A metagenomic-driven investigation of the pathobiome in anaerobic digestion environments

Abstract Anaerobic digestion processes organic wastes like manure and sludge, producing biogas and digestate fertilizer. Since these feedstocks can contain animal and human pathogens, understanding their fate during digestion is important for assessing the safety of digestate reuse. Past microbial surveys of digesters often used 16S rRNA gene sequencing, which offers broad taxonomic insight but typically cannot characterize pathogens at species level or identify their virulence genes. This study applies whole-metagenome sequencing to achieve higher resolution, aiming to map and analyze microorganisms with pathogenic potential across industrial biogas plants. Our analysis used a unified genomic database. This database combined two key resources: a newly curated collection of 1,361 high-quality reference genomes for known bacterial pathogens, and 4,568 metagenome-assembled genomes that were previously reconstructed from over 300 anaerobic digestion samples across multiple countries. We screened metagenomic sequencing data from digesters located in different countries against this combined database using an optimized approach to ensure the detection method performed consistently across the geographically diverse samples. This approach allowed us to screen for known pathogens while simultaneously examining the genetic relatedness of environmental microbial genomes to those pathogens. The analysis revealed that genomes from digesters can be highly similar to those of known opportunistic pathogens. For instance, one environmental MAG clustered with reference genomes for Escherichia coli and all four major Shigella species, a group known for its clinical relevance. A phylogenetic analysis was used to better characterize this MAG within a clade containing pathogenic E. coli and Shigella strains. We found that operational temperature had a clear association with pathogen signal. The abundance and diversity of microorganisms from genera containing opportunistic pathogens were highest in mesophilic digesters and lower in thermophilic systems. This pattern aligns with the established principle that higher temperatures reduce survival of some microbial species associated with humans or animals. The overall microbial community structure, including pathogens, also varied with feedstock type and geographic location, suggesting these factors influence the ecological niche for such organisms. It is important to note that detecting DNA derived from pathogenic species does not confirm the presence of viable, infectious cells. However, the persistence of these genetic signatures is a prerequisite for risk. The results indicate that digestate from mesophilic plants may require careful management if used in agriculture. This work provides a genomic framework for monitoring pathogens in anaerobic digestion and supports the use of thermophilic operation as a measure to reduce pathogen loads.