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 H ₂ and formate. Our findings show that H ₂ 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 H ₂ concentrations. Importantly, no evidence of biochemical interconversion between H ₂ and formate was identified in the primary fermenting bacteria or in syntrophs enriched in this study. This segregation of H ₂ and formate metabolism likely benefits the anaerobic oxidation of butyrate and propionate with a higher tolerance to H ₂ 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.