Exploring the Role of Phages in the Anaerobic Digestion Microbial Community through Metagenomics and Single-Cell Analysis

Phages play a crucial yet underexplored role in shaping microbial communities within anaerobic digestion systems. This study investigates the role of phages on a simplified microbial community under various stress conditions, including UV light exposure, antibiotic treatment, heavy metals, and hydrogen peroxide (H2O2). Among these stressors, oxidative stress induced by H2O2 had the most significant effect as it reduced microbial growth by 30%. Metagenomic analysis of DNA extracted from both pellets and supernatants enabled the reconstruction of 33 metagenome-assembled genomes (MAGs) and 129 viral MAGs. The most abundant organism was Methanothermobacter thermautotrophicus, a hydrogenotrophic methanogen capable of capturing carbon dioxide (CO2) producing methane. The second most abundant organism was Caldanaerobacter subterraneus, a putative competitor of M. thermautotrophicus with metabolic capabilities of capturing CO2 producing acetate through Wood-Ljungdahl (WL) pathway. A phage-host interaction analysis revealed that C. subterraneus was infected by Caudoviricetes sp. 124 which carried auxiliary metabolic genes involved in WL. Single-cell analysis enabled the confirmation of 2 phage-host interactions predicted from metagenomics data. Furthermore, single-cell analysis allowed the discovery of new phage-host interactions between 9 MAGs and 28 virMAGs. The integration of metagenomics and single-cell analysis offers a powerful approach for elucidating the dynamics of phage-host relationships and their influence on microbial community structure in carbon-dioxide methanation systems.