A panoramic view of the virosphere in three wastewater treatment plants by integrating viral-like particle-concentrated and traditional non-concentrated metagenomic approaches

Wastewater biotreatment systems harbor a rich diversity of microorganisms, and the effectiveness of biotreatment systems largely depends on the activity of these microorganisms. Specifically, viruses play a crucial role in altering microbial behavior and metabolic processes throughout their infection phases, an aspect that has recently attracted considerable interest. Two metagenomic approaches, viral-like particle-concentrated (VPC, representing free viral-like particles) and non-concentrated (NC, representing the cellular fraction), were employed to assess their efficacy in revealing virome characteristics, including taxonomy, diversity, host interactions, lifestyle, dynamics, and functional genes across processing units of three wastewater treatment plants (WWTPs). Our findings indicate that each approach offers unique insights into the viral community and functional composition. Their combined use proved effective in elucidating WWTP viromes. We identified nearly 50,000 viral contigs, with Cressdnaviricota and Uroviricota being the predominant phyla in the VPC and NC fractions, respectively. Notably, two pathogenic viral families, Asfarviridae and Adenoviridae, were commonly found in these WWTPs. We also observed significant differences in the viromes of WWTPs processing different types of wastewater. Additionally, various phage-derived auxiliary metabolic genes (AMGs) were active at the RNA level, contributing to the metabolism of the microbial community, particularly in carbon, sulfur, and phosphorus cycling. Moreover, we identified 29 virus-carried antibiotic resistance genes (ARGs) with potential for host transfer, highlighting the role of viruses in spreading ARGs in the environment. Overall, this study provides a detailed and integrated view of the virosphere in three WWTPs through the application of VPC and NC metagenomic approaches. Our findings enhance the understanding of viral communities, offering valuable insights for optimizing the operation and regulation of wastewater treatment systems. Our comparative analysis unveiled the distinct advantages of two methods in capturing the complexity of viral communities and their functions in wastewater treatment plants. The non-concentrated (NC) metagenomic approach identified a higher number of viral contigs, though it had a lower yield of high-quality contigs. In contrast, the viral-like particle-concentrated (VPC) metagenomic method outperformed in recovering a larger proportion of high-quality viral contigs, and significantly enriched free eukaryotic viruses. Notably, the phage-derived auxiliary metabolic genes and antibiotic resistance genes were predominantly located in the NC metagenomes rather than the VPC metagenomes. Overall, the integration of both VPC and NC metagenomic approaches offers a robust framework for a thorough and nuanced exploration of viral communities in wastewater environments, contributing valuable insights for the advancement of wastewater treatment technologies. image This study comprehensively compared viral-like particle-concentrated (VPC) and non-concentrated (NC) metagenomic approaches in virome investigation, highlighting their respective efficacies in detecting different viral communities and functional elements in wastewater treatment plants (WWTPs). Eukaryotic viruses belonging to Cressdnaviricota were the most prevalent in VPC metagenomes, while bacterial viruses belonging to Uroviricota were the most abundant in NC metagenomes. Diverse phage-born auxiliary metabolic genes (AMGs) were discovered in the virome of WWTPs, and a large array of AMGs involved in biogeochemical cycles like carbon, sulfur, and phosphorus cycling showed transcriptional activation in wastewater treatment systems. A tiny proportion (less than 0.08% of viral operational taxonomic units) of viruses was discovered to harbor antibiotic resistance genes (ARGs) (29 phage-born ARGs) only in the cellular fraction, and phage-born ARG types primarily included macrolide-lincosamide-streptogramin, tetracycline, and aminoglycoside resistance.