Glycol/glycerol-fed electrically conductive aggregates suggest a mechanism of stimulating direct interspecies electron transfer in methanogenic digesters

The possibility of stimulating direct interspecies electron transfer (DIET) within aggregates of methanogenic digesters respectively with ethanol, glycol, and glycerol as a primary substrate was investigated to better understand the mechanisms of alcohol compounds stimulating DIET. Aggregates fed with ethanol, glycol, and glycerol were electrically conductive (10.4-19.4 uS/cm), with a temperature dependence of metallic-like conductivity. Close examination of transmission electron microscope images observed the potential interspecies connected networks assembled by filaments within these aggregates. Further investigations via metatranscriptomics found that, genes for electrically conductive pili (e-pili) (Log2FPKM, 9.39-10.96) and c-type cytochromes (8.90-9.64) were highly expressed within aggregates. Glycerol-fed aggregates exhibited the highest gene expression for e-pili, while glycol-fed aggregates exhibited the highest gene expression for c-type cytochromes. Methanothrix species were dominant and metabolically active within aggregates. Genes encoding the enzymes involved in carbon dioxide reduction were highly expressed in Methanothrix species, suggesting that they participated in DIET. In addition, transcript abundance of genes encoding alcohol dehydrogenase and NADH-quinone oxidoreductase in alcohol dehydrogenation closely associated with NADH/NAD+ transformation within glycol- and glycerol-fed aggregates was generally higher than that within ethanol-fed aggregates. These results, and the fact that NADH/NAD+ transformation was very linked to the ATP synthesis complex that further supported the formation of extracellular electrical connection components, e-pili and membrane-bound multi-heme c-type cytochromes (MHCs), provided a possibility that alcohol compounds comprised of hydroxy groups could stimulate DIET and more hydroxy groups comprised were better for this stimulation.