Synergistic association between cytochrome bd-encoded Proteiniphilum and reactive oxygen species (ROS)-scavenging methanogens in microaerobic-anaerobic digestion of lignocellulosic biomass

Intermittent (every other day) microaerobic [picomolar oxygen by oxidation-reduction potential (ORP) set at +25 mV above anaerobic baseline] digestion of lignocellulosic biomass showed higher digestibility and better stability at a high organic loading rate (OLR) of 5 g volatile solids (VS)/L/d than that under strict anaerobic conditions. However, the microbial mechanisms supporting the delicate balance under microaeration remain underexplored. On the basis of our previous findings that microbial communities in replicate experiments were dominated by strains of the genus Proteiniphilum but contained diverse taxa of methanogenic archaea, here we recovered related genomes and reconstructed the putative metabolic pathways using a genome-centric metagenomic approach. The highly enriched Proteiniphilum strains were identified as efficient cellulolytic facultative bacterium, which directly degraded lignocellulose to carbon dioxide, formate, and acetate via aerobic respiration and anaerobic fermentation, alternatively. Moreover, high oxygen affinity cytochromes, bd-type terminal oxidases, in Proteiniphilum strains were found to be closely associated with such picomolar oxygen conditions, which has long been overlooked in anaerobic digestion. Furthermore, hydrogenotrophic methanogenesis was the prevalent pathway for methane production while Methanosarcina, Methanobrevibacter, and Methanocorpusculum were the dominant methanogens in the replicate experiments. Importantly, the two functional groups, namely cellulolytic facultative Proteiniphilum strains and methanogens, encoded various antioxidant enzymes. Energy-dependent reactive oxygen species (ROS) scavengers (superoxide reductase (SOR) and rubrerythrin (rbr) were ubiquitously present in different methanogenic taxa in response to replicate-specific ORP levels (-470, -450 and -475 mV). Collectively, cytochrome bd oxidase and ROS defenders may play roles in improving the digestibility and stability observed in intermittent microaerobic digestion.