Electrochemically Assisted Biohydrogen Production from Acetate

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Sep 11, 2007 - Electrochemically Assisted Biohydrogen Production from Acetate†. Wen-zong Liu,‡ Ai-jie Wang,‡ Nan-qi Ren,‡ Xun-yu Zhao,‡ Li-hong Liu,‡ ...
Energy & Fuels 2008, 22, 159–163

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Electrochemically Assisted Biohydrogen Production from Acetate† Wen-zong Liu,‡ Ai-jie Wang,‡ Nan-qi Ren,‡ Xun-yu Zhao,‡ Li-hong Liu,‡ Zhen-guo Yu,‡ and Duu-jong Lee*,‡,§ School of Municipal and EnVironmental Engineering, Harbin Institute of Technology, Harbin 150090, China, and Department of Chemical Engineering, National Taiwan UniVersity, Taipei, 10617 Taiwan ReceiVed May 28, 2007. ReVised Manuscript ReceiVed July 11, 2007

Little information exists on how various process parameters determine the success of a novel process first proposed in 2005, called the electrochemically assisted microbial cell process. This study attempts to identify the essential process parameters controlling the hydrogen production rates in this electrochemically assisted process. At an applied voltage of 600 mV, microbes in an anode chamber can effectively degrade acetate but produced no hydrogen. The control of neutral pH at the anode chamber was critical to the formation of an active biofilm on the anode surface; consequently, the generated protons and electrons were transferred to the cathode and formed hydrogen gas. Up to 11.6% of electrons produced were transferred to hydrogen in the present mass flow controller system at pH 7.0 and an applied voltage of 540 mV. Competition among other cations through the proton-exchange membrane and the presence of suspended-growth microbial populations correspond to the observed low recovery efficiency of hydrogen from acetate. Cell starvation can result in biofilm deterioration and, thus, system failure.

Introduction Hydrogen is a clean energy source and feedstock used in numerous industries.1–5 Kapdan and Kargi6 analyzed current literature on biohydrogen production techniques. The wastefermenting liquor from a biohydrogen dark fermentation reactor contains high levels of organic matter that is a carbon source for producing hydrogen. Currently available means of using residual organic matter in waste-fermenting liquor include photobiological7–9 and bioelectrochemical approaches.10 Hydrogen production by the electrochemically assisted microbial process extracts electricity using particular bacterial strains, such as Geobacter sp. and Shewanella sp.11–14 Liu et al.10 proposed a process for producing hydrogen via an electrochemically assisted microbial cell (EAMC) using the following two reactions in separated chambers: † Presented at the International Conference on Bioenergy Outlook 2007, Singapore, April 26–27, 2007. * To whom correspondence should be addressed. Telephone: +886-223625632. Fax: +886-2-23623040. E-mail: [email protected]. ‡ Harbin Institute of Technology. § National Taiwan University. (1) Li, Y. F.; Ren, N. Q.; Yang, C. P.; Wang, A. J.; Zadsar, M.; Li, J. Z.; Hu, L. J. J. EnViron. Sci. Health, Part A: Toxic/Hazard. Subst. EnViron. Eng. 2005, 40, 1929–1938. (2) Ren, N. Q.; Li, J. Z.; Li, B. K.; Wang, Y.; Liu, S. R. Int. J. Hydrogen Energy 2006, 31, 2147–2157. (3) Wu, K. J.; Chang, J. S.; Chang, C. F. J. Chin. Inst. Chem. Eng. 2006, 37, 545–550. (4) Wang, C. C.; Chang, C. W.; Chu, C. P.; Lee, D. J.; Chang, B. V.; Liao, C. S. J. Biotechnol. 2003, 102, 83–92. (5) Wang, C. C.; Chang, C. W.; Chu, C. P.; Lee, D. J.; Chang, B. V.; Liao, C. S.; Tay, J. H. Water Res. 2003, 37, 2789–2793. (6) Kapdan, I. K.; Kargi, F. Enzyme Microb. Technol. 2006, 38, 569– 582. (7) Shi, X. Y.; Yu, H. Q. Process Biochem. 2005, 40, 2475–2481. (8) He, D. L.; Bultela, Y.; Magnin, J. P.; Roux, C.; Willison, J. C. J. Power Sources 2005, 141, 19–23. (9) Kondo, T.; Arakawa, M.; Wakayama, T.; Miyake, J. Int. J. Hydrogen Energy 2002, 27, 1303–1308. (10) Liu, H.; Grot, S.; Logan, B. E. EnViron. Sci. Technol. 2005, 39, 4317–4320.

on anodes:

C2H4O2 + 2H2O f 2CO2 + 8H+ + 8e-

on cathodes: 8H+ + 8e- f 4H2 The potential difference between functional microbes and the anode, reduction of NAD+ to NADH, is near -310 mV. The potential difference of hydrogen production at the cathode is -420 mV. Hence, the standard potential difference between the anode and cathode is [-310 – (-420) mV )] 110 mV at pH 7.0. Restated, to produce hydrogen and carbon dioxide directly from acetate hydrolysis is not thermodynamically feasible. However, when a voltage difference >110 mV is applied on the two electrodes in the EAMC to overcome the energy barrier, 8 mol of protons can be potentially produced in the anode when 1 mol of acetate is degraded at the anode chamber, with generated protons transferred through the proton-exchange membrane (PEM) separating these two chambers. That is, 4 mol of hydrogen can be potentially produced at the cathode with a combination of generated protons (through the PEM) and electrons (through the conducting wire). Because the proposed applied voltage is significantly lower than the theoretical voltage for water electrolysis (1210 mV), this so-called “electrochemically assisted” microbial hydrogen production process should be a promising alternative to the direct water electrolysis process. Liu et al.10 attained a hydrogen yield of up to 2.9 mol of H2/mol of acetate in their feasibility study using an applied voltage difference of 850 mV. However, further data for this novel hydrogen-producing process are largely lacking. This work identified the process parameters affecting efficiencies of the electrochemically assisted biohydrogen produc(11) Bond, D. R.; Holmes, D. E.; Tender, L. M.; Lovley, D. R. Science 2002, 295, 483–485. (12) Bond, D. R.; Lovely, D. R. Appl. EnViron. Microbiol. 2003, 69, 1548–1555. (13) Kim, H. J.; Park, H. S.; Hyun, M. S.; Chang, I. S.; Kim, M.; Kim, B. H. Enzyme Microb. Technol. 2002, 30, 145–152. (14) Park, H. S.; Kim, B. H.; Kim, H. S.; Kim, H. J.; Kim, G. T.; Kim, M.; Chang, I. S.; Park, Y. K.; Chang, H. I. Anaerobe 2001, 7, 297–306.

10.1021/ef700293e CCC: $40.75  2008 American Chemical Society Published on Web 09/11/2007

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Figure 1. Photograph of the EAMC assembly. Left chamber, anode chamber; right chamber, cathode chamber. A PEM connects the two chambers.

tion process. The EAMC contains anode and cathode chambers separated by a PEM. A voltage difference substantially lower than the water electrolysis voltage was employed to assess the hydrogen production in the EAMC.

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Figure 2. Time courses of applied voltage and detected electric current in the reactor startup. No hydrogen was produced in this period. Fresh acetate medium was replaced on days 0, 8, and 16 in the anode chamber.

Experimental Section Electrochemically Assisted Microbial Cell. The EAMC is a two-chamber reactor with a PEM (NAFION 117, DuPont, Wilmington, DE) (Figure 1). Each reactor chamber, made of polymethylmethacrylic, comprised a cylinder 110 mm high with an inner diameter of 70 mm. The distance between PEM and the two electrodes were all 75 mm. Plain carbon cloth (without wet proofing; E-Tek Div, PEMEAS, Sommerset, NJ) was the anode, and carbon paper with a 0.35 mg cm-2 Pt catalyst (E-Tek Div, PEMEAS, Sommerset, NJ) was the cathode. The anode and cathode areas were 68 and 9 cm2, respectively. The PEM was sequentially boiled for 1 h in deionized water, 30% (w/w) H2O2 aqueous solution, deionized water, 0.5 M H2SO4 aqueous solution, and finally in deionized water.15 The Geobacter metallireducens medium for the anode chamber was prepared according to the following formula (per 1 L at pH 7.0): 0.1 g of KCl, 0.2 g of NH4Cl, 0.6 g of NaH2PO4, 2.5 g of NaHCO3, 10.0 mL of Wolfe’s vitamin solution, 10 mL of Wolfe’s mineral solution, and 10 mmol of sodium acetate as the electron donor. The medium was sterilized at 121 °C for 15 min before being filled into the anode chamber. The cathode chamber contained Tris-HCl buffer [24.2 g of Tris adjusted to pH 7.0 using 37% (w/w) HCl]. The seed sludge was sewage sludge collected from the Wen Chang Wastewater Treatment Plant in Harbin, China, which predominantly treats municipal sewage and some industrial wastewater. The sediment volume following 30 min settling of the seed sludge was approximately 20% of total sludge volume. The EAMC was operated in batch modes. That is, seed sludge was placed in an anode chamber with fresh, sterilized G. metallireducens medium. The fresh medium was added to replace the supernatant when the acetate level dropped below the prescribed value. All tests were anaerobic at 28–30 °C. The oxidation–reduction potentials (ORPs) of the medium were approximately -400 mV, thereby ensuring adequate anaerobe activity. Measurements. The pH and ORP were measured using a pH meter (pHS-25, Shanghai Precision & Scientific Instrument Co., Ltd., China). The electrode surfaces were scanned using a scanning electron microscope (SEM S-4700, HITACHI, Japan). An inductively coupled plasma mass spectrometry (ICP-OES, Optima 5300DV, Perkin Elmer, Waltham, MA) and Nessler’s reagent spectrometry (721, Shanghai, China) were employed to determine concentrations of Na+, K+, and Mg2+ ions and the NH4+ concentration, respectively. The acetate concentration was measured using (15) Liu, H.; Logan, B. E. EnViron. Sci. Technol. 2004, 38, 4040–4046.

Figure 3. Time course of pH in both anode and cathode chambers with no pH adjustment.

an ionic chromatograph (Dionex 4500i, Sunnyvale, CA). The hydrogen level in the reactor head space was determined using a gas chromatographer (GC122, Shanghai Precision & Scientific Instrument Co., Ltd., China). The test coulombs were calculated by the equation Q ) It. The electric recovery efficiency for hydrogen was calculated using the equation Eer ) CH/CT × 100%. The CH presents the coulomb of electrons used to produce hydrogen and is defined as CH ) 2nF, where F is Faraday’s constant (96 485 C mol-1) and n is the moles of hydrogen produced, calculated as n ) PV/(RT), where P is the atmospheric pressure (Pa), V is the hydrogen volume produced in the cathode chamber (m3), R is the gas constant (8.314 J mol-1 K-1), and T is the temperature (K). The theoretical coulomb is CT ) Fbm/M, where b is the number of moles of electrons produced per mol of acetate consumed and m and M are the mass consumed and molecular weight of acetate, respectively. Total DNA was extracted from 0.5 mL of biofilm samples with a bacterial genomic mini-extraction kit (Hua-shun, Shanghai, China) in accordance with the instructions of the manufacturer. The total DNA was suspended in 100 µL of 2 mM Tris-HCl (pH 8.0–8.5). The DNA extraction procedure was repeated once. For the singlestrand conformation polymorphism technique (SSCP) assessment of the bacterial community on all collected samples, the partial 16S rRNA gene (16S rDNA) fragments were amplified using primers BSF8/20 (5′-AGAGTTTGATCCTGGCTCAG-3′) and SRV3-2 (5′TTACCGCGGCTG CTGGCA-3′), synthesized by Invitrogen (Shanghai, China). These primer sets were used to amplify 16S rDNA from nucleotides 8 to 27 and nucleotides 533 to 515 (Escherichia coli numbering). The primer SRV3-2 was phospho-

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Figure 5. Water decomposition voltage versus current plot in the tested reactor without inoculum.

Tebbe.17 After being amplified, the PCR products were cloned into T-vector (pMD19-T, Takara). A total of 1–3 clones in one band were sequenced.

Results

Figure 4. Time course of voltage, current, pH, hydrogen production, and acetate consumption in the tested mass flow controller (MFC) assembly. pH in the anode chamber was adjusted using 1 M NaOH.

rylated at the 5′ end, and it can be identified and digested by lambdaexonuclease. Each polymerase chain reaction (PCR) for SSCP assessment was performed using a total volume of 50 µL in PCR tubes on the 9700 PCR system (PE) and started with an initial denaturation for 5 min at 94 °C. A total of 30 cycles, each including 40 s at 94 °C, 30 s at 55 °C, and 60 s at 72 °C, was followed by a final primer extension step of 10 min at 72 °C.The annealed temperature was reduced 0.1 °C every cycle. Lambda-exonuclease can specifically digest the 5′-terminal phosphorylated strand of the DNA molecule and not react with the nonphosphorylated strand. To obtain single-stranded DNA from PCR products and simplify the SSCP profile, the phosphorylated strand was removed by lambda-exonuclease digestion. For the digestion of the phosphorylated strand, 20 units of lambda-exonuclease (New England Biolabs, MA) was mixed with 5 µL of 10× buffer and 40 µL of the PCR product in a total volume of 50 µL. The reaction mixtures were incubated at 37 °C for 3 h. Protein was removed by phenolchloroform extraction, and finally, single-stranded DNA was resuspended in 20 µL of ddH2O. A total of 5 µL of denaturing loading buffer was added to 10 µL of sample (about 100 ng). Then, the samples were incubated at 95 °C for 5 min and immediately cooled on ice before being loaded onto the gel. The samples were then electrophoresed in 12% polyacarylamide gel plus 5% glycerol with 1× TBE buffer. Gel was run at 300 V for 18 h and then silverstained in accordance with the description of Bassam et al.16 Recovery of DNA molecules in the high intensity and variation of bands was based on the method described by Schmalenberger and (16) Bassam, B. J.; Caetano-Anolles, G.; Gresshoff, P. M. Anal. Biochem. 1991, 196, 80–83.

Startup. The reactor was started with a voltage difference of 600 mV (Figure 2). An electric current passing across the electrodes was immediately observed, peaked at 1.7 mA on day 3, and then dropped. On day 8, fresh sterilized G. metallireducens medium replaced the supernatant in the anode chamber and the electric current was increased to approximately 1.4 mA on days 9–11. The electric current started declining on day 12. With fresh medium added again on day 16, a steady current of 1.2 mA was again observed across the two electrodes up to day 24 (data on days 20–24 were not shown). No hydrogen gas was produced during this 24 day test period. Figure 3 presents the pH values of the suspension in the two chambers over days 8–16. The pH of the suspension in the anode chamber declined continuously from 7.08 to 6.30, whereas that for the cathode chamber solution remained stable at pH 6.90–7.10. The microbes in the anode chamber produced protons by consuming acetate and did not transfer the generated protons through the PEM to the anode chamber. No biofilm formed on the anode surface at this test stage. Hydrogen Production. Since day 30, the pH in the anode chamber was kept neutral using 1 mol L-1 NaOH titration (Figure 4). Hydrogen gas was then generated from the cathode surface, implying that the protons had been transferred from the anode through PEM to the cathode chamber. The pH of the cathode chamber increased with hydrogen production. Titration using 10 mol L-1 HCl adjusted the suspension of pH in the cathode chamber to neutral (Figure 4). Figure 4 also shows the applied voltage difference and electric current cross of the two electrodes and the consumption of acetate in the anode chamber and the accumulated amounts of hydrogen in the cathode chamber. The pH in the anode chamber dropped faster, and acetate levels decreased slower with hydrogen production than the startup stage (Figures 2 and 3) and, hence, acquired more frequent pH adjustments. A typical batch cycle required 240–260 h to consume 210 mg of acetate at a voltage of 540 mV, whereas the test with no hydrogen production required only roughly 100 h to consume all added acetate. In the typical cycle (Figure 4), 1.19 mmol of hydrogen was generated by the consumption of 210 mg of acetate. (17) Schmalenberger, A.; Tebbe, C. C. Mol. Ecol. 2003, 12, 251–262.

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Figure 6. SEM photographs of bacteria on the surface of the anode electrode.

Roughly 223 C of electrons were transferred from the anode to the cathode, corresponding to a recovery efficiency of 11.6% for hydrogen, a value lower than that obtained by Liu et al.10 (78%). Discussion Figure 5 presents the voltage versus current curve for the EAMC assembly with deionized water as the medium in both chambers. Owing to numerous internal resistances in the assembly, the decomposition voltage of water in this system was about 2.4 V, higher than the theoretical decomposition voltage (1.21 V). Hydrogen was produced at 560 mV (Figure 4); hence, the hydrogen produced was not of a purely electrochemical origin. The applied electrical field assisted the microbes in producing hydrogen. To produce hydrogen using microbe bioactivity in an EAMC assembly required close contact of microbes and electrodes for transferring electrons through the anode chamber to the conducting wire to the cathode chamber and short passage of protons to be transferred from the anode chamber to the PEM. The suspended-growth microbes degraded acetate to carbon dioxide and water and did not contribute to hydrogen production. The formation of an active biofilm is a prerequisite to the success of the proposed EAMC system. Figure 6 presents a scanning electron microscopy (SEM) image of the bacteria attached to the anode surface on day 46. These “rod-like” bacteria are likely the functional strains with electrochemical activity. Figure 7 presents the SSCP results for the biofilm sample collected on day 46. Band 1 is Pseudomonas sp., and band 2 is Shewanella sp.; both were enriched and dominant in the hydrogenproduction stage. Second, the protons, once produced at the anode surface, were transported effectively through the PEM to the cathode for hydrogen production. Other cations competed with the protons for transfer through the PEM under the applied voltage difference. Figure 8 presents the changes in NH4+ concentrations in both the anode and cathode chambers without inoculation. The NH4+ in the anode chamber dropped, whereas that in the cathode chamber increased over time. Other cations had similar trends (data not shown). Hence, during the hydrogen-production stage, numerous cations competed with protons through the PEM and reduced the recovery efficiency of hydrogen from acetate. To determine the fragility of the proposed EAMC system, fresh G. metallireducens medium stopped supplying to the anode chamber on day 60 for 72 h after the acetate level was nearly eliminated. No hydrogen was produced from the cathode chamber during this period, whereas the biofilm was largely detached from the anode surface. Fresh medium was added again after 72 h of starvation. However, hydrogen production was

Figure 7. SSCP profile of microbes in the anode chamber. The period was monitored from startup, the nonhydrogen period (days 1 and 31), and the hydrogen-producing period (days 36, 46, 48, 49, 53, and 54). Hydrogen ceases to be produced on days 61, 64, and 66. Band 1, Pseudomonas sp.; bands 2 and 7, Shewanella sp.

Figure 8. Changes of concentration of NH4+ in the anode and cathode chambers. The anode solution was medium without bacteria, and the cathode solution was deionized water only at the beginning. Wolfe’s mineral solution: 1.5 g of nitrilotriacetic acid, 3.0 g of MgSO4 · 7H2O, 0.5 g of MnSO4 · H2O, 1.0 g of NaCl, 0.1 g of FeSO4 · 7H2O, 0.1 g of CoCl2 · 6H2O, 0.1 g of CaCl2, 0.1 g of ZnSO4 · 7H2O, 0.01 g of CuSO4 · 5H2O, 0.01 g of AlK(SO4)2 · 12H2O, 0.01 g of H3BO3, 0.01 g of Na2MoO4 · 2H2O, and 1.0 L of distilled water.

not recovered. That is, the EAMC assembly lacked process robustness once the attached biofilm had deteriorated markedly.

Electrochemically Assisted Biohydrogen Production

Enhanced biofilm stability is an essential step to get stable hydrogen production by the proposed EAMC system. Proton transport was affected by a series of resistances in their path. The formation of biofilm is a prerequisite for the production of electric flow between electrodes. However, a “too stable” biofilm can yield high mass-transfer resistance to proton transport at an applied voltage difference.18 Optimization of biofilm growth and detachment may be another issue affecting the success of the proposed EAMC system. Conclusions This work identified the necessary process parameters impacting the efficiencies of an electrochemically assisted biohydrogen production process using a microbial fuel cell. Without control, the suspension pH in the anode chamber dropped in conjunction with acetate consumption. Such an inoculation stage cannot start the reactor. When the pH of the anode chamber (18) Ghigo, J. M. Res. Microbiol. 2003, 154, 1–8.

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suspension was maintained at neutral, an active biofilm developed on the anode surface and the cathode surface started generating hydrogen. At an applied voltage of 540 mV, 1.19 mmol of hydrogen was produced with an acetate consumption of 210 mg, equivalent to a recovery efficiency of 11.6% for hydrogen. This hydrogen yield was demonstrated to be of biological origin, whereas the stability of the active biofilm was proven essential to the success of the EAMC system. The SEM images demonstrated the presence of numerous rod-like bacteria on the anode surface during hydrogen production. However, 72 h of starvation deteriorated the biofilm from the anode surface. The following addition of acetate to the chamber was ineffective in restarting hydrogen production. Acknowledgment. We gratefully acknowledge the support of the National Natural Science Foundation of China (NSFC 50678049). EF700293E