Anaerobic conversion of organic wastes and biomass to methane is an important bioenergy strategy, which depends on poorly understood mechanisms of interspecies electron transfer to methanogenic microorganisms. Metatranscriptomic analysis of methanogenic aggregates from a brewery wastewater digester, coupled with fluorescence in situ hybridization with specific 16S rRNA probes, revealed that Methanosaeta species were the most abundant and metabolically active methanogens. Methanogens known to reduce carbon dioxide with H₂ or formate as the electron donor were rare. Although Methanosaeta have previously been thought to be restricted to acetate as a substrate for methane production, Methanosaeta in the aggregates had a complete complement of genes for the enzymes necessary for the reduction of carbon to methane, and transcript abundance for these genes was high. Furthermore, Geobacter species, the most abundant bacteria in the aggregates, highly expressed genes for ethanol metabolism and for extracellular electron transfer via electrically conductive pili, suggesting that Geobacter and Methanosaeta species were exchanging electrons via direct interspecies electron transfer (DIET). This possibility was further investigated in defined co-cultures of Geobacter metallireducens and Methanosaeta harundinacea which stoichiometrically converted ethanol to methane. Transcriptomic, radiotracer, and genetic analysis demonstrated that M. harundinacea accepted electrons via DIET for the reduction of carbon dioxide to methane. The discovery that Methanosaeta species, which are abundant in a wide diversity of methanogenic environments, are capable of DIET has important implications not only for the functioning of anaerobic digesters, but also for global methane production.

Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter metallireducens . Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. metallireducens isolates to share electrons with M. barkeri , demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H ₂ -producing Pelobacter carbinolicus , incapable of DIET, M. barkeri utilized H ₂ as an electron donor but metabolized little of the acetate that P. carbinolicus produced. This suggested that H ₂ , but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H ₂ transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H ₂ or electrons derived from DIET for CO ₂ reduction. Furthermore, M. barkeri is genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.

Granular activated carbon (GAC) is added to methanogenic digesters to enhance conversion of wastes to methane, but the mechanism(s) for GAC's stimulatory effect are poorly understood. GAC has high electrical conductivity and thus it was hypothesized that one mechanism for GAC stimulation of methanogenesis might be to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens. Metabolism was substantially accelerated when GAC was added to co-cultures of Geobacter metallireducens and Geobacter sulfurreducens grown under conditions previously shown to require DIET. Cells were attached to GAC, but did not aggregate as they do when making biological electrical connections between cells. Studies with a series of gene deletion mutants eliminated the possibility that GAC promoted electron exchange via interspecies hydrogen or formate transfer and demonstrated that DIET in the presence of GAC did not require the electrically conductive pili and associated c-type cytochrome involved in biological interspecies electrical connections. GAC also greatly stimulated ethanol metabolism and methane production in co-cultures of G. metallireducens and Methanosarcina barkeri. Cells were attached to GAC, but not closely aggregated, suggesting little opportunity for biological electrical contacts between the species. GAC also enhanced methane production in samples from a methanogenic digester in which Methanosaeta were the predominant methanogens. The results demonstrate that GAC can promote DIET and suggest that stimulation of metabolism in methanogenic digesters can be attributed, at least in part, to the high conductivity of GAC providing better interspecies electrical connections than those that can be forged biologically.

Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.