Mechanistic characterization of microbiome shifts under methanogenesis inhibition reveals pathways for circular carbon valorization

Background Arrested anaerobic digestion (AAD) technologies can leverage methanogenesis inhibitors such as iodoform to redirect carbon flux from methane to higher-value fatty acids (FAs), a strategy that aligns with circular bioeconomy goals. However, the effects of iodoform on microbial community structure and function remain poorly understood, limiting the ability to optimize microbiomes for enhanced VFA production. Results Microbiome responses to iodoform in AAD reactors treating food waste with anaerobic sludge inocula were characterized via 16S rRNA gene amplicon sequencing, metagenomics, and metabolite profiling. Iodoform addition eliminated detectable methane and significantly increased the accumulation of butyric acid and medium-chain FAs, including pentanoic, hexanoic, and heptanoic, compared to controls, which favored acetic and propionic acids. Microbial communities shifted toward hydrogen- and butyric acid-producing taxa such as Prevotella , Clostridium sensu stricto 1 , and Peptostreptococcaceae , while methanogens and Romboutsia were suppressed. Metagenomic analyses revealed suppression of oxidative phosphorylation and vitamin B₁₂-dependent pathways associated with methanogenesis and increased odd-chain fatty acid synthesis. Concurrently, iodoform treatment enriched genes related to reverse β-oxidation, substrate-level phosphorylation, assimilatory sulfur metabolism, and fatty acid biosynthesis. Conclusions This study provides the first integrated metagenomic framework for understanding how methanogenesis inhibition via iodoform perturbs microbiome structure and function in AAD systems. By revealing targeted and collateral effects of iodoform, these findings advance microbiome-guided strategies to redirect carbon flux toward valuable end products, informing scalable interventions in waste management and climate-resilient biotechnologies.