Metagenomic analysis reveals abundance of mixotrophic, heterotrophic, and homoacetogenic bacteria in a hydrogen-based membrane biofilm reactor.

Heterotrophic microorganisms are frequently observed in hydrogenotrophic denitrification systems and are presumed to contribute to their improved performance. However, their roles and metabolic pathways in the hydrogen-based membrane biofilm reactor (HMBfR) system remain unclear. The objective of this study was to elucidate the underlying mechanisms driving heterotrophic denitrification. For this purpose, metagenomic analysis was conducted on an HMBfR showing higher denitrification performance, focusing on the metabolic function of the microbial community. Functional genes related to H oxidation, organic carbon metabolism, and denitrification were the major targets of interest. This analysis revealed a substantial number of genes associated with the oxidation of organic carbon compounds in the biofilm, suggesting its potential for heterotrophic denitrification. Investigation of the genes of interest in metagenome-assembled genomes (MAGs) has demonstrated a predominance of mixotrophs or heterotrophs rather than obligate autotrophs. Notably, MAGs exhibiting the highest abundance of genes of interest were affiliated with Hydrogenophaga and Thauera, implying their significant role in denitrifying the HMBfR as mixotrophs utilizing both H and organic substrates. The identification of 11 MAGs, presumed to originate from homoacetogens suggested that acetate might contribute to the proliferation of heterotrophs. Based on these metagenomic findings, possible metabolic pathways were identified to explain heterotrophic denitrification within the HMBfR biofilms.