Viral auxiliary roles in hydrolytic and biosynthetic metabolism regulate prokaryotic microbial interactions in anaerobic digestion.

Anaerobic digestion (AD) viruses have gained recognition as significant regulators of microbial interactions within AD communities, yet their ecological roles remain largely unexplored. In this study, we investigated the ecological roles of AD viruses in regulating microbial interactions among syntrophic hosts. We recovered 3921 diverse viral sequences from four full-scale anaerobic digesters and confirmed their widespread presence across 127 global metagenomic sampling sites (with >95 % sequence similarity), underscoring the ubiquity of prokaryotic viruses in AD-related systems. Through the construction of virus-prokaryote interactions (66.8 % validated at the transcriptional level) and analysis of viral-host transcriptional abundances, we identified significant associations between AD viruses and key processes, including hydrolysis, acidogenesis, and methanogenesis. Notably, polyvalent viruses were found to interact with both hydrolytic and fermentative communities. We further characterized viral auxiliary metabolism, hydrolytic substrate spectra, and microbial auxotrophy, showing that viruses not only could enhance the breakdown of complex substrates (e.g., cellulose, chitin, peptidoglycan) but also potentially supported the biosynthesis of essential nutrients (e.g., cysteine, methionine, heme, and cobalamin). These activities were proposed to regulate resource fluxes through alternating lysogenic and lytic cycles. Phylogenetic analysis of viral gene and horizontal gene transfer (HGT) identification suggest that AD viruses employ promiscuous infection on syntrophic hosts, potentially as an adaptive evolutionary strategy in the AD ecosystem. This study provides new insights into the ecological roles of AD viruses, highlighting their potential impact on the stability and functionality of AD systems.