GCEP Technical Progress Report April Project: Capturing Electrical Current via Microbes to Produce Methane

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1 GCEP Technical Progress Report April 2014 Project: Capturing Electrical Current via Microbes to Produce Methane Alfred M. Spormann, Professor, Department of Chemical Engineering, and of Civil and Environmental Engineering, Stanford University; Bruce Logan, Professor, Department of Civil and Environmental Engineering, The Pennsylvania State University Svenja T. Lohner, Joerg S. Deutzmann, postdoctoral researcher,s Department of Civil and Environmental Engineering, Stanford University Michael Siegert, Xuifen Li, Matthew Yates, postdoctoral researchers, Dept. of Civil and Environmental Engineering, The Pennsylvania State University Abstract The proposed microbial electromethanogenesis cell (MEMC) process will allow for the cost effec- tive production of methane from CO2 produced, for example, from gas/coal powered power plants or anaerobic digestion of bio- waste. The MEMC technology used will allow for production of CH4 with high efficiency relative to the electrical input. The technology is currently at the concept stage, with the scientific feasibility proven. However, the underlying science fundamentals such as the molecular and microbiological mechanisms of electron transfer from cathodes via intermedi- ates to cells and of the stability of the cathodic microbial communities are only insufficiently un- derstood. This lack of understanding presents the most significant bottleneck that needs to be solved before such technology can be deployed. The proposed research will advance both the sci- ence and the technology of this process, and the outcome should enable commercialization of this technology. Background Recently, it has been discovered that CO2 can be directly converted to methane by microorganisms that harvest electrical energy from a cathode of a microbial electrolysis cell (Cheng et al. 2009). While the mechanism(s) behind electromethanogenesis are not fully understood, the experiment has been repeated by independent laboratories and is currently under development for industrial applications. The central issue is the potentials needed for this process. Direct electron transfer via electromethanogenesis has so far required cathode potentials more negative than approximately 0.8 V. However, indirect methanogensis can occur at much more positive potentials, resulting in less energy input. Our hypothesis for this GCEP project is therefore that in situ hydrogen production improves methane generation rates (mediated route using H2) compared to electromethanogenesis (direct transfer). Biofilm formation on the cathode can enhance methane production, and therefore one of the central questions examined here is which materials are best for methane production in these bioelectrochemical reactors. 1

2 Results 1. Hydrogenase- independent Uptake and Metabolism of Electrons by the Archaeon Methanococcus maripaludis Direct, shuttle- free uptake of extracellular, cathode- derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may be also involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (< 414 mv) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen- independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase- deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase- independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2- bromo- ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism. Figure 1 Bioelectrochemical methane formation in M. maripaludis wild type cells. (a) Potential dependent methane formation was observed in the wt strain ( ) but not in the abiotic control ( ) when tested in bioelectrochemical reactors with cathode potentials of 600 and 700 mv. (b) Hydrogen concentrations in the abiotic control ( ) were much higher and potential dependent compared to the wt strain ( ). Results shown are a representative example of replicate experiments (n = 4). The potential was decreased from 600 mv to 700 mv at t = 73 h. Electron recovery in form of methane and hydrogen, i.e. the coulombic efficiency under those conditions was in the range of %. 2

3 Figure 2. Hydrogen independent methane formation in the M. maripaludis strain MM1284. (a) Bioelectrochemical methane formation was observed in mutant strain MM1284 (solid symbols) but not in the abiotic control (open symbols), and it was independent of the set cathode potential in bioelectrochemical reactors at cathode potentials of 600 and 700 mv. (b) Hydrogen did accumulate in tests with either strain MM1284 (solid symbols) or the abiotic control (open symbols), and in both cases formation rates were dependent on the set cathode potential. The potential was decreased from 600 mv to 700 mv at t = 73 h. Results shown are a representative example of replicate experiments (n = 2). Figure 3. Inhibiting electron flow towards methanogenesis at a cathodic potential of 600 mv results in the formation of other reduced compounds. (a) When inhibited with 7 mm BES (solid line), wt M. maripaludis ceased forming methane ( ), increased hydrogen formation ( ) compared to the abiotic control ( ), and produced formate (r ). (b) Mutant strain MM1284 also showed inhibition of methane formation ( ) but no significant change in rate of hydrogen production ( ). Formate was observed when cells were inhibited with 7 mm BES (r ). Results shown are a representative example of replicate experiments (n = 2) 3

4 Figure 4. Bioelectrochemical methane formation seems to be linked to cells associated with the cathode rather than planktonic cells. Methane production rates at 600mV are not affected significantly by replacing the entire medium and washing planktonic cells out of the cathode chamber. Wild type reactors continued to produce methane at almost unchanged rates (q ) and strain MM1284 still produced methane at 50% of its previous rate ( ). In the abiotic control methane was not detected ( ). Figure 5. Biocathode with wt M. maripaludis cells exhibits a lower overpotential for cathodic reactions. The wt biocathode outperformed the abiotic and MM1284 cathode with respect to current consumption over the complete measured voltage range of 350 to 700mV. 4

5 2. One methanogenic archaeon dominates biocathodes The microbial communities that develop on the cathodes in methanogenic microbial electrolysis cells (MMCs) were investigated using two different inocula and ten different cathode materials. We discovered that one archaeal genus (Methanoarchaeon 1.1) dominated all cathodes that were poised at a potential of 600 mv. These communities were much different than those on controls (open circuit) except for one of the treatments, which had poor reproducibility (C- brush 1), and the Pt- catalyzed cathode which produces substantial hydrogen gas. Figure 6: Distribution of archaeal operational taxonomic units (OTUs, 97% sequence cutoff) across cathodes doped with different metals or minerals. Archaea identified here were obtained from cathodes poised at 600 mv. TMG = Terrestrial Miscellaneous Group 5

6 When Methanoarchaeon 1.1 was present in the inoculum (bog sediment) the performance of applied voltage single chamber MMCs was better compared with an inoculum that was dominated by another methanogenic genus (Methanoarchaeon 2.1) (anaerobic digester sludge, AD sludge) as observed by current uptake over time (Figure 7). Despite its absence in the inoculum (out of 700 sequences), Methanoarchaeon 1.1 was also the prevailing cathodic genus when AD sludge was used as inoculum. This indicates that the conditions were highly selective for Methanoarchaeon 1.1. The presence of Methanoarchaeon 1.1 also correlated with performance in some reactors. Methanoarchaeon 1.1 was also found on cathodes of open circuit controls but shared the habitat with other Archaea as well. Growth of the two bacterial genera was observed while they were absent in the AD inoculum. While our discovery that Methanoarchaeon 1.1 is the dominant methanogen in MMCs agrees well with microbial species observed in other MMCs 1 and iron corroding communities the presence of the two bacterial generawas typical only for iron corroding communities. The proportion of methanogenic Archaea increased from less than 4% of the microbial population in the inocula to 31% during incubation showing that the conditions were selective primarily for methanogenic Archaea and not for Bacteria. In context of previous reports this suggests at this time that the presence of the two bacterial genera was rather random and may not be a result of particular MMC conditions. The dominating methanogen with platinum, an efficient hydrogen evolution catalyst, on the cathode was Methanoarchaeon 1.2, a typical hydrogenotrophic methanogen. This indicates that the electron transferring process was different in reactors without platinum. However, it may also reflect differences in ph or hydrogen partial pressure. In conclusion, Methanoarchaeon 1.1 is indicative for methanogenesis on biocathodes of MMC reactors and is correlated with performance. At this time, the presence of Bacteria do not seem to be pivotal, although this issue is being further investigated. Figure 7: Current uptake in reactors that were inoculated with different amounts (0.01%, 0.1%, 1%, 10%, 25%) of bog sediment (left) or anaerobic sludge (right) in membraneless single chamber MMCs. Current that passed the electrical circuit was measured. 6