The Pennsylvania State University. The Graduate School. College of Engineering HYDROGEN PRODUCTION FROM CELLULOSE FERMENTATION END

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1 The Pennsylvania State University The Graduate School College of Engineering HYDROGEN PRODUCTION FROM CELLULOSE FERMENTATION END PRODUCTS USING MICROBIAL ELECTROLYSIS CELLS A Thesis in Environmental Engineering by Elodie Lalaurette 2008 Elodie Lalaurette Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2008

2 ii The thesis of Elodie Lalaurette was reviewed and approved* by the following: Bruce E. Logan Kappe Professor of Environmental Engineering Thesis Advisor John M. Regan Associate Professor of Environmental Engineering Rachel A. Brennan Assistant Professor of Environmental Engineering Peggy Johnson Professor of Civil Engineering Head of the Department of Civil and Environmental Engineering *Signatures are on file in the Graduate School

3 iii ABSTRACT Dark fermentation of cellulose has been well studied as a bio-hydrogen production method but its drawbacks include a low hydrogen yield and the presence of fermentation end products in the effluent. Electrohydrogenesis is a new hydrogen production method where bacteria in microbial electrolysis cells (MECs) oxidize an organic substrate at the anode producing electrons and protons which are combined at the cathode to produce hydrogen. This process has been used effectively with various substrates, including typical fermentation end products such as acetic and lactic acids. However, MECs have not been tested with mixtures of such substrates. It is shown here that good hydrogen yields, high maximum production rates, and high overall energy efficiencies can be obtained with either synthetic or cellulosic material fermentation effluents. MEC performance was tested with either a non-acclimated inoculum or an inoculum pre-acclimated to individual substrates in a synthetic fermentation effluent. MEC tests were then conducted using a synthetic fermentation effluent, and actual cellobiose and corn stover fermentation effluents. Hydrogen yields were between 800 ± 290 ml H 2 /g COD for reactors using non-acclimated inocula with the synthetic effluent, and 1000 ± 300 ml H 2 /g COD for reactors using acclimated inocula with the cellobiose effluent. The maximum hydrogen production rate ranged from 0.59 ± 0.21 to 1.11 ± 0.13 m 3 H 2 /m 3 reactor/d. Overall energy efficiencies decreased from 62 ± 7% with a synthetic effluent in the acclimated reactors to 44 ± 16% with a corn stover effluent. Pre-acclimation of the inoculum to the single substrates improved performance, and using a cellobiose fermentation effluent gave the most promising results among the actual fermentation effluents tested in this study.

4 iv TABLE OF CONTENTS LIST OF FIGURES...v LIST OF TABLES...vii ACKNOWLEDGEMENTS...viii CHAPTER 1 Introduction...1 CHAPTER 2 Literature Review...4 CHAPTER 3 Hydrogen Production from Cellulose Fermentation End Products Using Microbial Electrolysis Cells Introduction Materials and Methods Reactor Set-up MEC Inoculation and Operation Gas Analysis Calculations Hydrogen Yield and Production Rate Energy Recovery Results Hydrogen Production Hydrogen and Methane Yields Maximum Hydrogen Production Rates Hydrogen Recovery and COD Removal Electrical Energy Efficiency Overall Energy Recoveries Discussion Effect of Acclimation of Inoculum Predicted Behavior Actual Effluents Methanogenesis Comparison with Results Found in the Literature...36 CHAPTER 4 Conclusions...38 CHAPTER 5 Future Work...39 APPENDIX A MFC Results for Individual Substrates...40 APPENDIX B Gas Production and Analysis for Chapter APPENDIX C Results from Experiments to Reduce Methane Production...47 REFERENCES...48

5 v LIST OF FIGURES Figure 2.1: MEC standard reactor set up with the yellow circle representing the substrate, the green shapes on the anode the micro-organisms and the white circles on the cathode hydrogen bubbles produced [from 38]...7 Figure 3.1: MEC reactor with an 8 cm gas collection tube, an ammonia-treated brush anode (left), and a platinum carbon-cloth cathode (right)...12 Figure 3.2: MEC reactor hooked up to a power supply, a respirometer and gas bag and its computer interface...14 Figure 3.3: Gas production over the first 4 batches of synthetic fermentation effuent for a pre-acclimated reactor (A) and a non-acclimated reactor (B) Figure 3.4: Gas composition over the first 4 batches with the synthetic fermentation effluent for a pre-acclimated reactor (A) and a non-acclimated reactor (B) Figure 3.5: H, CH 2 4, and CO 2 yields for single substrates and fermentation effluents (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6)...24 Figure 3.6: Hydrogen maximum production rate for single substrates and fermentation effluents (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6)...25 Figure 3.7: Electrical efficiencies for single substrates and fermentation effluents (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6)...28 Figure 3.8: Overall efficiencies for single substrates and fermentation effluents (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6)...29 Figure A.1: Voltage output for cubic MFCs fed with 1g/L of acetic acid (A)...40 Figure A.2: Voltage output for cubic MFCs fed with 1g/L of ethanol (B) and succinic acid (C)...41 Figure A.3: Voltage output for cubic MFCs fed with 1g/L of lactic acid (D) and formic acid (E)...42 Figure B.1: Gas production (A) and analysis (B) for cellobiose fermentation effluent in MEC...43 Figure B.2: Gas production (A) and analysis (B) for corn stover fermentation effluent batch 1 in MEC....44

6 vi Figure B.3: Gas production (A) and analysis (B) for corn stover fermentation effluent batch 2 in MEC Figure B.4: Gas production (A) and analysis (B) for corn stover fermentation effluent batch 2 diluted in MEC....46

7 vii LIST OF TABLES Table 3.1: Concentration of individual substrates in the synthetic fermentation effluent...15 Table 3.2: Substrates characteristics...19 Table 3.3: Gas production and composition for different fermentation effluent in either non-acclimated or acclimated reactors (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6)...23 Table 3.4: Hydrogen recovery and COD removal (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6)...27 Table 3.5: Comparison between the results with synthetic effluent and the predicted behavior in MEC (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6)...31 Table 3.6: Comparison of COD removal for fermentation effluents with total COD (as measured) and with soluble COD ( scod* = COD contained in the synthetic fermentation effluent)...34 Table 3.7: Hydrogen recoveries calculated from the hydrogen produced (RH 2 ) and adding the hydrogen contained in the methane produced ( RH *) Table 3.8: Comparison of this study s results for acetic acid, lactic acid and synthetic fermentation effluents with data from Cheng et al., 2007 [22]...36 Table 3.9: Comparison between photo-fermentation [17] and electrohydrogenesis of acetic acid (this study)...37 Table C.1: Gas production and composition in MEC with lowering of the ph...47 Table C.2: Gas production and composition in MEC with increasing of the ph...47

8 viii ACKNOWLEDGEMENTS Many people have helped and guided me during my graduate studies at Penn State University. First, I would like to thank Dr Bruce E. Logan for his support and his help in making my graduate studies possible and a very positive and exiting experience in his research group. I would also like to thank Dr Jay Regan and Dr Rachel Brennan for accepting to serve on my committee. I want to thank all the researchers in Dr Logan s lab for their help and support in my research over the past year. In particular, I thank Doug Call for introducing me to the microbial electrolysis cell technology and patiently answering all my questions, Rachel Wagner and Dr Shaoan Cheng for their help in the course of my experiments and David Jones for constant technical support. I would like to thank Dr Tim Vogel and Dr Jean-Michel Monier who introduced me to microbial fuel cells. They encouraged and helped me find a career path and gave me the means to follow it by putting me in contact with Dr Logan. My parents and family have been there for me throughout this experience and I could not have done this without their constant love and support. I would like to thank them for believing in me and helping me realize this dream of studying in Penn State University. Thank you to Valerie Watson, Ahmed Moustafa, Hengjing Yan, Doug Call, Anne Savigny for reviewing my thesis and their encouragements. The work in this thesis was funded and done in partnership with the National Laboratory of Renewable Energies and especially the research group of Dr Pin-Ching Maness. A special thank you to Shivegowda Thammannagowda for the sending me the fermentation effluents.

9 1 CHAPTER 1 Introduction Energy is one of the key issues in our modern world as Donald F. Fournier and Eileen T. Westervelt write in a report for the US Army: The days of inexpensive, convenient, abundant energy sources are quickly drawing to a close. [1]. Primary energy consumption increased from 347 Quadrillion (10 15 ) BTU worldwide in 1980 to 463 Quadrillion BTU in 2005 with the United State consumption accounting for more than 20% [2]. This increase is predicted to continue in the future up to 695 Quadrillion BTU by 2030 [3]. Fossil fuels are the main source of energy used today with 85% of the United States energy produced from fossil fuels in 2007 [4]. Oil is especially needed for transportation which accounts for 29 Quadrillion BTU in the 102 Quadrillion BTU total energy consumption in the US [5]. However, oil availability is finite and the world s peak production is expected to be reached in the next couple of decades with predictions for peak production ranging from 2008 to 2040 based on different consumption growth rates [6]. OPEC is the Organization of Petroleum Exporting Countries and includes only 13 countries. In addition to the foreign dependency this creates, the use of fossil fuels leads to large and increasing carbon dioxide emissions in the atmosphere (28 Billion metric tons of carbon dioxide in 2005 a 10,000 times increase since 1980) [7]. Climate change is not only a reality but a very pressing problem with ecosystems survival at stake [8]. This is slowly being tackled by different countries with such agreements as the Kyoto protocol which has been signed by 37 countries to globally reduce emissions of carbon dioxide by 5% (compared with the emissions in 1990) and encourages a shift of the fossil fuel based economy to a more sustainable and renewable energy based economy. Already countries are trying to diversify their energy

10 2 resources: for example, the United States share of renewable energy is up to 7% in 2007 with 53% of the renewable energy coming from biomass [4]. A hydrogen-based economy is being investigated as a potential sustainable alternative to a fossil fuel based economy [1, 9-11]. Hydrogen has the highest energy content per weight for fuels with 120 MJ/kg compared with 44 MJ/kg for gasoline. It is therefore a good candidate as the energy carrier of the future. Hydrogen fuel cell automotive engines are being developed to replace gasoline in automotive applications [12]. Hydrogen can be used as fuel in either combustion engines or fuel cells with only water as the product. The major issues with hydrogen as an energy carrier are the costs of production and storage. Hydrogen is the most abundant element in the universe but it must be produced from hydrogen-containing compounds such as biomass, water or fossil fuels. Currently, the most cost effective method to produce hydrogen is from natural gas by steam reforming which releases green house gases [9-11]. Renewable methods to produce hydrogen include water electrolysis and biological processes such as biophotolysis and dark- or photo-fermentation [13-14]. Water electrolysis requires electricity input and its energy efficiency is typically 56-73% [15]. Biomass is the substrate for micro-organisms in bio-hydrogen production. Dark-fermentation can use various types of biomass including agricultural wastes and cellulose to produce hydrogen. However, this process recovers at best 23-25% of the hydrogen present in the substrate [26, 27] and leaves many hydrogen-containing components as end-products (for example acetic acid, butyric acid and ethanol) [16-18]. In 2005, a new process was discovered to produce hydrogen from various organic substrates called electrohydrogenesis [19]. In a microbial electrolysis cell (MEC), bacteria on the anode oxidize the organic matter in the effluent and send electrons through the circuit to the anaerobic cathode where hydrogen is formed using protons in the water with the help of a catalyst. This reaction needs an electrical input provided by a power source. However, the

11 3 efficiency relative to the electrical input can reach over 400% [20] and the theoretical electrical input needed is smaller than for water electrolysis (0.14V vs 1.23V with water electrolysis) [20, 21, 15]. Hydrogen can be produced in MECs from many different substrates including acetic acid, butyric acid, lactic acid, glucose, cellulose, and waste-water [21-23]. This technology could be an alternative method to produce an increasingly needed fuel (hydrogen) in a renewable manner while removing compounds from waste effluents in an energy efficient way [24, 25]. Although MECs can produce hydrogen from cellulose, the efficiency and hydrogen production is lower than with volatile fatty acids (VFAs) [22]. The aim of this research was to produce hydrogen in a membraneless single chamber MEC from cellulose fermentation end products by Clostridium thermocellum: acetic acid, ethanol, succinic acid, lactic acid, and formic acid. As most of these substrates had been used before separately to produce either electricity in MFCs [21, 48-50] or hydrogen in MECs [20, 36, 44], it is believed that a mixture of those VFAs could produce hydrogen. We wanted to show that using the mixture of the fermentation end products could produce hydrogen at a higher production rate and hydrogen recovery compared with directly using cellulose as in Cheng et al. s study [22]. In addition to using an unacclimated inoculum, we tested inoculum from mixed cultures enriched to the various single substrates found in the cellulose fermentation end products. Given the MFC microbial community enrichment processes already tested in the literature [51-52], we believed that cultures acclimated to the different single substrates would be a better inoculum which would increase performance of the MECs fed with the fermentation effluent. Showing that hydrogen production in an MEC is possible by using cellulose fermentation end products would improve the efficiency of biohydrogen production from cellulose using a 2-process system which would combine a darkfermentation system with an MEC system. This would be an effective way to further process fermentation effluent while still producing hydrogen.

12 4 CHAPTER 2 Literature Review Bio-hydrogen production from biomass has received considerable attention recently because it is a carbon neutral way of producing hydrogen from renewable resources and/or waste with simultaneous biodegradation of the biomass. Cellulose is one of the most abundant biopolymers on earth and the main component of plant biomass. It is available in many humancreated wastes such as straw, wood-chips, grass residue, and paper waste. Cellulosic material is the largest source of hexose and pentose sugars and can be degraded in nature by microorganisms. Using cellulose and cellulosic materials to produce energy and especially hydrogen could be a sustainable and cheap substrate especially if the cellulose is recovered from waste materials. There are multiple ways of producing hydrogen from biomass and cellulose using microorganisms with no external energy input, such as biophotolysis, photo-fermentation, and darkfermentation [13-14, 16-18]. Dark fermentation is currently one of the most promising of those technologies. The hydrogen production rate is highest, compared with the other methods, with around 2.4 to 3 mol H 2/mol glucose [26-27] or a maximum of 6 mol H2/mol sucrose [13] or up to 75% hydrogen recovery based on 4 mol H 2 /mol glucose [26-27]. Bacteria such as Bacilli or Clostridia can produce hydrogen by dark fermentation using glucose, sucrose, waste containing sugars, or cellulose [13-14, 17]. Dark-fermentation occurs when anaerobic bacteria are grown on carbohydrate-rich substrates[17]. With glucose as the model hexose representing a typical carbohydrate, the reactions which occur in dark fermentation are for example (2-1) or (2-2):

13 C CO (2.1) 6H12O6 H 2O 2CH 3COOH + 4H with acetic acid as the main by-product, or C CO (2.2) 6H12O6 H 2O CH 2CH 2CH 2COOH + 2H with butyric acid as the main by-product [14]. The highest theoretical yields of hydrogen occur when most of the substrate is fermented using the pathway producing acetic acid as the byproduct [13, 14]. To improve the yields of hydrogen produced from dark-fermentation, a variety of ways have been investigated including the optimization of ph [27-31], hydraulic retention time [32], pre-treatment of the substrate [26, 28-29], substrate concentration [17, 33-34], substrate composition [30] and the use of pure cultures [29, 34-36]. In particular, the genus Clostridium has been the focus of many studies [27, 32-33]. This genus has been found to be present in most mixed cultures producing hydrogen through dark-fermentation [17, 29]. Clostridia are sporeformers and can therefore survive pre-treatment (such as heat-treatment) of the substrate and/or inoculum before dark-fermentation. Different Clostridium species have been used as pure cultures to produce hydrogen with yields up to 3 mol H 2 /mol glucose [27]. Levin et al. [11] showed that Clostridium thermocellum could produce a hydrogen yield of 1.6 mol H 2 /mol glucose on average with by-products of acetate, ethanol, lactate and formate. Lin et al. [27] and Datar et al. [26] obtained similar high efficiencies in terms of hydrogen recovery with Clostridium beijerinckii L9 and corn stover biomass (70%) or with anaerobic sludge inoculum and glucose (71-75%). Their fermentation effluents contained by-products which included acetate, butyrate, ethanol, lactate and formate. These fermentation by-products could still react further to produce more hydrogen. If the full conversion of the carbohydrates to hydrogen and carbon dioxide took place, the theoretical hydrogen yield could be increased from 4 mol H 2/mol glucose by fermentation to 12 mol H2/mol glucose. However, the chemical reactions where fermentation end-products are converted into

14 6 hydrogen are not spontaneous as is shown in the equations (2.3) to (2.7). Gibbs free energy, at standard conditions of temperature and pressure (P=1 atm, T= 25C) and ph=7, is positive for acetate, ethanol, lactate, succinate and formate [35]. CH 2 + 3COO + 4 H 2O 2HCO3 + 4H + H (2.3) 0 ΔG = kj r + CH 3 CH 3OH + 5H 2O 2HCO3 + 6H 2 + 2H (2.4) 0 ΔG = kj r + C3 H 5O3 + 6H 2O 3HCO3 + 6H 2 + 2H (2.5) ΔG r 0 = kj 2 + C4 H 6O4 + 8H 2O 4HCO3 + 10H 2 + 3H (2.6) ΔG 0 = kj r HCOO + H + 2O HCO3 H 2 (2.7) ΔG 0 =+1.3 kj r These reactions can only occur if a small input of energy is given to the system. Studies have been made on coupling photo-fermentation (using solar energy to power the fermentation of the fermentation end products by algae [13,14,16-18]) with dark-fermentation to increase the hydrogen yield and use the dark-fermentation end-products [13,17,18]. Manish et al. [18] showed a low overall energy efficiency of only 27% if the waste materials are not used to produce heat. Photo-fermentation has a lower hydrogen production rate maximum 0.64 m 3 H /m 3 2 /d compared with up to m 3 H /m 3 /d with dark fermentation of glucose [17]. 2 Another process which could be used to produce hydrogen from these fermentation end products is electrohydrogenesis in microbial electrolysis cells (MECs). MECs, which are also known as biocatalysed electrolysis cells (BECs) or bioelectrochemically assisted microbial reactor (BEAMR), have been developed for a couple of years based on the design of microbial

15 7 fuel cells (MFCs) [21]. MECs are based on electrohydrogenesis [20, 21, 36-37] where electrons released by the bacteria at the anode are combined with protons at the cathode to produce hydrogen gas instead of electricity as in an MFC. At the anode, the substrate is oxidized by the bacteria on a biofilm which releases protons and electrons. For example, for acetate as a model substrate, the anode reaction is: + CH 3 COO + 4H 2O 2HCO3 + 9H + 8e (2.8) At the cathode which is kept anaerobic, protons and electrons combine to produce hydrogen: + 8H + 8e 4H 2 (2.9) However, as shown previously (2.3), this process is not spontaneous and MEC reactors require a power input to make the overall reaction thermodynamically favorable. This is done by the input of a voltage via a power supply (Figure 2-1). Figure 2.1: MEC standard reactor set up with the yellow circle representing the substrate, the green shapes on the anode the micro-organisms and the white circles on the cathode hydrogen bubbles produced [from 38]

16 8 The minimum external voltage or equilibrium voltage that should be applied to convert biomass into hydrogen can be calculated from the reaction s Gibbs free energy [36, 39-40], n the number of moles exchanged in the reaction and F Faraday s constant (96,485 C/eq) using the following equation: ' Δ E rg0 ap = (2.10) nf ( kj. mol) E ap = = 0. 14V (acetate reaction: (2.3)) (2.11) 8 ( mol / eq) ( C / eq) Therefore the minimum potential that should be applied to an MEC fed with acetate in order to observe the production of hydrogen is +0.14V. However, over-potentials both at the anode and cathode mean in practice that a minimum of 0.2V is needed to observe hydrogen production in such a system [20, 21, 23, 36, 39]. There have been only a few papers on MECs since the discovery of this technology in 2005 by Liu et al. [19]. Most of the studies were done in two chambers reactors with a membrane. Liu et al. [19] used bottle reactors, a proton exchange membrane, acetate as substrate and waste water inoculum and reported over 90% hydrogen recovery. Rozendal et al. [40] obtained 57% hydrogen recovery with a system using a cation exchange membrane (CEM), pre-acclimated inoculum, acetate as the substrate and electrodes pressed against the membrane. Ditzig et al. [23] showed that hydrogen production in an MEC using waste water as the inoculum and the substrate was possible although at lower recovery rates (42%). Cheng et al. [22] tested acetic acid, lactic acid, butyric acid, valeric acid, propionic acid, glucose, and cellulose in two-chambers MECs with an applied voltage of 0.6V, and obtained hydrogen recoveries between 67% and 91%. Most MEC reactors have used membranes to separate the cathode and anode chambers [19, 21-23, 39-44]. The main reason behind this was to avoid hydrogen leaking into the anode chamber where it could potentially be used by hydrogenotrophic micro-organisms [21, 36, 39]. A

17 membrane also kept the hydrogen production separate from the carbon dioxide produced at the anode therefore ensuring higher hydrogen gas purity at the cathode. However, Rozendal et al [39] showed that the presence of a membrane led to a high ph gradient across the membrane. Usually a CEM membrane was used because of higher hydrogen production rates and better overall efficiencies than with an anion exchange membrane (AEM) and cations crossed the membrane instead of protons lowering the ph at the anode and increasing the ph at the cathode. Also a major disadvantage of the MEC with membranes is the cost of the membrane can be quite high [20-21, 37-39]. Recently, two published studies have investigated the removal of membranes in MEC reactors [20, 37]. Call et al. [20] showed that higher hydrogen production rates and similar overall efficiencies could be obtained with a single chamber membraneless reactor, with up to 3.12 ± 0.02 m 3 H / m 3 2 reactor/d with an applied voltage of 0.8V, and 78% overall energy efficiency. Hu et al. [37] also proved that a membraneless MEC system could produce hydrogen with 0.53 m 3 H / m 3 2 reactor/d with an applied voltage of 0.6V. The main issue with these systems is the decrease in the gas s purity: hydrogen is mixed with carbon dioxide. Furthermore, methanogens can grow and remove some of the hydrogen being produced because no membrane separates the anode chamber from the cathode chamber, [20-21, 36, 39]. Call et al. [20] and Hu et al. [37] showed that by exposing the system to air and oxygen in between batches and using pure cultures methane production could be reduced or removed. MEC studies generally use acetate as model substrate for fermentation end product [19-23, 37-44], or glucose as a fermentable substrate [22, 44]. Tartakovsky et al. [44] showed that glucose was fermented in the MEC to produce volatile fatty acids which were then used as substrate by the biofilm. Cheng et al. [22] tested different fermentable and non-fermentable substrates (glucose, cellulose, acetic acid, lactic acid and others) in a two-chamber reactor with AEM and a graphite granule anode. They obtained lower results for fermentable substrates in 9

18 terms of hydrogen recovery: 71% and 68% for glucose and cellulose instead of 91% for acetic and lactic acids. Overall energy efficiencies were 64% to 63% with glucose and cellulose, compared with 82% for acetic and lactic acids. Cellulose had the lowest hydrogen production rate with 0.11 m 3 H 2 /m 3 /d compared with over 1 m 3 H /m 3 2 /d for acetic acid, lactic acid and glucose. This is consistent with MFC studies comparing glucose and acetate as the substrates where glucose produced a lower power density 9.8 mw/m 2 compared with 360 mw/m 2 with acetate [45]. Cellulose in an MFC can produce 143 mw/m 2 with a defined binary culture [45], or 100 mw/m 2 with a mixed culture in the presence of cellulase [46]. In MFC studies, formic acid and ethanol have also been successfully used as substrates [48-49] and could therefore potentially be used as substrates in MEC systems since the same reaction occurs at the anode as in an MFC if the correct potential is applied to the system. The microbial community in an MEC has so far been studied by only two papers. Liu et al. [41] found the presence of Shewanella and Pseudomonas on the anode, while Chae et al. [42] found Pelobacter and Geobacter, and observed a significant decrease in the diversity of the microbial community compared to the microbial community in MFCs. These results are consistent with microbial community analyses in MFCs which have been studied more extensively [21, 50]. Microbial community varies from study to study based on inoculum, conditions and substrate. Different substrates enrich the microbial biofilm on the anode with different micro-organisms but with major common species such as Pseudomonas, Shewanella or Geobacter (which are exoelectrogens [21, 50]). For example, Ha et al. s [49] study of biofilms using acetate or formate as the substrate showed some differences in the communities after enrichment. They also showed that MFC performance depended on inoculum. Kim et al. [48] enriched MFCs with ethanol as the substrate, found Protobacteria but only one Geobacter clone and no Shewanella in the biofilm and showed that using inoculum from an already enriched reactor reduced the enrichment time for an MFC. 10

19 11 CHAPTER 3 Hydrogen Production from Cellulose Fermentation End Products Using Microbial Electrolysis Cells 3.1 Introduction Fermentation of cellulose can produce hydrogen although fatty acids such as acetic acid, succinic acid, lactic acid, and formic acid as well as ethanol remain in the effluent. Microbial electrolysis cells (MECs) are a new method of producing hydrogen that has been demonstrated using different substrates such as acetic acid, lactic acid, and cellulose. MECs are related to microbial fuel cells (MFCs) which produce electricity instead of hydrogen and that have been studied more extensively. Different substrates can be used in MFCs including fermentation end products or fermentable substrates. For this thesis, my main hypothesis was that cellulose fermentation effluent containing a mixture of acetic acid, ethanol, succinic acid, lactic acid, and formic acid could be used in MECs to produce hydrogen at production rates, hydrogen recoveries, and overall energy efficiencies comparable to, or better than those in Cheng et al. s study [22] with cellulose as a substrate in MECs. They obtained 0.11 m 3 H 2 /m 3 /d, 68% H 2 recovery, and 63% overall energy efficiency. My secondary hypothesis was that inoculation of the MECs with a mixture of cultures acclimated to individual substrates would improve performance.

20 Materials and Methods Reactor Set-up The MEC used in this study was a single-cell cubic reactor as designed by Call et al. [20]. This reactor is a polycarbonate 4 cm cubic reactor with a cylindrical chamber (4 cm long, 3 cm diameter, empty bed volume of 28 ml) (Figure 3.1). The anode was an ammonia-treated graphite brush (25 mm diameter x 25 mm length; 0.22 m 2 surface area; fiber type: PANEX K, ZOLTEK), with a specific surface area of 18,200 m 2 /m 3 and porosity of 95% [20], placed in the center of the reactor s cylindrical chamber. The cathode was wet-proofed (30%) carbon cloth (type B; E-TEK), with a surface area of 7 cm 2 and a platinum (Pt) catalyst (0.5 mg/cm 2 ), placed on the opposite side of the reactor. Figure 3.1: MEC reactor with an 8 cm gas collection tube, an ammonia-treated brush anode (left), and a platinum carbon-cloth cathode (right)

21 13 The hydrogen gas produced at the cathode was collected into a gas collection tube (anaerobic glass tube 8 cm high, 1.6 cm in diameter and 15 ml in volume) glued on top of the cubic reactor. The gas-collection tube was closed by a butyl rubber stopper and an aluminum crimp cap. The MEC reactor was connected to a respirometer (AER-200; Challenge Environmental) [20, 22] which recorded continuously the gas production over a batch cycle (Figure 3.2). The gas produced was then collected in a gas bag (0.5 L capacity; Cali-5-Bond, Calibrated Instruments Inc.). Before each cycle, the anode was exposed to air for 20 to 30 minutes to limit the growth of methanogens [20]. The MEC reactors were sparged with ultra high purity (UHP) nitrogen (99.998%) [20] for 15 to 20 minutes to remove O 2 from the reactor. The gas bags were also sparged with UHP N 2 and vacuum sealed between each batch cycle. All batch experiments were conducted in a temperature controlled room (30ºC) and the pressure was assumed constant and equal to 1 atm. A constant voltage of 0.5 V was applied to the MEC reactor with an external power source (model 3645 A; Circuit Specialists, Inc.) with the positive lead connected to the anode and the negative lead serially connected to a 10 Ω resistor and then to the cathode. A multimeter (model 2700; Keithley Instruments, Inc.) [23] was used to record the voltage across the resistor (Figure 3.2). Current was calculated using Ohm s law (I=V/R).

22 14 Computer Interface Keithley Power Supply 10 Ω Resistor H 2 Gas Bag Respirometer Figure 3.2: MEC reactor hooked up to a power supply, a respirometer and gas bag and its computer interface MEC Inoculation and Operation The brush anodes were first enriched in single-chamber cubic MFCs with flat cathodes [53]. The MFCs were fed a 1:1 ratio of inoculum and substrate-buffer solution. When the reactor s voltage output exceeded V, the inoculum was omitted from subsequent cycles. The buffer solution contained a 50 mm PBS phosphate buffer (4.58 g/l Na 2 HPO 4, and 2.45 g/l NaH2PO 4 H2O, ph=7.0) and nutrient solution (0.31 g/l NH4Cl; 0.13 g/l KCl; trace vitamins and minerals [53]). The substrate was either a synthetic mix simulating a cellulose fermentation effluent from Clostridium thermocellum (Table 3.1): 1.56 g/l acetic acid, 0.64 g/l ethanol, 0.66

23 15 g/l succinic acid, 0.16 g/l lactic acid, and 0.03 g/l formic acid (which will be referred to as synthetic fermentation effluent in the rest of this thesis) or one of the following pure substrates: acetic acid (1 g/l), lactic acid (1 g/l), formic acid (1 g/l), succinic acid (1 g/l) or ethanol (1 g/l). Table 3.1: Concentration of individual substrates in the synthetic fermentation effluent gcod/g substrate concentration (g/l) concentration (mm) % (mm) acetic acid ethanol succinic acid lactic acid formic acid The fermentation of cellulose by Clostridium thermocellum was performed at NREL by Dr. P.-C. Maness. NREL also provided the composition used to make the synthetic fermentation effluent, and samples of actual fermentation effluents. The MFCs were inoculated with a solution of 1:1 buffer-substrate solution and inoculum where the inoculum was either unacclimated (primary clarifier effluent from the Penn State University s Wastewater Treatment Plant) or preacclimated (combination of equal volumes of suspended mixed culture of bacteria from the MFCs fed with the pure single substrates at 1g/L for about 2 months: see Appendix A for MFC data). After a reactor gave a reproducible maximum voltage for at least 3 cycles, the anode was considered acclimated and was transferred to an MEC. The MECs were fed the same buffer-substrate solution: 50 mm PBS buffer and synthetic fermentation effluent (conductivity=8; ph=7) or each single substrate at 1 g/l. MECs operated in duplicate. The MEC reactor with the anode acclimated to the synthetic mixed substrate from a pre-acclimated inoculum were then fed with effluent from cellulosic material fermentation: cellobiose (conductivity=8 ms; ph=7.3) or corn stover (conductivity=7 ms; ph=7.2) from 2 different batches provided by the National Renewable Energy Laboratory (NREL). Effects of

24 16 dilution of the fermentation effluents were studied for one of the corn stover effluent which was diluted two-fold with PBS. The end of the batch was determined by the end of the gas production and the sharp decrease in current production. New cathodes were used for each new type of substrate. Internal resistances were obtained by electrochemical impedance spectroscopy (EIS) using a potentiostat (model PC4/750, Gamry Instruments Inc.) [20]. Total chemical oxygen demand (COD) was measured at the beginning and end of each batch (TNT plus COD Reagent; HACH Company) Gas Analysis Gas chromatography was used to analyze the gases both in the anaerobic tube headspace and the gas bag [23]. Samples (200 μl) were taken with a gas-tight syringe (250 μl, Hamilton Samplelock Syringe) in duplicate for the head space of the reactors and in triplicate for the gas bags. N 2, H 2, and CH 4 were analyzed with one gas chromatograph (GC) (argon carrier gas; model 2610B; SRI Instruments), and CO 2 with a separate GC (helium carrier gas; model 310, SRI Instruments). Standards were prepared with pure N 2, H 2, CH 4, and CO 2 samples. N2 served as a diluting gas therefore it was removed from the calculations to find the concentrations of H 2, CH4, and CO produced by the system. 2

25 Calculations The performance of the reactors was measured and compared on the basis of hydrogen recovery, hydrogen production rate, and the energy recovery (electrical and overall energy) as described in Call et al. [20] and Logan [21, 36] Hydrogen Yield and Production Rate Hydrogen yield was calculated based on the maximum hydrogen production from COD consumption. This theoretical hydrogen yield is calculated in moles hydrogen produced, n th as follows: n th 2 LΔCOD = υ M O2 (3.1) where υ L = 28mL is the volume of liquid of the reactor, ΔCOD (gcod/l) is the change in COD over a batch cycle, M O2 is the molecular weight of the oxygen (32 g/mol) and 2 is the ratio of electron equivalent in oxygen to the electron equivalent in hydrogen [36]. The number of moles of hydrogen that can be recovered based on the current produced over one batch can be calculated: n CE t Idt t= 0 = (3.2) 2F where I=V/R ex is the current calculated from the voltage measured across the 10 Ω resistor with Ohm s law, dt is the time interval between two data collection points (in this case, 20 minutes), 2 the number of mole of electrons used per mole of hydrogen produced, and F is Faraday s constant (96,485 Coulombs/mole electron). The Coulombic hydrogen recovery, r, is: CE

26 n = C (3.3) CE r CE = nth E 18 where C is the Coulombic efficiency (i.e., the number of electrons recovered in the circuit over E the number of electrons theoretically available from the substrate consumed). The cathodic hydrogen recovery (r cat ), or the moles of hydrogen produced by the reactor over the moles of hydrogen that could have been produced from the current, is given by: n = (3.4) H 2 rcat nce where n is the number of moles of hydrogen produced by the system during a batch cycle. The H2 overall hydrogen recovery was calculated as: n 2 = (3.5) n H 2 RH th The maximum volumetric hydrogen production rate Q in m H/ m reactor/ d is calculated as: 5 Q = I V Tr cat (3.6) where I (A/ m ) is the volumetric current density averaged over 4 hours of maximum current V production for a batch cycle normalized by the liquid volume of the reactor, T is the temperature in Kelvin and 3.68*10 is a constant for unit conversion assuming the ideal gas law with hydrogen produced at 1 atm Energy Recovery the resistor, is: The electrical energy added to the system by the power source, accounting for losses in

27 19 W E = n 1 2 ( IE Δt I R Δt) (3.7) ap ex where I is the current calculated as before, E ap is the voltage applied to the reactor, R ex is the external 10 Ω resistor, and Δt is the time interval between two data points (20 minutes). The amount of energy provided by the substrate was calculated based on heats of combustion as: W = ΔH * n (3.8) S S S where ΔH s is the heat of combustion of the substrate (see Table 3.2 for specific values) and ns is the number of moles of substrate consumed in one batch cycle based on COD removal (n th / b H2,s, with b H2,s the stoichiometric number of moles that can be produced per mole of substrate see Table 3.2 for values). Table 3.2: Substrates characteristics substrate (mole H 2 / mole of substrate) ΔHs (kj/mole) acetic acid ethanol succinic acid lactic acid formic acid synthetic fermentation effluent b H2/s The substrate characteristics for the synthetic effluent were calculated based on the molar percentage of each single substrate in the synthetic effluent: 54% acetic acid, 29% ethanol, 12% succinic acid, 4% lactic acid and 1% formic acid (Table 3.1). The amount of energy recovered as hydrogen is: where ΔH W = ΔH n (3.9) H 2 H 2 H 2 H2 is kj/mol the heat of combustion of hydrogen. The energy efficiency η relative to the electrical input is the energy recovered as hydrogen over the electrical energy input and can be over 100% because it does not take into account the energy input coming from the substrate: E

28 20 W η H E = 2 (3.10) WE The overall energy efficiency which takes into account both the electrical and the substrate energy input is calculated as follows and is always under 100%: W H 2 η E+ S = (3.11) WE + WS 3.4 Results Hydrogen Production Hydrogen production was increased and stabilized by using pre-acclimated cultures for the inoculation of the reactors fed with synthetic effluent (Figure 3.3). 110 ± 10 ml of gas was produced per batch containing an average of 79.1 ± 2.5% of hydrogen (Table 3.3). The preacclimated reactor produced between 120 and 99 ml of gas for the first 4 batches, and stabilized at 101 ± 4 ml of gas produced after the first batch cycle (Figure 3.3 A). The non-acclimated reactor s gas production showed a greater variability, decreasing from 159 to 89 ml over the first 4 batches (Figure 3.3 B).

29 21 gas production (ml) Time (days) (A) gas production (ml) Time (days) (B) Figure 3.3: Gas production over the first 4 batches of synthetic fermentation effuent for a preacclimated reactor (A) and a non-acclimated reactor (B). When the inoculum was pre-acclimated to the different single substrates, gas composition was stable at 83% to 75% hydrogen, and 8% to 15% methane (Figure 3.4 A). This was an improvement over the non-acclimated reactors where the gas composition showed a decrease in hydrogen from 86% to 62% of the total gas production, combined with an increase in methane production from 1% to 29% of the total gas production (Figure 3.4 B).

30 H2 CH4 CO2 (A) gas produced (ml) batch gas produced (ml) H2 CH4 CO2 (B) batch Figure 3.4: Gas composition over the first 4 batches with the synthetic fermentation effluent for a pre-acclimated reactor (A) and a non-acclimated reactor (B). Actual fermentation effluents produced comparable or slightly lower amounts of gas per batch cycle than when the reactor was fed with synthetic effluent (Table 3.3). Cellobiose fermentation effluent produced a comparable amount of gas to the synthetic effluent (105 ± 17 ml vs. 110 ± 10 ml synthetic effluent), but it contained a lower proportion of hydrogen (69 ± 4% vs. 79 ± 3% synthetic effluent). The corn stover fermentation effluents produced 97 ± 16 and 90 ±

31 23 29 ml of gas compared with 110 ± 10 ml produced with the synthetic effluent. The hydrogen proportion was about 10% lower and more variable than when synthetic effluent was used: 69 ± 6% and 66 ± 8% compared with 79 ± 3%. This is closer to the hydrogen ratio obtained with the cellobiose effluent 69 ± 4%. The corn stover diluted by 50% gave better hydrogen ratio results, with 73 ± 5% of 49 ± 11 ml of gas produced. All fermentation effluents, apart from the corn stover diluted by 50%, showed decreasing gas production for each subsequent batch. See Appendix B for additional figures. Table 3.3: Gas production and composition for different fermentation effluent in either nonacclimated or acclimated reactors (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6) Fermentation effluent Total gas Gas composition (%) production (ml) H2 CH4 CO2 Synthetic (Acclimated) 110 ± ± 3 10 ± 2 11 ± 1 Corn stover 1 97 ± ± 6 12 ± 3 19 ± 3 Corn stover 2 90 ± ± 8 15 ± 5 19 ± 3 Corn stover 2 diluted 50% 49 ± ± 5 12 ± 4 15 ± 1 Cellobiose 105 ± ± 4 16 ± 4 14 ± Hydrogen and Methane Yields Hydrogen yields (ml H 2 /g COD consumed) varied between 1400 ± 170 and 700 ± 260 ml H 2 /g COD depending on the substrate (pure single substrate, synthetic effluent or real fermentation effluent) and on the type of reactor (pre-acclimated or non-acclimated) (Figure 3.5). Acetic acid was the best pure substrate for the production of hydrogen, with 1400 ± 170 ml H 2 /g COD compared to hydrogen production between 1100 ± 130 ml H 2 /g COD and 810 ± 260 ml H /g COD obtained with other pure substrates. 2

32 H2 CH4 CO2 ml gas/g COD Acetate Ethanol Succinate Lactate Formate Non-Accl substrates Accl Cellobiose Corn Stover 1 Corn Stover 2 Corn stover 2 dil Figure 3.5: H 2, CH 4, and CO2 yields for single substrates and fermentation effluents (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6) Pre-acclimation of the inoculum increased and stabilized the hydrogen yield at 980 ± 110 ml H 2 /g COD, whereas non-acclimated reactors fed synthetic effluent yielded 800 ± 290 ml H 2 /g COD. Cellobiose fermentation effluent had the largest hydrogen yield of all the fermentation effluents, with 1000 ± 300 while corn stover effluent produced 750 ± 180 and 700 ± 260. The yield increased to 810 ± 260 when the corn stover effluent was diluted by 50%. Comparing the methane yields for the different substrates, reactors and fermentation effluents, we see that single substrates tended to produce less methane, with little to no methane for acetic acid, succinic acid and lactic acid. There was 49 ± 24 ml CH 4 /g COD with ethanol as substrate, and 54 ± 32 ml CH 4 /g COD with formic acid. Synthetic and real fermentation effluents produced on average more methane than the pure substrates between 120 ± 14 ml CH 4 /g COD for the diluted corn stover fermentation effluent to 240 ± 70 ml CH 4 /g COD for the cellobiose fermentation effluent.

33 Maximum Hydrogen Production Rates The maximum hydrogen production rate varied between 1.17 ± 0.07 m 3 H /m 3 2 reactor/d with acetic acid and 0.59 ± 0.21 m 3 H /m 3 2 reactor/d with synthetic effluent in the non-acclimated reactors. The highest maximum hydrogen production rates were obtained for acetic acid (1.17 ± 0.07 m 3 H 2 /m 3 reactor/d), ethanol (0.92 ± 0.14 m 3 H 2 /m 3 reactor/d) and lactic acid (1.11 ± 0.13 m 3 H /m 3 2 reactor/d). Formic acid and succinic acid had significantly lower rates of 0.62 ± 0.20 and 0.64 ± 0.02 m 3 H /m 3 2 effluent/d respectively. Pre-acclimation of the inoculum almost doubled the maximum hydrogen production rate from 0.59 ± 0.21 m 3 H 2 /m 3 effluent/d in non-acclimated reactors, to 1.06 ± 0.14 m 3 H 2 /m 3 effluent/d in pre-acclimated reactors fed with the synthetic effluent. 1.5 Q (m3/m3/d) Acetate Ethanol Succinate Lactate Formate Non-Accl Accl substrates Cellobiose Corn stover 1 Corn stover 2 Corn stover 2 dil Figure 3.6: Hydrogen maximum production rate for single substrates and fermentation effluents (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6)

34 When feeding the reactors the actual fermentation effluents, the first batch of corn stover fermentation effluent and the cellobiose effluent gave the best results with 1.0 ± 0.19 and 0.96 ± 0.16 m 3 H 2 /m 3 effluent/d. However, the second corn stover effluent s production rates were comparable to the single substrates rates with 0.71 ± 0.27 for the non-diluted effluent and 0.83 ±0.22 m 3 H /m 3 2 effluent/d with a 50% dilution of the effluent. The effluent was diluted to reduce the COD loading in the MECs but this did not significantly improve the results Hydrogen Recovery and COD Removal Hydrogen recovery was between 45 ± 17% for the second batch of corn stover effluent, and 66 ± 19% for cellobiose effluent (Table 3.4). Cellobiose fermentation effluent had the best hydrogen recovery and Coulombic efficiency of all the fermentation effluents with 66 ± 19% hydrogen recovery and 93 ± 23% Coulombic efficiency. Acclimation with the synthetic effluent improved the hydrogen recovery from 51 ± 19% for the non-acclimated reactors with synthetic substrates to 63 ± 7%. This behavior is consistent with an improvement in the cathodic recovery from 49 ± 16% to 86 ± 7%, although the coulombic efficiency decreased from 108 ± 22% to 73 ± 3%. Corn stover effluent had the lowest hydrogen recoveries between 45 ± 17% and 52 ± 17%. Dilution of the first corn stover batch effluent increased the hydrogen recovery by 7%. Overall, the fermentation effluents did not produce hydrogen recoveries as high as those for acetic acid, which had the best hydrogen recovery of 93 ± 11%. The best results were close to those obtained with succinic acid (69 ± 9%) and lactic acid (67 ± 25%). The worst results (for corn stover) were close to those obtained with ethanol and formic acid, with only 55 ± 10% and 52 ± 17%.

35 27 Table 3.4: Hydrogen recovery and COD removal (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6) substrate r CE (%) r cat (%) RH 2 (%) COD (%) Synthetic Non-Acclimated 107 ± ± ± ± 5 Synthetic Acclimated 73 ± 3 86 ± 7 63 ± 7 91 ± 2 Corn stover 1 62 ± ± ± ± 3 Corn stover 2 79 ± 2 57 ± ± ± 4 Corn stover 2 diluted by 50% 76 ± 3 69 ± ± ± 3 Cellobiose 93 ± ± 8 66 ± ± 10 Acetic acid 97 ± ± 7 93 ± ± 5 Ethanol 71 ± 8 76 ± 8 55 ± ± 2 Succinic acid 89 ± 8 77 ± 3 69 ± 9 89 ± 3 Lactic acid 81 ± ± ± ± 2 Formic acid 59 ± ± 5 52 ± ± 45 COD removal was high for the synthetic fermentation effluent (89 ± 5% and 91 ± 2%) and similar to the COD removal obtained with single substrates between 89 ± 3 for succinic acid and 93 ± 2% for ethanol. Cellulose fermentation effluents showed lower COD removal with only 59 ± 4% for corn stover to 65 ± 10% for cellobiose. Formic acid was the only single substrate with a very low COD removal: only 59 ± 45%.

36 Electrical Energy Efficiency The electrical energy efficiency only takes into account the electrical energy input and not the substrate energy input, thus its values are often above 100%. Electrical energy efficiency was highest for acetic and formic acid with respectively 280 ± 40% and 270 ± 20% in the pure single substrates and for the synthetic effluent in pre-acclimated reactors with 270 ± 20% (Figure 3.7). 350 energy recovered (%) Acetate Ethanol Succinate Lactate Formate Non-Accl Accl substrates Cellobiose Corn stover 1 Corn stover 2 Corn stover 2 dil Figure 3.7: Electrical efficiencies for single substrates and fermentation effluents (Standard deviations are calculated for duplicate reactors over 3 cycles: n=6) The pure substrates had similar electrical energy efficiencies that were well above 200% ranging from 240 ± 10% to 280 ± 40%. For the fermentation effluents, the highest electrical efficiency of 270 ± 20% obtained with the synthetic effluent in the pre-acclimated reactors, was comparable to the electrical efficiencies obtained with the pure single substrates. The lowest electrical energy efficiency was obtained for the synthetic effluent in the non-acclimated reactors (150 ± 50%). The actual fermentation effluents performed better, with the highest electrical