A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane

Abstract

a 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 H 2 or formate as the electron donor were rare. AlthoughMethanosaeta 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. Broader contextIn this study we report a fundamentally new concept for the microbial ecology of anaerobic digestion, one of the oldest bioenergy strategies. The reliance of methanogenic communities on interspecies electron transfer has been recognized for over forty years, but it has been thought that only H 2 or formate served as the interspecies electron carriers. However, the nding that Methanosaeta species can make direct electrical connections with Geobacter species, accepting electrons for the reduction of carbon dioxide to methane, demonstrates that direct interspecies electron transfer (DIET) is an alternative to interspecies H 2 / formate transfer. DIET appears to predominate over interspecies H 2 /formate transfer in upow anaerobic digesters converting brewery waste to methane, and the metatranscriptomic approach described here provides a tool to discriminate between pathways for interspecies electron transfer in other digester designs, treating other types of wastes or biomass. Methanosaeta species are also ubiquitous in methanogenic soils and sediments, suggesting that a substantial portion of global methane production could be derived from DIET.

The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer

“…These truncated pilA genes are predicted to encode mature pilin structural proteins with only 60-90 amino acids, compared with the >120 amino acid residues typically found in PilA subunits of most other bacteria (Table 1). In the few instances in which pili conductivity has been directly measured, truncated pilA genes code for proteins that give rise to conductive pili (Geobacter sulfurreducens and Geobacter metallireducens), whereas longer pilA genes yield pili with poor conductivity (Geobacter uraniireducens and Pseudomonas aeruginosa) (Liu et al, 2014;Tan et al, 2016). Therefore, the truncated pilA genes were designated e-pilin to distinguish them from longer type IVa pilA genes that are more commonly found in bacteria.…”

“…In contrast, many lines of evidence support the concept of long-range electron transport along the e-pili of Geobacter sulfurreducens. This includes the findings that: (1) deleting the gene for PilA, the pilus monomer, inhibited electron transport to Fe(III) oxide, interspecies electron exchange, and the development of thick electrically conductive biofilms Nevin et al, 2009;Reguera et al, 2005;Summers et al, 2010); (2) genetically modifying pilA to yield pili with poor conductivity inhibited Fe(III) oxide reduction and reduced biofilm conductivity (Vargas et al, 2013); (3) a strain of Geobacter sulfurreducens expressing the poorly conductive pili of Pseudomonas aeruginosa was ineffective in Fe(III) oxide reduction and current production (Liu et al, 2014); (4) the individual pilin filaments are electrically conductive Malvankar et al, 2011;Reguera et al, 2005); and (5) the pili propagate charge similarly to carbon nanotubes .…”

“…The concept that electrically conductive pili (e-pili) can enable long-range electron transfer to insoluble minerals (Reguera et al, 2005), to other cells (Rotaru et al, 2014;Shrestha et al, 2013;Summers et al, 2010) and through electrically conductive biofilms Reguera et al, 2006) is a paradigm shift in microbial electron transport. The distance over which e-pili can conduct electrons and the apparent competitive advantage of this capability in a number of anaerobic environments distinguish electron transfer via e-pili from more typical forms of short-range biological electron transfer (Lovley & Malvankar, 2015;Malvankar et al, 2015;Vargas et al, 2013).…”

Enhanced methane production in an anaerobic digestion and microbial electrolysis cell coupled system with co-cultivation of Geobacter and Methanosarcina

“…1c). As DIET has been proved between Geobacter and Methanosarcina in a methane production environment (Rotaru et al, 2014a), it was hypothesized that the reducing energy may derived from the electrons transferred from Geobacter, leading to part of the carbon dioxide being reduced to methane. Moreover, Fig.…”

Microbial nanowires: an electrifying tale

“…MNWs have been believed to play a role in methane production in syntrophic microbial communities (Morita et al, 2011;Rotaru et al, 2014;Summers et al, 2010;Wegener et al, 2015), which can be exploited further for improved methane production in anaerobic digesters. Interested readers are referred to specific reviews on this topic (Lovley, 2011;.…”