Sulfide-Driven Microbial Electrosynthesis - American Chemical Society

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Sulfide-Driven Microbial Electrosynthesis Yanming Gong,† Ali Ebrahim,† Adam M. Feist,† Mallory Embree,† Tian Zhang,‡ Derek Lovley,‡ and Karsten Zengler*,† †

Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, United States Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003, United States

‡

S Supporting Information *

ABSTRACT: Microbial electrosynthesis, the conversion of carbon dioxide to organic molecules using electricity, has recently been demonstrated for acetogenic microorganisms, such as Sporomusa ovata. The energy for reduction of carbon dioxide originates from the hydrolysis of water on the anode, requiring a sufficiently low potential. Here we evaluate the use of sulfide as an electron source for microbial electrosynthesis. Abiotically oxidation of sulfide on the anode yields two electrons. The oxidation product, elemental sulfur, can be further oxidized to sulfate by Desulfobulbus propionicus, generating six additional electrons in the process. The eight electrons generated from the combined abiotic and biotic steps were used to reduce carbon dioxide to acetate on a graphite cathode by Sporomusa ovata at a rate of 24.8 mmol/day·m2. Using a strain of Desulf uromonas as biocatalyst on the anode resulted in an acetate production rate of 49.9 mmol/day·m2, with a Coulombic efficiency of over 90%. These results demonstrate that sulfide can serve effectively as an alternative electron donor for microbial electrosynthesis.

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INTRODUCTION Microbial electrosynthesis, the bacterial reduction of carbon dioxide and formation of multicarbon molecules using electrical current, has recently garnered attention in the fields of microbiological and electrochemical engineering.1,2 Nevin et al. demonstrated for the first time the microbial production of acetate from carbon dioxide with external electricity as the only energy source in a bioelectrochemical system.3 A process originally described for the bacterium Sporomusa ovata, microbial electrosynthesis has now been reported for a set of different acetogens attached to a graphite electrode.4 In previous reports, water served as the electron donor on the anode. The electrolysis of water generates molecular oxygen, protons, and electrons. The protons are then delivered to the cathode through a cation exchange membrane and utilized together with electrons generated on the anode for the microbial reduction of carbon dioxide. The generation of molecular oxygen can be detrimental to the electrosynthetic reduction of carbon dioxide due to oxygen sensitivity of the microbes described thus far. Herein, we describe the use of hydrogen sulfide (eqs 1 and 2) as an electron source alternative to water for microbial electrosynthesis. Sulfide represents a superior electron source for microbial electrosynthesis due to its very low redox potential. Therefore, the energetic input required to promote carbon dioxide reduction with sulfide as an electron donor is significantly less than for carbon dioxide with water as an electron donor. Previous research demonstrated that sulfide © 2012 American Chemical Society

reacts abiotically with inexpensive, yet durable, graphite electrodes to produce elemental sulfur and two electrons (eq 1).5−7 This reaction yields two moles of electrons for each mole of sulfide oxidized (Figure 1). Further, it has been proposed that the oxidation of sulfide to sulfur at the anode of microbial fuel cells could be an industrially important process for removing waste sulfide.7,8 Such a process could generate electricity for a treatment plant while also producing elemental sulfur, which could be harvested. However, this process is difficult to sustain because the sulfur that is deposited on the anodes eventually passivates (i.e., covers rendering unreactive) the anode, thereby limiting further oxidation. H 2S → S0 + 2H+ + 2e −

(1)

S0 + 4H 2O → SO4 2 − + 8H+ + 6e−

(2)

4H 2S + 2CO2 → 4S0 + C2O2 H4 + 2H 2O

(3)

H 2S + 2CO2 + 2H 2O → H 2SO4 + C2O2 H4

(4)

Studies have demonstrated that elemental sulfur passivation of the anode can be alleviated microbiologically, a phenomenon first discovered in benthic microbial fuel cells harvesting electricity from highly sulfidic marine sediments.9 Molecular Received: Revised: Accepted: Published: 568

September 20, 2012 December 6, 2012 December 10, 2012 December 19, 2012 dx.doi.org/10.1021/es303837j | Environ. Sci. Technol. 2013, 47, 568−573

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for the Desulf uromonas strain consisted of 13.7 g/L ferric citrate, 0.25 g/L NH4Cl, 0.1 g/L KCl, 0.5 g/L NaH2PO4, 2.5 g/ L NaHCO3, 1 mL of 1 mM NaSeO4, 1 mL 1000× Trace Mineral and 1000× Vitamins, and 0.1 g/L CaCl2. In addition, 0.1 mL 1 M acetate and 0.5 mL extra salts solution (180 g/L of NaCl, 54 g/L MgCl2 and 2.7 g/L of CaCl2) was added in every 10 mL of medium.12 S. ovata was cultivated as previously described.14 The medium used in bioreactors for both anode and cathode chambers consisted of 0.25 g/L NH4Cl, 0.1 g/L KCl, 0.5 g/L NaH2PO4, 2.5 g/L NaHCO3, 1 mL/L of 1 mM NaSeO4, 1 mL/ L 1000× Trace Mineral Mix, 1 ml/L 1000× Vitamins, and 0.1 g/L CaCl2. The pH was adjusted to 7.0 prior to autoclaving at 121 °C for 25 min. Chemical Analysis. Composition and concentration of products produced by microbial electrosynthesis was quantified by high performance liquid chromatography, using an Aminex HPX-87-H Ion Exclusion column in a Waters system. HPLC samples were prepared by filtering 1 mL of medium obtained from the reactor through a 0.22 μm filter. If not analyzed immediately, the filtered medium was kept at −20 °C in closed vials to prevent loss of volatile compounds. Gases in the headspace of the bioreactor were analyzed and monitored by gas chromatography (GC-2014, Shimadzu) with high purity nitrogen as carrier gas as previously described to measure H2 production.15 Quantitative determination of sulfide in growth experiments and bioreactors was performed calorimetrically by the previously described methylene blue method.16 Sulfate production was determined by a modified version of a turbidimetric assay described by Jackson et al.17 A 1 mL sample was added to 1 mL acetate solution (2%) and incubated for 15 min at room temperature. A total of 220 μL of the solution was transferred to a 15 mL falcon tube containing 880 μL water, followed by the addition of 1.2 mL of an 8% trichloroacetic acid solution and 600 μL of 0.01% agarose solution containing 25 mM barium chloride. After the turbidity stabilized, the absorbance of the sample (in the range of 1 to 15 mM) was measured at 500 nm using a spectrophotometer against a standard of Na2SO4. Setup of Bioreactors. Glass bioreactors (H-Cell devices) with an internal volume of 250 mL in both the anode and cathode chamber were employed in this study. For each of the enumerated experimental steps, multiple identical reactors were set up and run. A magnetic stir plate (Thermo Scientific Variomag Poly 15) set at 130 rpm was installed beneath the bioreactor to ensure mixing and to maintain a homogeneous electrolyte. The anode and cathode chambers were separated by a Nafion 117 membrane with an effective working area of 6.25 cm2. Both electrodes were made of graphite plates with a normalized surface area of 46 cm2. The applied potential for sulfide-mediated electrosynthesis was set to +300 mV vs a Ag/ AgCl reference electrode on the anode using a potentiostat (Gamry G-350 and multiplexer). The temperature was maintained at 30 °C. Microscopy. Cells of D. propionicus attached to the surface of the electrode were imaged using confocal laser scanning microscopy (Olympus FV1000 confocal on an IX81 inverted microscope). Cells on the graphite surface were stained with a LIVE/DEAD backlight bacterial viability kit (Molecular Probes) following an established protocol.18,19

Figure 1. A schematic of the microbial electrosynthesis process for production of organic compounds from carbon dioxide with sulfide as the electron source.

analysis demonstrated that microorganisms closely related to Desulfobulbus propionicus, a sulfate-reducing Delta Proteobacterium, were able to oxidize elemental sulfur to sulfate, (eq 2) with a graphite anode serving as the sole electron acceptor. Pure culture studies demonstrated that D. propionicus oxidized sulfur that was deposited on the anode as the result of abiotic sulfide oxidation to sulfate (which is more soluble), with accompanying transfer of six electrons per molecule sulfur to the anode (eq 2). This biological oxidation is beneficial as the soluble sulfate is easier to remove from a bioreactor system. Studies of microbial fuel cells (MFCs) and microbial electrolysis systems (MESs) have traditionally focused more on either the biological process on the anode or the cathode. However, in recent years the study of integrated bibioelectrodes in the same electrochemical system has received considerable attention.10,11 Here we investigate the integration of biological processes on both the anode and the cathode for sulfide-driven microbial electrosynthesis.

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MATERIALS AND METHODS Sources of Microorganisms. Desulfobulbus propionicus (DSM 2023) and Sporomusa ovata (DSM 2662) were obtained from the Deutsche Sammlung von Mikroorganismen and Zellkulturen, GmbH (Germany) and propagated in the laboratory as recommended. Additionally, a newly isolated strain belonging to the genus Desulfuromonas12 was grown with ferric citrate as electron acceptor and acetate as electron donor at 30 °C. Strains were grown anaerobically in 125 mL serum bottles to an optical density at 600 nm between 0.3 and 0.4. At this point, 80 mL of culture was harvested by centrifugation, washed and resuspended in fresh bioreactor medium and inoculated anoxically into the bioreactors. Cells of D. propionicus and Desulfuromonas sp. were added to the anode chamber, while S. ovata was applied to the cathode. Media and Cultivation. D. propionicus medium consisted of 1.17 g/L NaCl, 0.19 g/L MgCl2, 0.30 g/L KCl, 0.15 g/L CaCl2·2H2O, 0.27 g/L NH4Cl, 0.23 g/L KH2PO4·H2O, and 2.84 g/L Na2SO4. Two milliliters each of 0.025 g/L Riboflavin, 0.1 g/L Thiamin, 0.05 g B12, 0.004 g/L Na2SeO3·5H2O and 0.008 g/L Na2WO4·2H2O, 50 mL 1 M NaHCO3 and 0.4 mL 1 M H2SO4. Vitamins and trace minerals were prepared and added as previously described.13 The pH was adjusted to 7.5 and the medium was autoclaved at 121 °C for 25 min. Medium 569

dx.doi.org/10.1021/es303837j | Environ. Sci. Technol. 2013, 47, 568−573

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Experimental Approach. Step 1: Abiotic Oxidation StepCoupling oxidation of sulfide to elemental sulfur with electrosynthesis. S. ovata was cultured in serum bottles with hydrogen as the electron donor, then harvested during log phase by centrifugation and added to the cathode chamber containing bicarbonate-buffered medium without any additional carbon sources. An abiotic anode with a constant potential of +300 mV vs Ag/AgCl was then coupled together with the cathode containing the S. ovata cells. After the addition of sulfide to the anode (7.3 mM), formation of acetate in the cathode chamber was monitored by HPLC over the next 24 h. Step 2: Biotic Oxidation StepMicrobial oxidation of sulfur to sulfate with the electrode as the electron acceptor. A potential of +300 mV vs Ag/AgCl, favorable for conversion of sulfur to sulfate,20 was applied to the anodes of two sterile bioreactors. Sulfide (1.6 mmol) was injected into both systems at 20, 113, and 253 h and current production was monitored over the course of the experiment. One bioreactor was kept sterile, accounting only for the chemical oxidation of sulfide (eq 1). The other bioreactor was inoculated with D. propionicus at 90 h (prior to the second sulfide injection), to allow for both the abiotic and biotic oxidation reactions to occur (eqs 1 and 2). Step 3: Integrated Abiotic and Biotic OxidationSulfidedriven electrosynthesis. Sterile chambers for both the anode and cathode were filled with anoxic sterile media and inoculated with D. propionicus and S. ovata respectively. The electrochemical cell was held at an applied potential of +300 mV vs Ag/AgCl on the anode. After 25 h, sulfide solution was added to the anode chamber. The head space in the cathode chamber was monitored constantly for production of hydrogen throughout the experiments. The solution in the cathode chamber was monitored by HPLC for acetate production, and the solution in the anode chamber was monitored for sulfate production and sulfide consumption using the previously described assays.

Figure 2. Abiotic oxidation of sulfide to elemental sulfur and current generation on the anode coupled to carbon dioxide reduction using electrical current on the cathode. A batch bioreactor with an abiotic anode and a cathode chamber containing Sporomusa ovata was set up with a potential of +300 mV. Sulfide oxidation in the anode chamber (triangles) and production of acetate by electrosynthesis in the cathode chamber (rectangles) was monitored over time.

efficiency of S. ovata (around 90%).3 Previously it was reported that the strain can also produce small amounts of 2-oxobutyrate in addition to acetate during electrosynthesis.3 However, no production of 2-oxobutyrate or other chemicals during electrosynthesis was observed by HPLC at significant concentration during our experiments. For the duplicates performed (Figure 1, Supporting Information (SI) Figure S2), the columbic efficiency of the microbial electrosynthesis system using S. ovata and electrons from abiotic sulfide oxidation was measured at 83% and 93% respectively. Step 2: Biotic Oxidation StepMicrobial Oxidation of Sulfur to Sulfate with the Electrode As Electron Acceptor. Sulfate-reducing bacteria belonging to the family Desulfobulbaceae have been observed as the predominant species during enrichment studies on the anodes of benthic microbial fuel cells.22,23 This finding suggests a role of these microbes in harvesting electricity under sulfidic conditions. Furthermore, a pure culture of D. propionicus (a member of the Desulfobulbaceae) was shown to oxidize elemental sulfur with a subsequent transfer of electrons to a graphite electrode.6 Therefore, we used D. propionicus for the biological oxidation of sulfur to sulfate following a chemical oxidation of sulfide to sulfur (as described in the previous section). The rate of electricity generation from the chemical oxidation of sulfide alone, and the complete oxidation of sulfide to sulfate mediated by microorganisms was compared. The electrical charge (integration of current over time) for each of the three reaction cycles in the sterile control showed that the electricity generated was roughly equivalent (±8%) to electrons being generated solely from sulfide oxidation in each cycle (Figure 3B), with the base current excluded from the integrated total charge. The total charge in the second bioreactor, which was inoculated with D. propionicus prior to the second cycle, increased from 270 coulombs in the first cycle to 349 coulombs in the second cycle and to 420 coulombs in the third cycle (Figure 3A). The additional electric charge for the biotic reactor over the solely abiotic reactor (+30% after second and 56% after third sulfide amendment) was likely generated from

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RESULTS AND DISCUSSION Step 1: Abiotic Oxidation StepCoupling Oxidation of Sulfide to Elemental Sulfur with Electrosynthesis. We first examined if electrons generated by sulfide oxidation on an anode could be used for bacterial-mediated carbon dioxide reduction in the cathode chamber using S. ovata, as it had previously been shown to accept electrons and use them for carbon dioxide reduction.3 Hydrogen sulfide provided in the anode chamber was known to be oxidized abiotically to elemental sulfur at this potential, generating two moles of electrons per mole of sulfide according to eq 1. The sulfide concentration in the anode chamber steadily decreased over 120 h, while the amount of acetate increased accordingly in the cathode chamber (Figure 2). Acetogenic bacteria such as Sporomusa and Acetobacterium species have been shown to require hydrogen concentrations above 400 ppm for reduction of CO2.21 Monitoring of hydrogen concentrations in MES experiments did not reveal production of hydrogen. The final concentration of sulfide after 120 h was 3.7 mM, whereas 0.75 mM acetate was produced on the cathode, representing a ratio of sulfide to acetate of 4.8:1. This ratio is in close agreement with the theoretical stoichiometry of 4:1 based on the optimal conversion of electrons from sulfide oxidation to carbon dioxide reduction (eq 3). The slightly higher amount of sulfide oxidized in the experiment can be explained by the columbic 570

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multiple additions of sulfide (SI Figure S1), whereas little coloration was observed in the biotic reactor containing D. propionicus cells. Formation of sulfate over time was determined using a turbidimetric assay. As expected, sulfate was not detected after the first reaction cycle in either reactor. After addition of D. propionicus, the sulfate concentration in the anode chamber of the biotic reactor increased gradually over time, reaching 8 mM at the end of the third reaction cycle. No sulfate was detected in the sterile (abiotic) reactor throughout the experiment. Complete conversion of sulfide to sulfate would yield 24 mM sulfate, indicating that around 30% of sulfur was converted to sulfate by the end of the third reaction cycle. Biofilm formation by D. propionicus on the anode was investigated using confocal laser scanning microscopy. Figure 4A shows an image of cells stained with LIVE/DEAD stain after sulfide amendment. The image reveals multiple layers of cells on the anode that are metabolically active, confirming the active role of D. propionicus in the oxidation of sulfur with the electrode as the sole electron acceptor. Although dense patches of cells were observed on the electrode, the overall biofilm formation was not as pronounced as it has been shown for other current generating bacteria, such as Geobacter sulf urreducens.24 Overall, the results demonstrated that electrons combined from both the chemical oxidation of sulfide and the sequential oxidation of sulfur by D. propionicus are available to contribute to the reduction of carbon dioxide in an electrosynthesis system. Step 3: Integrated Abiotic and Biotic Oxidation Sulfide-Driven Electrosynthesis. An anode containing D. propionicus was coupled to a cathode chamber with S. ovata cells. Prior to the addition of sulfide, neither sulfate (on the anode) nor acetate (on the cathode) was detected in the system. The current increased immediately after sulfide supplementation. Formation of acetate in the anode chamber was detected 15 h after sulfide addition to the anode chamber, and the concentration of acetate increased over time (Figure 5A). However, production of sulfate was not immediately detected in the anode chamber upon sulfide addition, and was first detectable at 89 h, a delay of around 50 h. This lag was also observed in an experimental replicate (SI Figure S3), and could be due to the fact that an appropriate interface for electron

Figure 3. Continuous current production by sulfide oxidation on an anode poised at +300 mV and a sterile cathode. D. propionicus cells were added at 60 h to the anode (A) compared to a sterile control (B). Sulfide (1.6 mmol) was added to the anode chamber at 20, 113, and 253 h.

oxidation of elemental sulfur to sulfate by D. propionicus. The difference in oxidation product between both reactors could also be visually observed by a change in color of the electrolyte. Accumulation of elemental sulfur in the sterile (abiotic) bioreactor resulted in yellow coloration that intensified with

Figure 4. (A) Image of Desulfobulbus propionicus on the anode surface after 25 days. (B) Image of Desulfuromonas on the anode surface after 25 days. Cells of D. propionicus and Desulf uromonas were stained using a LIVE/DEAD BacLight viability-stain. 571

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a recently isolated strain of Desulf uromonas12 that is capable of oxidizing elemental sulfur to sulfate with the electrode as the sole electron acceptor. Cells of Desulf uromonas formed dense biofilms when supplied with sulfide on an anode (Figure 4B). Similar to the previous experiment using D. propionicus, an equal number of Desulfuromonas cells were added to the anode. S. ovata was present in these experiments in the cathode chamber. The conversion of sulfide and subsequent formation of sulfate by Desulfuromonas is shown in Figure 5B. In contrast to sulfide-mediated electrosynthesis with D. propionicus where production of sulfate was detected after 89 h, sulfate production occurred after 20 h when Desulf uromonas cells were used (Figure 5B). The overall sulfide oxidation rate using Desulf uromonas cells was 97.8 mmol/day·m2, while sulfate was produced at a rate of 40.3 mmol/day·m2. The overall acetate production rate by electrosynthesis was 49.9 mmol/ day·m2, which is twice the rate of the reactor with D. propionicus. The combined oxidation of sulfide and sulfur on the anode resulted in formation of electrons at a rate of 436 mmol/day·m2. Utilization of these electrons for the formation of acetate from carbon dioxide on the cathode occurred at a rate of 400 mmol/day·m2. Accordingly, the Coulombic efficiency of sulfide-mediated electrosynthesis over a period of 120 h was >90%. These results demonstrate that sulfide can be used effectively to drive microbial electrosynthesis.

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ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details and figures. This material is available free of charge via the Internet at http://pubs.acs.org

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Figure 5. (A) Sulfide-driven electrosynthesis by Desulfobulbus propionicus and Sporomusa ovata. Oxidation of sulfide (circles) and production of sulfate (triangles) on the anode, and acetate (rectangles) on the cathode is monitored over time. (B) Sulfide-driven electrosynthesis by Desulfuromonas sp. and Sporomusa ovata. Oxidation of sulfide (triangles) and production of sulfate (circles) on the anode, and acetate (rectangles) on the cathode is monitored over time.

AUTHOR INFORMATION

Corresponding Author

*Phone: (858) 534-5758; fax: (858) 822-3120; e-mail: [email protected]. Author Contributions

Y.G., A.E., A.M.F., and M.E. performed the abiotic experiments. Y.G. carried out experiments involving both microbial anode and cathode. T.Z. isolated and cultivated the Desulfuromonas strain. K.Z. directed the experiments. Y.G. prepared figures and Y.G. and K.Z. wrote the manuscript with contributions from all authors.

transfer by D. propionicus to the electrode needed to be established. The chemical oxidation of sulfide to sulfur appeared to precede the oxidation of sulfur to sulfate by D. propionicus. The initial rate of sulfide oxidation (from 25 to 89 h) was 80.2 mmol/day·m2 (Figure 5A), supporting a maximal production of acetate of 20.1 mmol/day·m2. The sulfide oxidation rate after 89 h decreased to 24.8 mmol/day·m2, with a corresponding rate of sulfate production of 23.7 mmol/day·m2, indicating the source of electrons provided to the anode changed around this point in time. According to the half-cell reaction for acetate production on the cathode, 8 mol electrons are needed to reduce carbon dioxide to 1 mol acetate, requiring electrons from 4 mol of sulfide or 1.25 mol of sulfur. It can be concluded that the abiotic oxidation of sulfide and biological production of sulfate from sulfur cocontributed to the production of acetate. The production of acetate at a rate of 24.0 mmol/day·m2 was enabled by the generation of two electrons from sulfide oxidation and six electrons from sulfur oxidation (eqs 3 and 4). The biological conversion of sulfur accompanied by electron transfer to the anode by D. propionicus is the rate-limiting factor in this system. This could due in part to limited accessibility of sulfur that is formed in direct contact with the anode as well as the limited capability of D. propionicus to form dense biofilms (Figure 4A). To identify if these limitations are due to the choice of organism, sulfide-driven electrosynthesis was investigated using

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Gerard Norwich (Bioengineering Department, UCSD) for support with CLSM imaging. The work presented herein was funded by the Advanced Research Projects AgencyEnergy (ARPA-E), U.S. Department of Energy, under Award Number DE-AR0000087 and DE-AR0000159

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