Multi-omics approaches insight into the assembly, dynamics and interactions of microbial and viral communities in anaerobic digestion

Abstract

The efficient biotransformation of organic matter in anaerobic digestion depends on diverse microbes, their metabolic activities and trophic cooperation. Despite technological advances, the function of uncultivated anaerobic lineages and their interactions in carbon mineralization remain unclear. In addition, bacteriophages exert a crucial impact on microbial populations and ecosystem by modulating host metabolism. However, the diversity of anaerobic viral communities and their potential interactions with key microbial functional guilds during anaerobic digestion remain largely unexplored. Firstly, I explored fresh leachate treatment with anaerobic membrane bioreactor (AnMBR) based on the on-site investigation of the characteristics of fresh leachate. Temperature-related profiles of fresh leachate properties were observed. In addition, AnMBR achieved a high COD removal of 98% with a maximum organic loading rate (OLR) of 19.27 kg-COD/m3/d at the shortest hydraulic retention time (HRT) of 1.5 d. Then, to investigate the key anaerobes and their metabolic functions involved in fresh leachate degradation, a combination of long-read sequencing and metatranscriptomics-guided metabolic reconstruction was utilized to provide a genome-wide perspective on carbon mineralization flow from polymers to methane. Results showed that hybrid assembly recovered 132 high-quality genomes and over 50% more prokaryotic genes compared to the short-read-only assembly. Metatranscriptomics-guided metabolic reconstruction revealed the metabolic flexibility of several novel Bacteroidales-affiliated bacteria and populations from Mesotoga sp. in scavenging amino acids and sugars. In addition to recovering two circular genomes of previously known but fragmented syntrophic bacteria, two newly identified bacteria within Syntrophales were found to be highly engaged in fatty acids oxidation via syntrophic relationships with dominant methanogens. Moreover, metagenomics, metatranscriptomics, metaviromics and Hi-C sequencing were integrated to explore the diversity of anaerobic DNA and RNA virome, as well as their role in carbon transformation. Long-read sequencing technology not only improves the assembly quality of viral genomes but also facilitates the recovery of 7,417 anaerobic-specific viral species. A total of 736 virus-to-host linkages uncovered that virus impacts anaerobic processes by regulating the metabolism of keystone anaerobic species. Moreover, a wide range of auxiliary metabolic genes (AMGs) and their transcriptional patterns were observed in different HRTs, suggesting their ecological role in carbon mineralization. Finally, multi-omics approaches were utilized to yield significant insights into the diversity and potential host interactions of anaerobic viral communities in practical engineering anaerobic scenarios. By mining metagenomics, metaviromics and metatranscriptomics, a total of 25,125 anaerobic DNA viral genomes and 1,697 RNA viruses in full-scale anaerobic digesters were recovered. The utilization of Hi-C sequencing and in silico approaches contributed to the retrieval of 2,993 virus-host connections. A total of 5,816 viral-encoded AMGs and their transcriptional expressions revealed that phages may influence host metabolism and carbon flux in anaerobic processes via the transcription of AMGs in engineering environment. Overall, this study utilized multi-omics approaches to uncover the metabolic functions and interactions of microbial and viral communities in both lab-scale and practical engineering anaerobic ecosystems. These findings advance the understanding of metabolic activities and trophic interactions between key anaerobic guilds and viral genomes, providing foundational insights into carbon transformation in both engineered and natural ecosystems.