Novel phenazine crystals enable direct electron transfer to methanogens in anaerobic digestion by redox potential modulation

With one billion tons of methane produced annually by microorganisms, biogas production can be appreciated both for its role in global organic matter turnover and as an energy source for humankind. The importance of electron transfer from electrically conductive surfaces or from bacteria to methanogenic Archaea has been implicated in widespread commercial anaerobic digestion processes, though a mechanism for reception of electrons from conductive surfaces or pili by methanogens has never been demonstrated. Here we describe a novel crystalline form of the synthetic phenazine neutral red that harvests electrons from reduced inorganic and organic microbial sources in anaerobic environments and makes them available to methanogenic Archaea. The novel crystalline form is so effective at harvesting reducing equivalents because it displays a potential for reduction 444 mV higher than the soluble form (E' = 70 mV). Neutral red molecules solubilised in the reduced state by protonation at the point of methanogen cell contact with the crystal surface deliver electrons to methanogens at a negative midpoint potential (E' = -375 mV). We demonstrate that soluble neutral red delivers reducing equivalents directly to the membrane bound HdrED heterodisulfide reductase of Methanosarcina, replenishing the CoM-SH and CoB-SH pool for methanogenesis and generating proton motive force. An order of magnitude increase in methane production is recorded in pure acetate fed Methanosarcina and coal and food waste fed mixed cultures in the laboratory. The phenomenon is also demonstrated at field scale in a sub-bituminous coal seam 80 m below ground level.