Insights Gained by High-throughput Chromosome Conformation Capture (Hi-C) into the Viral Modulation of Methane Production in Anaerobic Digestion.

Methanogenesis is a critical driver of global carbon cycling and bioenergy recovery, yet how bacteriophages modulate methane production remains poorly understood. Here, we integrated high-throughput chromosome conformation capture (Hi-C) with multi-omics to map in situ phage-host interactions through an over 440 day anaerobic digestion experiment. We captured 6100 active physical linkages, revealing that 16.3 ± 2.1% of interactions involved auxiliary metabolic genes (AMGs). Notably, we observed a dynamic community-level compositional shift in viral life strategies driven by operational stress. Under mesophilic conditions, lysogenic piggyback-the-winner dynamics prevailed, with AMGs enhancing host competitiveness. Conversely, thermophilic conditions with high total solids stress triggered lytic kill-the-winner strategies. During this phase, AMGs supported rapid phage replication targeting overproliferating bacteria to restore the disrupted balance between acidogenesis and methanogenesis. Furthermore, we identified 529 DNA viral operational taxonomic units (vOTUs) directly infecting methanogens, alongside broad-host-range phages spanning bacterial and archaeal domains. Importantly, we detected four RNA vOTUs exclusively under thermophilic conditions, providing the first omics-based evidence of RNA phages actively infecting methanogenic archaea. These findings highlight phages as important modulators of methane production, offering a foundational framework for developing targeted phage-engineering strategies to optimize bioenergy systems and mitigate methane emissions.