Metabolic Segregation and Characteristic Genes of the Key Functional Populations in Anaerobic Digestion Consortia

Enrichment experiment integrated with genome-centric meta-omics analysis revealed that metabolic specificity, rather than metabolic flexibility, governs the anaerobic digestion (AD) ecosystem. This study provides new insights into interspecies electron transfer in the AD process, highlighting a segregation in the metabolism of H₂ and formate. Our findings indicate that H₂ serves as the primary electron sink for recycling redox cofactors, NAD⁺ and oxidized ferredoxin (Fdox), during primary fermentation. In contrast, formate predominates in the co-occurring secondary fermentation, particularly under conditions of elevated H₂ concentrations. Notably, no evidence of biochemical interconversion between H₂ and formate was identified in the primary fermenting bacteria or in syntrophs enriched in this study. This segregation of H₂ and formate metabolism likely benefit the anaerobic oxidation of butyrate and propionate with a higher tolerance to H₂ accumulation. Furthermore, this study underscores the functional partitioning among microbial populations within key niches of the AD bioprocess: primary fermentation, secondary fermentation (syntrophic acetogenesis), hydrogenotrophic methanogenesis, and acetoclastic methanogenesis. Through genome-centric analysis of the AD microbiome, we identified several key functional gene clusters, highlighting the potential for improving genome-centric genotype-phenotype correlations, which is particularly valuable for strict anaerobes that remain challenging to isolate and characterize in pure culture.