Coupling of stable isotope probing and metagenomics reveals the diversity of viruses infecting formatotrophs during anaerobic digestion process

Abstract

Viruses play a major role in regulating microbial community composition and activity, yet their diversity and host associations in anaerobic digestion (AD)—a complex biotechnological process converting organic waste into biogas—remain poorly characterized. Linking viral sequences to metabolically active hosts in such complex communities is particularly challenging. Here, we applied an integrative approach combining 13 C-stable isotope probing (SIP) with shotgun sequencing of viromes and microbiomes to identify and characterize viruses infecting the active formatotrophic guilds in batch AD microcosms. Microcosms fed with 13 C-labeled formate as sole carbon source selectively enriched two primary formate-consuming guilds: hydrogenotrophic methanogens (Methanobacteriales, predominantly Methanobacterium subterraneum ) and acetogenic bacteria (family Natronincolaceae, genus Andreesenella ). From cross-assembly of six viromes on the one hand, and six 13 C-SIP microbiome fractions on the other, we assembled a catalogue of 2,368 vOTUs, of which 261 were selected for detailed analysis. The striking enrichment of vOTUs associated with methanogenic archaeal hosts, specifically in the heavy ( 13 C-enriched) SIP fractions, contrasted with their near-absence in total viromes—demonstrates that DNA-SIP successfully resolved viruses of active formatotrophs at the viral community level. Novel viruses were identified for both primary guilds, including archaeal viruses of Methanobacteriales (family Anaerodiviridae and a possible novel family) and two Andreesenella viruses carrying diversity-generating retroelements. A provirus predicted to infect Methanothrix —a strictly acetoclastic methanogen—further illustrates the capacity of SIP-viromics to capture viral associations across trophic levels. Analysis of auxiliary viral genes (AVGs) revealed numerous defense-associated genes ( dcm , metK , queC ) and genuine metabolic AVG candidates, such as cysH (assimilatory sulfate reduction) and, a contiguous cluster including uxe, galU and serB (surface polysaccharide biosynthesis), which may influence on host metabolism. This study demonstrates the power of SIP-viromics for resolving virus-host associations in complex anaerobic communities, linking viral diversity directly to metabolically active formatotrophic and methanogenic guilds. The identification of novel viral lineages infecting key AD microorganisms, combined with AVGs with potential consequences for carbon and sulfur cycling, provides a foundation for understanding the functional role of viruses in AD process performance.