Metabolic Segregation and Functional Gene Clusters in Anaerobic Digestion Consortia

A combined enrichment experiment and genome-centric meta-omics analysis demonstrated that metabolic specificity, rather than 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 show that H₂ acts as the primary electron sink for recycling redox cofactors, including NAD⁺ and oxidised ferredoxin (Fd_ox), during primary fermentation, while formate is the dominant electron carrier in secondary fermentation, especially under conditions with elevated H₂ concentrations. Importantly, 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 benefits the anaerobic oxidation of butyrate and propionate with a higher tolerance to H₂ accumulation. Moreover, this study highlights the functional partitioning among microbial populations in key AD niches: primary fermentation, secondary fermentation (syntrophic acetogenesis), hydrogenotrophic methanogenesis, and acetoclastic methanogenesis. Genome-centric analysis of the AD microbiome identified several key functional gene clusters, which could enhance genome-centric genotype–phenotype correlations, particularly for strict anaerobes that are difficult to isolate and characterise in pure culture.