fatty acids as indicators of

process

imbalance

in

anaerobic

digestors.

Applied

Microbiology and

Biotechnology, 43:559-565. 2. APHA, AWWA and WPCF (2005) Standard Methods for Examination of Water and Wastewater. 22nd ed., APHA Washington, DC. 3. Behera S.K., Park J.M., Kim K.H. and Park H.S. (2010) Methane production from food waste leachate in laboratory-scale simulated landfill. Waste Management,30:1502-1508. 4. Gustavsson J., Cederberg C., Sonesson U., Otterdijk R. and Meybeck A. (2011) Global food losses and food wastes. Food And Agriculture Organization Of The United Nations, pp. 1-23. 5. Han S. K. and Shin H.S. (2004) Biohydrogen production by anaerobic fermentation of food waste. International Journal of Hydrogen Energy, 29:569-577. 6. Holechek J. L. (2013) Global trends in population, energy use and climate: implications for policy development, rangeland management and rangeland users. The Rangeland Journal, 35:117-129. 7. http://www.cpcb.nic.in/divisionsofheadoffice/pcp/MSW_Report.pdf 8. Kesari K.K., and Behari J. (2008) Ultrasonic impact on bacterial population in sewage sample. International Journal of Environment and Waste Management, 2: 233–244. 9. Krishna R. H. (2013) Review of Research on Production Methods of Hydrogen: Future Fuel. European Journal of Biotechnology and Bioscience,1:84-93. 10. Layne E. (1955) Biuret method. In: S. P. Colowick & O. Kaplan (Ed.), Enzymology, Academic Press, New York, 3, pp. 450–451. 11. Lewis N.S. and Nocera D.G. (2006) Powering the planet: chemical challenges in solar energy utilization. PNAS,103:15729-15735. 12. Li X.M., Cheng K.Y., Selvam A. and Wong J.W.C. (2013) Bioelectricity production from acidic food waste leachate using microbial fuel cells: Effect of microbial inocula. Process Biochemistry, http://dx.doi.org/10.1016/j.procbio.2012.10.001.

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Recent Advances in Bioenergy Research Vol. III 2014 13. Logan B.E., Hamelers B., Rozendal R.A., Schrorder U., Keller J., Freguia S., et al. ( 2006 ) Microbial fuel cells: methodology and technology. Environmental Science Technology,40:5181–5192. 14. Mohan S.V., Mohanakrishna G., Reddy B.P., Saravanan R. and Sarma P. N. (2008) Bioelectricity generation from chemical wastewater treatment in mediatorless (anode) microbial fuel cell (MFC) using selectively enriched hydrogen producing mixed culture under acidophilic microenvironment. Biochemical Engineering Journal,39:121–130. 15. Mohan S.V., Mohanakrishna G., Velvizhi G., Lalit Babu V. and Sarma P. N. (2010) Bio-catalyzed electrochemical treatment of real field dairy wastewater with simultaneous power generation. Biochemical Engineering Journal,51:32–39. 16. More T.T. and Ghangrekar M.M. (2010) Improving performance of microbial fuel cell with ultrasonication pre-treatment of mixed anaerobic inoculum sludge. Bioresource Technology, 101: 562–567. 17. Nam J.Y., Kim H.W. and Shin H.S. (2010) Ammonia inhibition of electricity generation in single-chambered microbial fuel cells. J. Power Sources, 195: 6428–6433. 18. Öztürk S., Sözdemir A. and Ülger O. (2013) The Real Crisis Waiting for the World: Oil Problem and Energy Security. International Journal of Energy Economics and Policy, 3:74-79. 19. Rifkin J. (2002) The Hydrogen economy: the creation of worldwide energy web and the redistribution of power on earth. New Work, NY, US: Penguin Putnam, pp.15-17. 20. Rikame S.S., Mungray A.A. and Mungray A.K. (2012) Electricity generation from acidogenic food waste leachate using dual chamber mediator less microbial fuel cell. International Biodeterioration & Biodegradation,75:131-137. 21. Scott T.A. and Melvin E.H. (1953) Determination of dextran with anthrone. Anal. Chem.,25:1656–1661. 22. Wang Y., Zhang Y., Wang J. and Meng L. (2009) Effects of volatile fatty acid concentrations on methane yield and methanogenic bacteria. Biomass and Bioenergy, 33:848–853. 23. Xie B., Liu H. and Yan Y. (2009) Improvement of the activity of anaerobic sludge by low-intensity ultrasound. Journal of Environmental Management,90: 260– 264.

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Recent Advances in Bioenergy Research Vol. III 2014 24. Zhang Y.P., Sun J., Hou B. and Hu Y.Y. (2011) Performance improvement of aircathode single-chamber microbial fuel cell using a mesoporous carbon modifiied anode. J. Power Sources, 196: 7458–7464. 25. Zhu H. and Beland M. (2006) Evaluation of alternative methods of preparing hydrogenproducing seeds from digested wastewater sludge. International Journal of Hydrogen Energy, 31: 1980–1988.

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Part VI Hybrid Systems

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 40 DEVELOPMENT OF NANO BASED THERMIC FLUID: RHEOLOGICAL ASPECTS OF NEW ENERGY SYSTEM Vijay Juwar, Shriram Sonawane

Abstract Nanofluids are emerging as highly efficient thermic fluids due to its enhanced thermal conductivity, to used nanofluids as heating or cooling media on industrial level it becomes mandatory to study the flow related aspects of nanofluids. The viscosity of nanofluid plays decisive role in economic viability nanofluids as thermal fluid. Variation of viscosity of nanofluid with temperature and concentration of nanoparicle directly affects pressure drop which in turn affects pumping cost in heat exchange equipments. Present study deals with measurement of viscosity nanofluid, prepared by dispersing Fe3O4 nanoparticles in ethylene glycol. Viscosity is measured both as function of volume concentration and temperature. Pure base fluid displays Newtonian behaviour in the experimental temperature range. After addition of nanoparticles nanofluid retains Newtonian behaviour. Our study shows that viscosity of nanofluid increases with increase in volume concentration and decreases with increase in temperature Key Words: Nanofluid, Thermic fluid Nanofluid viscosity 40.1

Introduction Nanotechnology is emerging field, to which entire world is looking forward as a

solution to many issues. Amongst those, energy and environmental issues are occupying the topmost attention. Fossil fuels are depleting and creating environmental problems like air pollution, green house effect etc. so need of the hour is to design the system which can efficiently utilize the conventional fuel with least environmental issues like air pollution or search of alternative which replace the existing fuel which can be economic and environmental friendly. Nanofluid is one such potential alternative which can be used to reduce the dependence on the conventional fuel and also reduce load of pollution. Nanofluid is colloidal

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Recent Advances in Bioenergy Research Vol. III 2014 suspension of nanoparticles in base fluid. Nanofluid shows high thermal conductivities which attracted great attention of many researchers across the globe. Due to high thermal conductivity there are numerous potential application in microelectronics, energy supply, transportation. Nanofluid is proposed as next generation heat transfer fluid .At constant Nusselt number convective heat transfer coefficient is directly proportional to thermal conductivity. In convective heat transfer process flow properties of heat transfer media such as viscosity plays important role. Till the date very few research works are done on the viscosity of nanofluids. Al2O3 nanoparticles dispersed in propylene glycol were studied by Prasher et al (2006). Viscosity of copper oxide nanoparticle in engine oil was studied by Koley and Dey (2010). Viscosity of Fe3O4 nanoparticles was investigated by Sundar et al (2013). The behavior of nanofluid viscosity with changing temperature is one of the important aspects of studies in nanofluid. Aldag et al (2012) studied effect temperature on Al2O3 water and CNT water nanofluids. Namburu et al (2007) and Koley and Dey (2010) studied effect temperature on viscosity of copper oxide nanoparticle dispersed in ethylene glycol water mixture and car engine coolant respectively. Duangthongsuk et al (2009) studied TiO2 and water nanofluid. Lee et al (2011) investigated SiC nanofluid. Ferrous nanofluids possess distinct importance due its magnetic property. Magnetic property of nanoparticle can particularly important for the recovery of nanoparticles from nanofluid. Thermal conductivity acquires direct importance in convective heat transfer process. Viscosity and its temperature dependence plays vital role in design of heat exchange equipments. Change of properties such as shear stress, shear strain with change concentration of nanoparticle and temperature of nanofluid are important in understanding of ferrous nanofluid as heat transfer fluid. In present work the effect of changing concentration and temperature on shear rate and viscosity of Fe3O4 ethylene glycol are studied. 40.2

Materials and Method In our experiment we used Fe3O4 nanoparticles with average size of 16nm with

density of 5.17gm/cc. Nanofluids with different volume concentration of 1%, 2%, 3% & 4% were dispersed in ethylene glycol. Ethylene glycol of Fisher Scientific of SQ grade was used in experiment. While making samples high precision mass balance of Shimadzu was used for weighing nanoparticles. To ensure uniform dispersion of nanoparticles in the mixture ultra sonication treatment was given to the samples for 30 minutes. A ChromTech sonicator (Taiwan) with 40 kHz and 1200W was used for ultra sonication treatment. Measurement of 434

Recent Advances in Bioenergy Research Vol. III 2014 viscosity was done with the help of AR G2 Rheometer. Plate cone geometry with diameter 40 mm and A 1 was used. A gap of 32

was maintained when geometry acts as parallel

plate.It has facility to raise temperature with help of peltier plate that offers temperature range of -40 C to 200 C with typical heating rates up to 50 C/min and accuracy of 0.1 C. They incorporate four Peltier heating elements to cover an 80mm diameter plate surface. These Peltier elements are placed directly in contact with thin copper disc with an externally rugged, hardened chrome surface. A platinum resistance thermocouple, PRT is placed at exact centre ensuring accurate temperature measurement and control. The unique design provides for rapid, precise and uniform temperature control over entire 80mm diameter surface allowing standard geometries up to 60mm in diameter.The viscosity of nanofluid was measured with increasing shear rate in the range of 1-500

. Each sample of different volume concentration

was taken for measurement of viscosity in temperature range 100-500 C. 40.2.1 Modeling of Viscosity After experimentation it becomes necessary to correlate results with the effective viscosity of nanofluid. In theoretical point of view to understand the properties of nanofuid is another challenge. Since nanofluid is two phase fluid, must have common features of solid liquid mixture. Application of relation of solid liquid mixture to nanofluid is still doubtful to predict the properties of nanofluid. Some of the widely used models are mentioned below: Einstein’s model is used for relatively low volume fraction ( ≤0.02) which is given as = Where,

(1 + 2.5 )

is base fluid viscosity.

Brinkman et al (1952) extended Eienstein’s model for the use of moderate volume fraction as =(1 − )

.

Batchelor (1977) considered Brownian motion of particles on the bulk stress of an isotropic suspension of spherical particles and the expression for viscosity is =1+2.5 +6.5 Maiga et al (2004) proposed another model

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Recent Advances in Bioenergy Research Vol. III 2014 = 306

-0.19 + 1

44.2.2 Temperature dependence of viscosity Generally it is observed that fluids are having low viscosity near to their boiling point and high viscosity near freezing point. To study the effect of temperature on viscosity of nanofluid various models are proposed. Some widely used are given in a table1 Table 1 Models for temperature dependence of viscosity. Authors

Equations

White(2004)

ln

Reid et al (1987)

= a + b( ) + c(

)

=Aexp(BT)

Yaws (1977)

Log(

Kulkarni et al (2006)

ln

Namburu et al (2007)

Log

) =A + B =A

+ CT +D

-B = A!

"

44.3 Results and Discussion The viscosity of nanofluid as function of shear rate is shown in figure 1. It may be seen that that viscosity of nanofluid remains independent of shear rate as the shear rate is increased; at lower shear rate viscosity sharply declines and for further increase in shear rate remains constant under experimental temperature range of 100C to 500C. This indicates the Newtonian behavior of nanofluid in experimental temperature and shear rate range of 01/s to 500 l/s. Figure 2 shows relationship between shear rate and shear stress of nanofluid, a linear relationship is observed with zero intercept in experimental range, indicates Newtonian behavior of nanofluid. In figure 3 the effect of concentration of nanoparticles and temperature on the viscosity of nanofluid is shown. The viscosity of nanofluid increases with increase in concentration of nanoparticle and decreases with increase in temperature of nanofluid.

436

Recent Advances in Bioenergy Research Vol. III 2014 A

0.05 0.04 Nanofluid Viscosity (Pa.s)

0.03 0.01 0 0

100

200

300

400

500

600

Shear Rate (1/S) 283 K

293 K

303 K

313 K

323

0.06

Nanofluid Viscosity, (Pa.s)

B

0.02

0.05 0.04 0.03 0.02 0.01 0 0.1

100.1 283K

Nanofluid Viscosity (Pa.s)

C

200.1 293K

313 K

323 K

0.2 0.15 0.1 0.05 0 100 283 K

Nanofluid Viscosity (Pa.s)

303 K

500.1

0.25

0

D

300.1 400.1 Shear Rate, (1/S)

200 300 400 Shear Rate (1/S) 293 K

303 K

500 313 K

600 323 K

4.50E-01 4.00E-01 3.50E-01 3.00E-01 2.50E-01 2.00E-01 1.50E-01 1.00E-01 5.00E-02 0.00E+00 0 283 K

100

200 293 K

300

400

Shear303 Rate K (1/s) 313 K

500

600 323 K

Figure 1 Nanofluid viscosity as a function of shear rate: (A)1%; (B) 2%; (C)3%(D)4%

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Recent Advances in Bioenergy Research Vol. III 2014 A

14 SHEAR STRESS (Pa.)

12 10 8 6 4 2 0 0

200

400

600

SHEAR RATE (1/S) 283 K

Shear Stress (Pa.s)

B

293 K

303 K

313 K

323 K

16 14 12 10 8 6 4 2 0 0

100

200

300

400

500

600

Shear Rate (1/S) 293 K

293 K

303 K

313 K

323 K

1.20E+01

C

1.00E+01

Shear Stress (Pa)

8.00E+00 6.00E+00 4.00E+00 2.00E+00 0.00E+00 0

100 283 K

200 300 400 Shear Rate (1/S) 293 K

303 K

500 313 K

600 323 K

Figure 2 Relation between shear stress and shear strain : (A) 1% (B) 2% (C) 4%

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Recent Advances in Bioenergy Research Vol. III 2014 0.03

0.025

Nanofluid viscosity (pa.s)

0.02 4% 3%

0.015

2% 1%

0.01

0.005

0 280

290

300 310 Temperature (K)

320

330

Figure 3 Effect of concentration and temperature on nanofluid viscosity Figure 4 discuss model studies of Brinkman, (1952) ,Batchelor

(1977)and

Maiga,(2004). All of these models consider the effect of nanoparticle concentration on viscosity of nanofluids. At lower temperature all models over predict the viscosity of nanofluid and higher temperature all models under predicts the viscosity of nanofluid. In figure 5 temperature dependence of nanofluid viscosity is shown. Models shown in table 1 are proposed for prediction of nanofluid viscosity at different temperatures. Out of all mentioned models only Namburu’s model fits well to the experimental data. Log

= A!

"

where A and B are constants, T is temperature in Kelvin and

is nanofluid viscosity in centipoises. Values of curve fit constants A and B are shown in table 2.

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Recent Advances in Bioenergy Research Vol. III 2014 Relative Viscosity

A

1.5 1 0.5 0 0

0.002

0.004

0.006

Experimental Brinkman Batchelor Maiga

Volume Fraction

B Relative viscosity

1.20E+00 1.00E+00 8.00E-01 6.00E-01 4.00E-01 2.00E-01 0.00E+00

experimental Brinkman Batchelor Maiga 0

Relative viscosity

C

Relative viscosity

0.006

1.50E+00 Experimental

1.00E+00

Brinkman 5.00E-01

Batchelor

0.00E+00

Maiga 0

D

0.002 0.004 volume fraction

0.002 0.004 volume fraction

0.006

1.20E+00 1.00E+00 8.00E-01 6.00E-01 4.00E-01 2.00E-01 0.00E+00

Experimental Brinkman Batchelor Maiga 0

E

0.002 0.004 volume fraction

0.006

Relative viscosity

2.00E+00 1.50E+00

Experimental

1.00E+00

Brinkman

5.00E-01

Batchelor Maiga

0.00E+00 0

0.002 0.004 volume fraction

0.006

Figure 4 Study of Models of viscosity at constant temperature: (A) 283 K (B) 293K (C) 303K (D)313K (E) 323K

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Recent Advances in Bioenergy Research Vol. III 2014 Table 2 Curve fit values NP volume conc. (v/v)

log( log(μ μf )

A

2%

3%

4%

A

54.49996

54.49996

56.77083

B

0.013077

0.013372

0.012931

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

Namburu's Experimental Linear (Namburu's) 280

300

320

340

Temperature (K)

log( log(μ μf )

B

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

Experimental Namburu's Linear (Namburu's) 280

300

320

340

Temperature (K)

log(μf )

C

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

Exprimental Namburu's Linear (Namburu's) 280

300

320

340

Temperature (K)

Figure 5 Effect of temperature on nanofluid viscosity by Naburu’s model: (A) 2%(B) 3% (C) 4%

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Recent Advances in Bioenergy Research Vol. III 2014 40.4 Conclusion F3O4 Nanoparicles in ethylene glycol shows Newtonian behavior in temperature range of 100C to 500C and in volume concentration of 1% to 4%. The shear rate was increased from 1s-1 to 500s-1. The viscosity of nanofluid is increased when volume concentration of nanoparticle is increased. At temperature of 500C approximately 63.1% increase in base fluid viscosity is observed at 4% volume concentration of nanoparticle. As the temperature increases the viscosity of nanofluid decreases exponentially.

References 1. Aladag B., Halelfadl S., Doner N., Maré T., Duret S. and Estellé P. (2012) Experimental investigations of the viscosity of nanofluids at low temperatures. Applied Energy., 97:876–880 2. Batchelor G.K. (1977) The effect of Brownian motion on the bulk stress in asuspension of spherical particles, J. Fluid Mech., 83:97–117. 3. Brinkman H.C. (1952) The viscosity of concentrated suspensions and solution, J. Chem. Phys. 20:571–581 4. Duangthongsuk W. and Wongwises S. (2009) Measurement of temperature-dependent thermal conductivity and viscosity of TiO2-water nanofluids Experimental Thermal and Fluid Science., 33:706–714 5. Kole M. and Dey T.K. Viscosity of alumina nanoparticles dispersed in car engine coolant, Experimental Thermal and Fluid Science 34 (2010) 677–683 6. Kulkarni D.P., Das D.K. and Chukwu G.A. (2006) Temperature dependent rheological property of copper oxide nanoparticles suspension, J. Nanosci. Nanotechnol., 61150– 1154. 7. Lee S.W., Park S.D., Kang S, Bang I , KimG.H. (2011) Investigation of viscosity and thermal conductivity of SiC nanofluids for heat transfer applications International Journal of Heat and Mass Transfer 54:433–438 8. Maiga E.B.,.Nguyen C.T, Galanis N. and G. Roy. (2004) Heat transfer behaviors of nanofluids in a uniformly heated tube, Super lattices and Microstructures,35:543 -557 9. Namburu P.K., Kulkarni D.P., Misra D. and D.K. Das. (2007) Viscosity of copper oxide nanoparticles dispersed in ethylene glycol and water mixture, Exp. Ther. Fluid Sci. 32:397–402

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Recent Advances in Bioenergy Research Vol. III 2014 10. Prasher R., Song D., Wang J. and Phelan P. (2006)Measurements of nanofluid viscosity and its implications for thermal applications, Applied Physics Letters., 89:133108– 133111 11. Reid R.C., Prausnitz J.M. and Sherwood T.K. (1987)The Properties of Gases and Liquids, forth ed., McGraw Hill, New York 12. Sundar L. S, Singh M.K, and Antonio C.M. Sousa , (2013) Investigation of thermal conductivity and viscosity of Fe3O4nanofluid for heat transfer applications International Communications in Heat and Mass Transfer, 44 7–14 13. White F., (2005) Viscous Fluid Flow, third ed., McGraw Hill, New York, 14. Yaws C.L. (1977) Physical Properties – A Guide to the Physical, Thermodynamic and Transport Property Data of Industrially Important Chemical Compounds, McGraw Hill, New York.

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Recent Advances in Bioenergy Research Vol. III 2014 CHAPTER 41 CONVERSION OF PLASTIC WASTES INTO LIQUID FUELS – A REVIEW Arun Joshi, Rambir and Rakesh Punia

Abstract Various technologies are being developed to overcome the drawback of plastics, namely, their non-biodegradability. Though work has been done to make futuristic biodegradable plastics, there have not been many conclusive steps towards cleaning up the existing problem. Recycling waste plastics into reusable plastic products is a conventional strategy followed to address this issue for years. However this technique has not given impressive results as cleaning and segregation of waste plastics was found difficult. Over a 100 million tones of plastics are produced annually worldwide, and the used products have become a common feature at overflowing bins. Plastics is placed in a landfill, it becomes a carbon sink, Incineration, blast furnace, gasification are not much appreciated solution to the problem, as toxic gases are produced and their cost of production is quite high. Pyrolysis of waste plastics into fuel is one of the best means of conserving valuable petroleum resources in addition to protect the environment. This process involves catalytic degradation of waste plastic into fuel range hydrocarbon i.e. petrol, diesel and kerosene etc. A catalytic cracking process in which waste plastic were cracked at very high temperature, the resulting gases were condensed to recover liquid fuels. Type of plastics also effect the rate of conversion of into fuel and the results of this process are found to be better than other alternate methods which are used for the disposal of waste plastic. Key words: waste plastics, thermal degradation, pyrolysis, catalyst degradation. 41.1

Introduction Plastics play an important role in day- today life. It is unique material because of their

toughness, light weight, resistance to water and chemicals, resistant to heat and cold, low electrical and thermal conductivity, ease of fabrication, remarkable color range, more design flexibility, durability and energy efficiency. Due to above properties it is used in packaging

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Recent Advances in Bioenergy Research Vol. III 2014 materials, agriculture, construction, insulation, automobile sector, electronic devices, textiles and sports equipment and toys. Plastics constitutes in two main categories. It is thermoplastics and thermoset plastics. Thermoplastics make up 80% of the plastics and thermoset plastics make up of remaining 20 % of plastics produced today (Birley et al, 1988), etc. Thermo plastics can re-melt or remould and therefore it recyclable easily but thermoset plastics cannot re-melt or reshape and therefore it is difficult to recycling. Use of different type of some thermo plastics is given in table1 below. Plastics are relatively cheap, easy available, easy to manufacture and their versatility replace to conventional materials. Plastic waste management is biggest problem now due to their non- biodegradability nature. Now plastics manage by plastics recycling technologies. Table 1:Uses of different types of plastics. Type of Plastics

Uses

Polyester

Textile fiber

PET

Carbonated drink bottles, plastics film

PE

Supermarket bags, plastics bottle

HDPE

Milk jugs, detergent bottles, thicker Plastics film, pipes

LDPE

Floor tiles, shower curtains, cling film

PVC

Agriculture (fountain) pipe, guttering Pipe, window frame, sheets for building material

PS

foam use for insulation of roofs and walls, disposal cups, plates, food Container, CD and cassette box.

PP

Bottle caps, drinking straws, Bumper, house ware, fiber carpeting and rope.

41.1.1 Plastics in environment The quantum of solid waste is ever increasing due to increase in population, developmental activities, changes in life style, and socio-economic conditions, Plastics waste is a significant portion of the total municipal solid waste (MSW). In India generation of

445

Recent Advances in Bioenergy Research Vol. III 2014 plastics are increased from about 2.6 MT in 2003 to about 3.6 MT in 2007(MOEF, 2007). Also it is estimated that approximately 10 thousand tons per day (TPD) of plastics waste is generated i.e. 9% of 1.20 lacks TPD of MSW in the India(CPCB, 2003). 32 million of plastics were generated in 2011 in America, representing 12.7 percent of total MSW (EPA, 2011). It is estimated that 100 million tones of plastics are produced each year with PE, PS, PVC and PP amounting to more than 65% of total produced. The average European throws away 36kg of plastics each year. Discarded plastic products and packaging materials make up a growing portion of municipal solid waste. Plastics packaging totals 42% of total consumption and very little of this is recycled (Vogler et al, 1984), etc. Only 8 percent of the total plastic waste generated in 2011 was recovered for recycling (EPA, 2011). Plastics waste may grow in India in future because more and other countries like as U.S, China and U.K will comes in Indian market. There is a much wider scope for recycling in developing countries mainly in India due to low labor cost, plastics consumption increase and therefore raw materials increase. 41.1.2 Environmental hazards due to mismanagement of plastics waste Plastics are no biodegradable material. It takes time to biodegrade is 300-500 years and therefore environmental hazards due to improper manage include following aspect: 1. Littered plastics spoils beauty of the city and choke drains and make important public places dirty. 2. Garbage containing plastics, when burnt may cause air pollution by emitting polluting gases. 3. Garbage mix with plastics gives problem in landfill operation. 4. Lack of recycling plant to posing unhygienic problem to environment 41.1.3 Side Effect of plastics in nature 1. Durability and chemical structure greatly influences the biodegradability of some organic compounds therefore an increased number of functional groups (groups of atoms) attached to the benzene ring in an organic molecule usually hinders microbial attack. 2. Instead of biodegradation, plastics waste goes through photo-degradation and turns into plastic dusts which can enter in the food chain and can cause complex health issues to earth habitants.

446

Recent Advances in Bioenergy Research Vol. III 2014 3. Plastics are produced from petroleum derivatives and are composed primarily of hydrocarbons but also contain additives such as antioxidants, colorants, and other stabilizers. 4. However, when plastic products are used and discarded, these additives are undesirable from an environmental point of view. 5. Burning of plastics give NOX, COX, SOX, particulate, dioxins, furans and fumes to increase air pollution with result acid rain and increase global warming. 6. Plastics in land fill area leaching of toxins into ground water. 41.2. Target of waste plastics into liquid fuel 41.2.1 Recycling Technologies 1. Mechanical Recycling of waste plastics into reusable product is difficult and unfeasible due to contamination of plastics, difficulty to identifying and separating different type of plastics. 2. Uncontrolled incineration of plastics at higher temp above 850 deg Celsius to produces polychlorinated dibenzo-p-dioxins, a carcinogen (cancer causing chemical). Open-air burning of plastic occurs at lower temperatures, and normally releases such toxic fumes and many oxide gases. So flue gases treatment use for protect environment and health problems in incineration plant. 3. Chemical recycling could lead to useful raw materials via by degradation and monomerization of plastics waste, but no method of this primary recycling currently available. The degradation of some plastics into chemicals has been reported in research level. Gasification and blast furnace of plastics waste to produce gases that are carbon dioxide, nitrogen, carbon mono oxide, hydrogen and methane at higher temp above 800 deg. Celsius. 41.2.2 Biodegradability Plastics are non biodegradable material that resists microbial attack. Though work has been done to make futuristic biodegradable plastics, there have not been many conclusive steps towards cleaning up the existing problem because prices of biodegradable plastics is more than petrochemicals based plastics. It may be due to high cost of production and low availability or high cost of raw materials. Some degradable plastics have been developed, but

447

Recent Advances in Bioenergy Research Vol. III 2014 none has proved compatible with the conditions required for most waste landfills. Thus, there is an environmental problem associated with the disposal of plastics. 41.2.3 Energy Demand Fossil fuel i.e. coal, petroleum and natural gas age is expected to span only 1000 years of human civilization (1700 AD to 2700 AD). It is limited sources which are likely to be exhausted in a few more decades or centuries. Increasing population and fuel consumption rates increase in petroleum prices and due to this the energy starvation is felt by every developing and less developed country. The Growing energy demand in table 1.2 is below. Some developing countries like as India have to import petroleum for transportation and chemical industry sector. The prices of petroleum are increasing due to increase prices in international market. Conversion of waste plastics into fuel is complete the some part of objectives in National Energy Strategy is: 1. To reduce petroleum Imports 2. To reduce the annual growth of total energy demand to 2 percent From 4 to 6% by conservation of energy. 3. To develop alternative sources of energy. Table 2: Growing Energy Demand. Year

World Primary Energy Demand (exajoules/year)

1972

270

1985

390

2000

590

2020

840 (S. Rao and Dr B.B. Parulekar, 2012)

41.3

Plastics Recycling Technologies Recycling of plastics should be carried in a manner to minimize pollution during the

process and enhance efficiency and conserve the energy. There is different type of technology include following aspect: 1. Mechanical Recycling- Recycling of plastics waste into reusable product. 2. Chemical Recycling – Gasification, blast furnace 3. Incineration- Burning of waste plastics to obtain energy. 4. Pyrolysis – Conversion of waste plastics into liquid fuels. 41.4

Process technology 448

Recent Advances in Bioenergy Research Vol. III 2014 41.4.1 Raw materials Type of Plastics as raw materials and its contents in table 3 is below. Table 3: Type of plastics and its content. Type of plastics

contents

PE (HDPE, LDPE), PP, PS

hydro carbons

PET, PVA, PF

hydro carbons with oxygen

PVC, PVCD

hydrocarbons with chlorine

Nylon (polyamide), PU

hydrocarbons with nitrogen

Polyphenylene sulfide

hydrocarbons with sulfur

41.4.2 Effect of raw material as plastics in production If PE, PS, PP with other plastics gives flue gas pollution and contaminated to reactor by making other unexpected compound. In contamination to reactor resulting liquid may contain alcohol, waxy hydrocarbons and inorganic substance. Type of plastics and their product in table 4 is below. Table 4: Effect of plastics in production. Type of plastics

Product

PET

terephthalic acid and benzoic acid

PVA

water and alcohol

PVC, PVDC

HCL gas and carbonous compound

PU, PF, NYLON

carbonous product

PE, PS, PP

liquid fuels (UNEP, 2009)

41.4.3 Pyrolysis It is thermal degradation process in the absence of oxygen. It prevent of formation of C0X, NOX, SOX due to absence of oxygen. It breaks large hydrocarbon chain into smaller ones, but this type of pyrolysis requires higher temperature and high reaction time. Also resulting fluid have low octane value, higher pour point of diesel and high residue content. 41.4.4 Catalytic Pryolysis Pyrolysis of waste plastics in presence of catalyst lower the pyrolysis temp and reaction time, increase conversion rate of waste plastics into fuel, increase the yield of fuel and satisfying diesel, petrol quality of fuel by increase octane value of petrol and decrease 449

Recent Advances in Bioenergy Research Vol. III 2014 pour point of diesel. Catalyst use for this purpose is solid acids such as silica, alumina, zeoliteβ, zeoliteY, mordenite, HZSM-5, MCM-41. Acidic catalysts (HZSM-5, Zeolitey, mordenite and so on) have greater efficiency than less acidic ones, for example amorphous alumina silicate. The pore size and structure of catalyst determine their performance on cracking reaction as well as production, for example mordenite size( about 7x8Ȧ) larger give large product molecules while HZSM-5 have smaller pore size(5x5Ȧ) give small product molecules.(P.A. Parikh and Y.C. Rotliwala, 2008) 41.4.5 Process of formation Collect waste plastics and separate that clean and recyclable. Store the waste plastics that can’t separate. Shredding of waste plastics to reduce volume of its. Shredded plastics is treated in a cylindrical reactor at temperature of 300ºC – 350ºC(Pawar harshal and Lawankar Shailendra, 2013).Plastics waste further cracked with catalyst and resulting hydrocarbons are condensed from water cool condenser and collected in receiver. Then liquid fuel fractionates to get diesel, kerosene, petrol etc. Gases produced are toxic, corrosive with non toxic gases. For example hydrogen chloride, hydrogen sulfide etc is toxic and non toxic is butanes, methane, ethane and propylene. So all the gases are treated from this process before it discharge into atmosphere. Therefore flue gas treated through scrubbers and water/ chemical treatment for neutralization i.e. Solution of methanol amine is use in hydrogen sulfide absorption. Treated flue gas can incinerate use in dual Fuel diesel-generator set for generation of electricity. After process remove the formed carbonous substance or residue in reactor to work as insulator for maintaining the efficiency of process. The block diagram of process is given in figure1. 41.4.6 Yield The average percentage yield of various fuel fractions by fraction distillation depending on composition of waste plastics are Gasoline (60% ) and Diesel (30%). The percentage of liquid distillate is mentioned in terms of weight by volume (Antony Raja and Advaith Murali 2011). 41.5. Advantages of process of fuel production 41.5.1 Eco-friendly The fuel satisfies quality of liquid fuel with low sulfur content and low carbon residue. The properties of waste plastic pyrolysis oil and diesel in table 5.

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Recent Advances in Bioenergy Research Vol. III 2014 collection and segregation of plastic waste

storing of plastic waste

shredding of plastic waste

feeding into hopper Flow of waste into heating vessel in absence of oxygen and presence of catalyst

movement of liquid-vapor into condenser

vessel tarry waste

Tapping of liquid fuel

Fractionation of liquid fuel to obtain diesel, petrol, kerosene etc.

Figure 1- Conversion waste plastics into liquid fuel (Pawar Harshal and lawankar, 2013) Table 5: Properties of Waste Plastic Pyrolysis Oil and Diesel. Sr. No.

Properties

WPPo

1.

Density(kg/m2)

2.

Ash content (%)

3.

Calorific value(kJ/kg)

41,800

42,000

4.

Kinematic viscosity @

2.149

3.05

793