Metabolic Adaptation of Methanogens in Anaerobic Digesters upon Trace Element Limitations

Anaerobic digestion is a complex multi-stage process relying on the activity of highly diverse microbial communities including hydrolytic, acidogenic and syntrophic acetogenic bacteria as well as methanogenic archaea. The aim of the present study was to understand how methanogens cope with trace element limitation and sustain their growth and metabolic activity. Amplicon sequencing of mcrA genes revealed Methanosarcina (72%) and Methanoculleus (23%) as the predominant methanogens in the undisturbed reactors. With increasing trace element limitation, Methanoculleus increased its relative abundance to 33%. A shift of the methanogenic pathways from acetoclastic towards hydrogenotrophic methanogenesis was seen in batch tests with 13C-methyl- or 13C-carboxyl-labelled acetate. Metaproteome analysis revealed abundance shifts of the enzymes involved in methanogenic pathways of the predominant families Methanosarcinaceae and Methanomicrobiaceae. In Methanosarcinaceae, proteins involved in methylotrophic and acetoclastic methanogenesis decreased in abundance while formylmethanofuran dehydrogenase increased, confirming our assumption hypothesis of a shift from acetoclastic to hydrogenotrophic methanogenesis by Methanosarcina. The efforts of both methanogenic families to stabilize their metabolism and energy balance by increasing the abundance of methyl-H4MPT:CoM methyltransferase and methyl:CoM reductase were seemingly more successful in Methanomicrobiaceae. In summary, our study revealed that mmethanogens use different strategies to stabilize their energy balance under trace element limitation. Methanosarcina switched from trace element expensive pathways (methylotrophic and acetoclastic methanogenesis) to hydrogenotrophic methanogenesis. Methanoculleus showed a higher robustness and was favored over the more fastidious Methanosarcina, thus stabilizing reactor performance under trace element limitation.