Bioenergy generation from components of a Continuous ... - IEEE Xplore

2013 Annual IEEE India Conference (INDICON)

Bioenergy generation from components of a Continuous Algal Bioreactor: Analysis of Lipids, Spectroscopic and Thermal properties Durga Madhab Mahapatra Energy and Wetlands Research Group, Centre for Ecological Sciences, Centre for Sustainable Technologies Indian Institute of Science (IISc), Bangalore, India [email protected]

H N Chanakya Centre for Sustainable Technologies, Indian Institute of Science (IISc), Bangalore, India [email protected]

Abstract— Influx of sewage into surface water results in nutrient enrichment and consequently leads to algal bloom and voluminous organic sludge production in urban areas as in case of Bangalore. The lack of utilities of algae and resulting sludge has lead to anoxia and GHG emissions. Environmental friendly ways of sludge and algal biomass utilities as well as disposal are scant. Testing for biofuel and thermal properties can be beneficial to meet the energy requirement to run treatment plants that might have better fuel value to increase the net energy gain in the system. As the volume of algal biomass and quantity of sludge has increased over the past few years, sustainable means of biomass and sludge utilization needs to be devised for beneficial purposes, to keep the surface waters clean and regulate the biomass productivity of such systems. Therefore the biomass and sludge characterization becomes imperative for any further utilities. In the present study the indigenous suspended algae and the algal bioreactor sludge were characterized for the different functionalities and presence of bio-chemicals (carbohydrates, proteins and lipids) through Infrared Analysis (ATR-FTIR). The total lipids and fatty acid methyl esters (FAME) composition were studied. The heat values and thermal decomposition pattern were analyzed by [Thermogravimetry (TGA)/Differential Thermal Analysis (DT) and Differential Scanning Calorimetry (DSC)]. The algae were found to have a better total lipid content of >34 % compared to 22% in case of wastewater sludge with quality FAME for better biofuel properties. However there were higher number of FAME in wastewater sludge (>30) where C16 and C18 members dominated. Algal biomass showed higher calorific value of 17.96 MJkg-1compared to 10.33 MJkg-1 of wastewater sludge.

Keywords—bioenergy, algal bioreactor, wastewater, biofuel, spectroscopy, thermal

I.

INTRODUCTION

Water and wastewater treatment constitute a vital component of urban ecosystem. Unprecedented urbanization during the post globalization period, has stressed prime resources such as water and energy. Spurt in urban agglomerations has

T V Ramachandra Energy and Wetlands Research Group, Centre for Ecological Sciences, Centre for Sustainable Technologies Indian Institute of Science (IISc), Bangalore, http://ces.iisc.ernet.in/energy [email protected]

necessitated exploration of viable options to treat wastes generated in the region. Last two decades has witnessed evolution of treatment technologies across various parts of the globe [1]. Bioreactors and lagoons with algae have drawn much attention with the prospects of bioenergy generation [2]. This option would create new avenues, through enhanced resource management with optimal wastewater treatment while also providing bioenergy to meet the growing demand of energy in India [3]. Algal biomass and sludge are solid byproducts of these reactors and efficient utilization of such products will permit recycling and reuse of nutrients. Greater Bangalore, capital of Karnataka state, India, with the population of over 8 million generates wastewater that is either partially treated or untreated and eventually reaches the water bodies. The sustained inflow of sewage, leads to nutrient enrichment with prolific algal growth with a mean biomass productivity of 12gm-2d-1. Algae removes 60-70 % of nutrients (C, N and P) during the retention period of ~4-6 days [4,5] with voluminous algal biomass and a substantial sludge production. Unmanaged sludge together with algal residues under anoxic conditions emit greenhouse gas (GHG). Earlier studies include utilities of algal biomass and sludge, such as dry and wet extraction of algae for biodiesel production, direct combustion of wet algal biomass for energy generation [6], generation and combustion of sludge bio-oil and pyrolysis of sewage treatment plant sludge etc [7]. However, there are no studies on the thermal and spectroscopic evaluation of algal bioreactor components. The present study for the first time investigates and characterizes the domestic wastewater fed bioreactor components to test its suitability for energy generation and lipid production. Treating wastewater through algae is attractive due to cost effectiveness and minimal energy requirements, with the scope for propagation at decentralized levels. The current investigation through the spectroscopic [ATR-FTIR] and thermal [using Differential Scanning Calorimetry (DSC), Thermogravimetry

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(TG) and Differential Thermal Analysis (DT)] characterizes algal biomass and reactor sludge produced in a continuous algal bioreactor that treats wastewater at a loading of ~5.4 gCODl-1d-1; ~0.48gNl-1d-1; 0.42 gPl-1d-1 with in a retention period of ~4 days and a feed rate of ~0.5 ld-1. In addition, this study also looks at the growth and lipid accumulation capability of indigenous algae grown in wastewater fed bioreactors.

Spectroscopic and thermal properties studies were performed in order to monitor the biochemical functionalities and thermal phenomena of the material respectively. The solid content of algae and sludge were determined using drying oven and muffle furnace following standard protocols (2540 G, APHA) [8]. Fig. 1. Continous Algal Bioreactor

II. MATERIALS AND METHOD A. wastewater sampling and characterization Wastewater was collected from the sewage treatment unit of 0.5 MLD capacities at Indian Institute of Science (IISc) campus. Table 1 provides the characteristics of wastewater. TABLE I. CHARACTERISTICS PHYSICO-CHEMICAL DOMESTIC WASTEWATERS USED FOR EXPERIMENTS

Physico-chemical Parameters

OF

Values

-1

Total Nitrogen (TN) mgL

45±1.88

Ammonium-Nitrogen (NH4-N) mgL-1

36.5±3.76

Nitrate-Nitrogen (NO3-N) mgL-1

0.8±0.041

Nitrite-Nitrogen (NO2-N) mgL-1

0.06±0.085

-1

Total Phosphorus (TP) mgL

31±4.26

Ortho-phosphates (OP) mgL-1

19.6±6.55

Total Organic Carbon (TOC) mgL-1 Chemical Oxygen Demand (COD) mgL -1

Total Solids (TS) mgL

Total Suspended solids(TSS) mgL

FAME extraction was carried out as shown in Fig. 2, by rapid transesterification using chloroform methanol solvent system.

153±12 -1

450±77 1277±130

-1

C. Lipid extraction, FAME composition and Quantification

Fig. 2. Protocol for Single step transesterification and lipid extraction

Freeze dried algal biomass and sludge (1 g)

569±84

Total Volatile Solids (TVS) mgL-1

374±66

pH

6.94±.1

Redox Potential (ORP) mV

-112±27

Taken in a vial and mixed with 1.7 ml of methanol, 0.1 ml H2SO4 and 2 ml chloroform Heated at 80 ºC for 50 min and then shaken and vortex thoroughly Cooled to room temperature by quick freezing

B. Bioreactor Design and Analysis Wastewater is fed at 0.5 lhr-1 and CO2 sparging was maintained at 16 mlhr1 to a continuous algal bioreactor 45 (15X3) litres capacity (Fig.1) with hydraulic retention time (HRT) of ~4days. The reactor was intermittently aerated with aerators at an average air flow rate of 54 lhr-1. The reactor was inoculated with selected algal species comprising of euglenoides and green algae. The reactor was run for more than 2 weeks for stabilization of the reactor environment. The algal and sludge biomass were collected manually and were dewatered by centrifugation.

Then added 2 ml of deionised water Mixed for 2 minutes Centrifuged at ~7000 g for phase separation (10 min) FAME containing solvent layer extracted Transferred to pre weighted glass vial for weighing (gravimetry) – Total lipids Solvent (chloroform) evaporation using liquid N2 Eluded in n-hexane and GC –MS Analysis

The FAME samples were loaded in silica column with helium gas as carrier in split less mode. The total run time was calculated to be 47.67 min. Fatty acids were identified by comparing the retention time to that of known standards. The composition of the fatty acid methyl ester (FAME) was assessed by gas chromatography mass spectrometry (Agilent Technologies 7890C, GC System; Agilent Technologies 5975C insert MSD with Triple-Axis Detector). Both the initial column temperature and the injection port temperature were maintained at 250 °C. Detector temperature was 280 °C and was increased to 300 °C at a temperature gradient of 10 °C min-1. The oven temperature was then raised to 230 °C at a ramp rate of 3 °C min-1, and finally, it was raised to 300 °C at a ramp rate of 10 °C min-1. This temperature was maintained for 2 min. The components were identified based on their retention time, abundance and fragmentation patterns by comparing with a known standard.

respectively. There were >30 different fatty acids identified in the FAME mixture extracted from sludge and 17 were from algal biomass grown in domestic wastewaters, among which palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2) were abundant. The order of the fatty acid methyl ester (FAME) in algae were C16:0(34.7%) >C18:0(19.01%)>C18:1(10.44%)>C18:2(9.95)>C16:1(5.7%). Compared to this, in sludge samples, palmitic acid (C16:0), linolenic acid (C18:2) were the major fatty acids. The order of the fatty acids in sludge are C16:0(33.81%)>C18:2(13.71%) >C16:2(11.37%)>C18:0(7.42%). The total FAME profile with the percentage composition of each fatty acid and their time of retention is provided in Table II. The GC-MS chromatograms are also depicted in Fig 3 and 4. The lipid profile shows a dominance of C16 and C18 methyl esters, i.e. ~90 % C16 and C18 methyl esters in case of both algal biomass and sludge emphasizing that the lipids produced are similar to vegetative oil and have good biofuel properties [11].

D. Spectroscopic Analysis (ATR-FTIR) Macromolecular biochemical compositions of algal cells were assessed using a FTIR spectroscope (Alpha Bruker). Dry algal biomass after freeze drying was analyzed with attenuated total reflectance (ATR) using an ATR–FTIR spectroscope in the absorbance mode (range 1,800–800 cm−1 wave numbers) with 128 scans at a spatial resolution of 2 cm−1. IR absorption spectra were collected. The data analysis was carried out with Origin Pro software with an initial base line correction and scaled up to Amide I max [9]. E. Thermal Analayis (DTA-TGA and DSC) TG and DTA experiments were carried out using a DTG-60 H thermo analyzer from room temperature to 800 ºC, under a nitrogen flowing rate of 50 mlmin-1 and constant heating rate of 10 ºCmin-1. Sample masses ranged around 5.00 mg. Thermal analysis was conducted in a METTLER TOLEDO DSC1 thermal analyzer where DSC profiles are recorded. Heating of samples (50 mg) was done with in a temperature range from 0 to 500 ºC, using a gas flow of 120 ml min-1 (N2 environment) and a constant heating rate of 10 ºC min-1. DSC profiles of the sludge samples collected across the decomposition gradient ware analyzed. DSC curves were corrected (Self-controlled calibration) by subtracting the DSC curve of the empty pan from the recorded sample curve. Enthalpies were calculated by integration of the area below the DSC curve between 0 and 500 ºC, drawing a horizontal baseline (heat flow 0) from 0 to 500 ºC.

N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 22 23 24 25 26 27 28 29 30 31 32 33

FAME

Formula

RT

Algae

Sludge

Butanedioic acid, diME Tridecanoic acid, 4,8,12-triME Tridecanoic acid, ME Methyl tetradecanoate Tetradecanoic acid, 12-ME Pentadecanoic acid, ME 7,10-Hexadecadienoic acid, ME 7-Hexadecenoic acid, ME 17-Octadecynoic acid, ME 9,12-Hexadecadienoic acid, ME Hexadecanoic acid, ME 7,10-Hexadecadienoic acid, ME 7,10,13-Hexadecatrienoic acid ME 9,10-methylene-hexadecanoic acid, ME Heptadecanoic acid, ME 11-Octadecenoic acid, ME 9-Octadecenoic acid (Z)-, ME 9,15-Octadecadienoic acid, ME Octadecanoic acid, ME 9,12-Octadecadienoic acid ME 9,12,15-Octadecatrienoic acid ME 5,8,11,14 Eicosatetraenoic acidME 8,11,14-Eicosatrienoic acid, ME Met 8,11,14,17-EicosatetraenoicacidME cis-11,14-Eicosadienoic acid, ME Eicosanoic acid, ME 17-Octadecynoic acid, ME Docosanoic acid, ME Tetracosanoic acid, ME Hexadecanoic acid, ME Octacosanoic acid, ME Saturates Unsaturates Monounsaturated FAME Polyunsaturated FAME C16-C18 FAME Unsaturated/Saturated FAME Mono/Poly FAME FAME (mg)

C8:0 C13:0;n C13:0 C14:0 C14:0;n C15:0 C16:2(7,10) C16:1(7) C18:1(Z) C16:2(9,12) C16:0 C16:2(7,10) C16:3(7,10,13) C16:2(9,10) C17:0 C18:1(11) C18:1(9) C18:2(9,15) C18:0 C18:2(9,12) C18:3(9,12,15) C20:4(5,8,11,14) C20:3(8,11,14) C20:4(8,11,14,17) C20:2(11,14) C20:0 C18:1(17) C22:0 C24:0 C26:0 C28:0

7.504 16.36 21.75 24.97 26.97 28.18 30.31 30.54 30.63 31.12 31.84 33.28 33.55 33.72 34.48 36.91 37 37.02 37.65 39.33 39.58 41.19 41.7 41.9 42.24 43.13 43.66 48.45 51.7 53.81 55.53

1.328 --1.49 -3.198 2.776 5.744 --34.78 ---2.662 4.885 10.44 -19.01 9.952 -0.576 ---0.789 -0.943 0.785 0.427 0.213 65.62 34.37 21.07 13.3 90.25 0.524 1.584 6

0.435 0.065 0.075 1.628 0.497 1.06 0.596 -5.228 11.37 33.81 1.328 0.309 0.265 1.162 --13.71 7.422 11 3.853 0.91 0.449 0.356 0.256 0.351 2.085 1.243 0.542 --48.29 51.78 7.313 43.8 90.05 1.072 0.167 1.33

*ME – methyl ester

TABLE II.

III. RESULTS AND DISCUSSION A. Lipid and FAME Analysis by GC-MS The quality of the biofuel depends on the composition of methyl esters obtained from lipids [10]. The total lipid content of algae and sludge ranged from 26-34 % and 14-22 %

FAME PROFILE FOR ALGAE AND SLUDGE

FAME with higher palmitate and oleate content show good biofuel properties with a quality ignition, higher oxidative stability and lubricity [11]. In case of sludge the PUFA content was ~44%, increasing the chances of susceptibility to oxidation. Algal suspension was more exposed to light resulting in high percentage of saturated fatty acids that was comparatively higher than unsaturated fatty acids.

Fig. 3. Gas Chromatogram of Algae with FAME assignments

were recognized as the most common fatty acids contained in bio-diesel [11]. In the present FAME study the percentage of saturated fatty acids were higher compared to PUFA. Furthermore the essential fatty acid from biofuel perspectives was found to be in higher percentages. The ATR–FTIR spectra of the algal biomass and sludge cells showed 7 distinct absorption bands from the wave numbers ranging from 1800-800 cm-1(Fig.5). These bands were assigned to specific functional groups following biochemical standards and published matter [9]. The assignments of bands corresponding to the functional groups are provided in the Table III. Out of all these bands three bands were most important for the study i.e. ~1740 cm-1 for ester/fatty acid, ~1655 cm-1 for proteins and ~1150-950 cm-1 for carbohydrates. The characteristic lipid peak is normally visualized in the FTIR spectra at ~1700 cm-1 during the final phases of the culture. The algal cells showed comparatively higher peaks in the Amide II region, while sludge showed higher composition of polysaccharides and hydrocarbons evident from the big and broad peak at 1000 cm-1. Fig. 5. ATR-FTIR spectra of algae and sludge

Fig. 4. Gas Chromatogram of Sludge with FAME assignments

Band Assignments

Functional groups

~1740 cm-1 ~1655 cm-1 ~1545 cm-1 ~1455 cm-1 ~1380 cm-1 ~1240 cm-1 ~1000 cm-1

ν(C=O)stretching of ester groups, from lipids and fatty acids ν(C=O)stretching of amides from proteins (Amide I) δ(N–H)bending of amides from proteins(Amide II) δas(CH2) and δas(CH3) bending of methyl from proteins δs(CH2) and δs(CH3) bending of methyl and νs(C–O) stretching COO– νas(>P=O) stretching, associated with phosphorus compounds ν(C–O–C) stretching from polysaccharides

TABLE III.

BAND ASSIGNMENTS FOR FTIR (FUNCTIONALITIES)

C. Thermal analysis by TGA/DTG and DSC B. Spectroscopic Analysis by ATR-FTIR The percentage FAME within C16-C18 was ~90%. C16-C18 fatty acids are the most essential fatty acids having desirable biofuel properties. Palmitic, stearic, oleic and linolenic acids

Thermogravimetric behavior of bioreactor algae and sludge at a heating rate of 10 ºCmin-1 is elucidated as thermograms in Fig. 6. The TGA and DTG curves revealed that there were broadly three stages in the process of thermal degradation. The first stage from 30ºC to 200ºC was characterized by a slight weight loss. During this stage, an initial slight weight loss of

~4% and ~7% occurred between 30 ºC and 120 ºC in algae and sludge samples respectively. This could be due to the elimination of water in the cells and external water bound by surface tension. The maximum rate of water loss in case of algae and sludge was at 68 ºC due to the production of carbon oxides (CO and CO2) besides the loss of water.

and ΔH of 18.09 Jg-1and transition temperature of 365 ºC. The presence of a thermal event on the DSC curve with endothermic characteristic close to 100 ºC indicates the presence of moisture in sample. Fig. 7. DSC curves of algae and sludge

Fig. 6. TGA-DTG curves of algae and sludge

In the second stage (200-560 ºC), a major weight loss of 67.12% and 35.6 % appeared, which corresponded to the main pyrolysis process in decomposition of algae and sludge respectively. Most of the volatiles were released in this stage. It proceeded at a high rate and led to the formation of the pyrolysis products (Fig. 6). The maximum rate of weight loss (DTGmax) was 0.42 and 0.18 %ºC-1, corresponding to a temperature of 326 and 271 ºC. During the third stage (560800 ºC), a slow further loss in weight of 5.9 % and 1 % in case of algae and sludge respectively showed that the carbonaceous matters in the solid residue continuously decomposed at a very slow rate and the solid residue reached an asymptotic value. In DTG a small peak was observed at 659 ºC. As a result of the whole process the final weight loss reached up to 73.01 % and 36.4 % in algae and sludge respectively. The average rate of weight loss was 0.093% and 0.046% for algae and sludge respectively. Lower values of mass decomposition in sludge compared to algae can be attributed to the presence of inert (silt and clay) particles. A progressive weight loss of 3.18% was observed up to 200 ºC. It has been reported that thermal decomposition of thermo labile components (proteins, esters and carboxyl groups) of the organic material produces substantial exothermic reactions at about 300 ºC, while exothermic reactions at higher temperatures (∼450 ºC) originate from the decomposition of C refractory’s of carbon, such as aromatic rings, N-alkyl long chain structures and saturated aliphatic chains [12]. The curve obtained from the DSC shown in Fig. 7 as a first scan (heating cycle), of the algae (red) and sludge (green), indicats the decomposition process, with its respective enthalpies. As shown in Fig. 7, for algal biomass the first event was an exothermic event with ΔH of 7.77 Jg-1 and a transition temperature of 165 ºC, followed by second exothermic event (ΔH = 31.84 Jg-1) at a peak temperature at 210 ºC. This was followed by another two exothermic events with ΔH of 26.87 Jg-1 and a transition temperature of 285 ºC

However, for the sludge the first exothermic event with ΔH of 52.83 Jg-1 was at a transition temperature of 125 ºC, followed by second exothermic event (ΔH = 13.63 Jg-1) with a peak temperature at 170 ºC. Thus in terms of the magnitudes of the exotherms, the algal biomass has superior heating properties compared to bioreactor sludge. It was observed that the algal biomass has a higher calorific value of 17.96 MJKg-1 compared to 10.33 MJKg-1 of wastewater sludge. The probable reasons for a lower calorific value in case of sludge can be due to the presence of minerals, and inert materials as silt, clay etc. unlike algal biomass that are highly organic being suspended in the system. Earlier reports for calorific values for digested anaerobic sludge show 13 MJkg-1 [13]. Higher calorific value of algae 22.22 MJkg-1 is reported to be dependent on nature, origin and composition of the biomass [5]. Higher calorific values of algal biomass and sludge in the present study compared to the earlier reported mean calorific values of 14 MJkg-1 [7] and 10 MJkg-1 [13] highlight the scope for algae based energy generation. D. Potential for bio-energy in Bangalore Bangalore city generates more than 1200 million litres per day (MLD) of wastewater that is primarily domestic [14]. There is a huge scope of resource recovery from the nutrient laden waters in the city [14-16]. The algal rich water bodies (urban algal ponds) help in 70-80 % nutrient and ~90 % C removal [4,15]. Algae proliferate due to nutrient enrichment [4,16] in the water bodies, and subsequently die after the growth cycle. Organic matter, dead algal matter and other debris in the lower strata of these water bodies decompose and critically reduce dissolved oxygen (DO) rendering the system anaerobic [17] favoring GHG emissions. The carbon captured through algal biomass with an average productivity of ~12 gm-2d-1 is useful as fuel. Considering harvest possibility to the tune of 40% from such systems, provides about >12,000 tonnes (t) of algal

biomass. These biomass can then be variably used either by production of ~2,400 tyr-1 lipid crude at an average lipid content of ~20 % [18,19]. In addition to that the sludge in such water bodies provide >4,000 tyr-1 of crude lipid. The spent biomass (i.e. ~10,000 tyr-1) that is left out of the system after lipid extraction that comprise mainly of protein and carbohydrate can be reutilized for bioenergy generation either through combustion or pyrolysis (>30 MJkg-1) or through anaerobic digestion. On the other hand a high nutrient recovery potential of ~3,600tCyr-1; ~720tNyr-1and ~120tPyr-1 can be achieved for Bangalore region. Such approaches would provide scope for integration of biomass and sludge management with present wastewater management practices for ensuring triple benefits of i) water purification ii) nutrients capture and iii) biofuel to meet the growing energy demand.

IV. CONCLUSION The algal bioreactor highlights the prevalence of myxotrophic algae in Indian domestic wastewater with high N and P concentrations. The lipid content of the major bioreactor components i.e. suspended algae and sludge ranged from 2634 % and 14-22 % respectively. The FAME analysis showed desirable fatty acids (i.e. with better biofuel quality) dominated by saturated fatty acids (C 16 and C 18). However algae showed a better constitution of desirable fatty acids. The FTIR results showed abundant carbohydrates in algae followed by proteins with minimal lipids. The sludge is mostly comprised of aliphatic chains with double bonds, as well as carbonyl, hydroxyl and N–H groups in organic compounds. Thermal analyses (TGA/DT/DSC) showed major decomposition of algal biomass in the bioreactor with a greater mass loss compared to sludge. The DSC studies revealed higher exothermic events in algae (compared to sludge). The algal biomass showed higher calorific value of 17.96 MJkg-1 compared to 10.33 MJkg-1 of wastewater sludge. The study highlights the potential of the two major treatment bi-products for better utilities in terms of deriving bioenergy that fosters sustainability. ACKNOWLGEMENT We are grateful to the Department of Biotechnology (DBT), The Ministry of Science and Technology (DST) and Indian Institute of Science for providing the financial and infrastructure support. REFERENCES [1] W. Zhou, M. Min, B. Hu, X. Ma, Y. Cheng and Chen. P, “Ahetero-photoautotrophic two-stage cultivation process to improve wastewater nutrient removal and enhance algal lipid accumulation,” Bioresou. Technol., vol.110, pp.448-455, 2012. [2] M.A. Borowitzka, “Commercial production of microalgae: ponds, tanks, tubes and fermenters,” J. Biotechnol., vol.70, pp. 313-321, 1999.

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