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Though there are multiple lines of evidence that point to a mode of extracellular electron transfer (EET) in Methanosarcina barkeri, there is incomplete understanding of the mechanistic basis for this process. The goal of this Master’s thesis is to investigate the protein basis of EET mechanisms in this microbe. In Chapter 1, the current evidence for EET in Methanogens and the state of knowledge for EET in M. barkeri are reviewed. Like other EET systems, it is predicted there is an extracellular protein conduit(s) that bridges the insulating cell envelope and connects the cell’s internal redox machinery to the external electron source. However, the biophysical basis of this protein remains unknown. Part of the challenge for identifying such a protein is that the methanogen extracellular proteome is poorly characterized, complicating the search for the unknown protein conduit. To address this issue, in Chapter 2, a whole cell biotin-labeling technique was used to investigate the composition of the extracellular proteome of M. barkeri under methanol growth, poised potential electrochemical conditions, and open circuit control conditions. Combined, a total of 209 proteins was recovered from ten biotin-labeled samples and 48 from five non-biotin-labeled control samples. After accounting for potential non-specific binding, a list of 52 putatively extracellular proteins was curated from this work. Of these proteins, Fe-S oxidoreductase (Q46E87), ferritin-like diiron domain protein (Q46C50), and copper binding protein, plastocyanin/azurin family protein (Q466T4) were the main redox-active proteins of interest and may be worth further investigation regarding a potential role in EET. However, several proteins of unknown function were also identified, with some containing putative S-layer-like protein domains. DUF 1699 family proteins in particular may be worth further investigation, as they are highly conserved within Archaea. While this approach provides evidence of extracellularity, this work was exploratory and may miss relevant extracellular proteins. It also does not allow the quantification of differential protein expression that would provide insight into the proteins that play an express role under electrochemical conditions. As such, in Chapter 3, the proteins involved in M. barkeri’s EET process were investigated using tandem mass tags (TMT) of differentially grown M. barkeri cells, which were analyzed via mass spectrometry to quantify differential protein expression across conditions. In total, 805 proteins were detected across all samples, and 425 unique proteins were identified. Of these, 84 are upregulated under EC conditions compared to MeOH growth. A list of 19 proteins with additional supporting evidence for a potential role in EET was further curated. These proteins were either previously identified in the examination of the extracellular proteome, were identified as putatively redox-active, and/or experienced a two-fold or greater upregulation under DIET conditions with Geobacter metallireducens. These are potentially interesting targets to be investigated in the future. Future work could include gene deletion studies that confirm a phenotype for these proteins in EET and/or confirmation of extracellularity, using GFP tagging to verify protein localization, and creating his-tag and over-expression mutants to assess the protein’s role in the mutant’s ability to perform direct cathodic electron uptake.

Abstract: The concept of electro-methanogenesis by combining the microbial electrolysis cell and anaerobic digestion (MEC-AD) has become a promising method for improving methane generation and improving the stability of digesters. Although the electro-methanogenesis process is often featured as a simple process of coupling MEC with an anaerobic digester, several fundamental and engineering bottlenecks are associated with their practical application. Most importantly, a streamlined roadmap for establishing an active microbiome, process design, optimization, and scale-up has not yet been achieved. Particularly, this doctoral thesis focuses on understanding the roles of microbial syntrophy, extracellular polymeric substances, and power supply schemes on electro-methanogenesis. First, we reported an experimental investigation of extracellular polymeric substances (EPS), reactive oxygen species (ROS), and the expression of genes associated with extracellular electron transfer (EET) in methanogenic biocathodes electrodes. The MEC-AD systems were examined using two cathode materials: carbon fibers and stainless-steel mesh. A higher methane generation was attained in MEC-AD with stainless-steel mesh as a cathode electrode. A higher abundance of hydrogenotrophic Methanobacterium sp. and homoacetogenic Acetobacterium sp. appeared to play a major role in superior methanogenesis from stainless steel biocathode than carbon fibers. Moreover, the higher secretion of EPS accompanied by the lower ROS level in stainless steel biocathode indicated that higher EPS perhaps protected cells from harsh metabolic conditions (possibly unfavorable local pH) induced by faster catalysis of hydrogen evolution reaction. In contrast, EET-associated gene expression patterns were comparable in both biocathodes. Thus, these results indicated hydrogenotrophic methanogenesis is the key mechanism, while cathodic EET has a trivial role in distinguishing performances between two cathode electrodes. These results provide new insights into the efficient methanogenic biocathode development. Second, previous studies for conventional anaerobic digestion systems have emphasized maintaining an optimum propionate/acetate (HPr/HAc) ratio. To date, the detrimental ratio of HPr/HAc concentrations towards the electro-methanogenesis process has not been examined yet. Thus, this study focused on understanding the impact of different VFAs concentrations with varied HPr/HAc ratios on the microbial community and methanogenesis process. The total cumulative methane production remained almost the same after increasing HPr/HAc ratio from 0.5 to 1.5. When HPr/HAc ratios further increased to 2.5 and 5, the total cumulative methane production markedly decreased. EET-associated gene expression reduced under high HPr/HAc ratios (2.5 and 5) indicates the partial inhibition of biofilm electroactivity. Geobacter and Methanobacterium species were abundant under lower HPr/HAc ratios, while their abundance decreased under higher HPr/HAc ratios. Therefore, this study demonstrated that higher HPr/HAc ratios would adversely impact methanogenesis rates in MEC-AD systems. Third, from the perspective of energy saving in the operation of MEC-AD, we focused on developing an intermittent power supply scheme. The applied potential was switched off for 12 and 6 hours/day during the operation of a laboratory-scale MEC-AD system fed with glucose. The results from the operation under continuous applied potential served as the control. The overall biomethane generation and net energy income from the process were unaffected when the applied potential turned off for 6 hours/day. Both quantitative and qualitative analyses of microbial communities suggested that a balanced microbiome could be maintained under short-term switching-off the applied potential. However, performance substantially deteriorated when the applied potential turned off for 12 hours/day. Overall, the results of this study suggest that MEC-AD operation does not need a continuous power supply, and higher energy efficiency can be effectively achieved by intermittently powering the reactor. However, previous efforts to optimize power supply schemes for MEC-AD systems were limited to the synthetic substrate only. However, conventional digesters are typically operated with more complex substrates. Hence, we investigated the impact of intermittent power supply in MEC-AD fed with mixed primary and sewage sludge. Overall, the electrocatalytic activity of the anode biofilm demonstrated a higher current density at 12 hrs ON mode. Also, the maximum methane generation attained when the applied potential switched ON for 12 hrs/day. The extracellular electron transfer-associated genes showed the highest expression at 12 hrs ON mode. Accordingly, the intermittent applied potential for 12 hrs/day could provide an attractive opportunity to saving electrical energy input in MECAD systems, thereby its economic benefits.