Deciphering the diversity of prokaryotic viruses in the activated sludge and anaerobic digester systems

Abstract

As the largest biotechnological application, the activated sludge (AS) system and the following anaerobic digester (AD) system in wastewater treatment plants (WWTPs) are the embodiment of artificially controlling the activity of microbial communities to perform services to humans. Viruses are important but poorly explored components in WWTPs, reaching 10-1,000-fold higher concentrations than their counterparts in other aquatic systems. Through specific interactions such as host lysis, viruses can directly impact prokaryotic communities. Considering the potential importance of prokaryotic viruses in AS and AD systems, there is a critical need to disclose the diversity and variation of prokaryotic viruses among different WWTPs and how viruses may impact prokaryotic taxa. Using the targeted metagenomic approach, I amassed a catalogue of ~50,000 prokaryotic viruses, which expands the current AS virome database (IMG/VR database v.2.0) by 12-fold at the genus-level and by 23-fold at the species-level. I discovered substantial uncharacterized viral diversity in AS communities, with only between 0.4-1.6% of total viral contigs being assigned to a known viral family. By predicting hosts, I demonstrated that viruses may impact functional microorganisms in biological nutrient removal and carbon cycle in WWTPs, linking viruses with aerobic/anaerobic heterotrophs, and other functional microorganisms controlling N/P removal. In addition to the AS system, prokaryotic viral diversity in the AD system was also investigated. I amassed a catalogue of over 26,000 DNA viruses, which expands the newest AD virome database (IMG/VR database v3) by 2.2-fold at the genus-level and by 3.4-fold at the species-level. I discovered substantial uncharacterized viral diversity in AD communities, with only between 0.3-1.7% of total viral contigs being assigned to a known viral family. By predicting hosts, I show that viruses may impact key methanogens in AD systems, thus affecting methane production and energy recovery efficiency. Further, I built a currently most comprehensive AS viral catalog of over 190,000 DNA viruses using targeted metagenomic sequencing for 32 virus-enriched samples covering 6 WWTPs in Hong Kong, China. Then I used this catalog to analyze the role of prokaryotic viruses after disturbance in 97 longitudinal AS samples over 9 years from the same WWTP. Notably, taken as a whole, AS viral community might follow the previously proposed royal family model after disturbance where though kill-the-winner fluctuations happened in opportunistic viruses like Actinobacteria-infecting viruses, members of the royal family like Proteobacteria-infecting viruses remained dominant due to optimized niches. Finally, with the methods of integrated metagenomics and metatranscriptomics, the viral transcripts from 12 temporal cellular microbial metatranscriptomics were analyzed. Correlation analysis showed that the relative abundance of dominant AS viruses in metagenomic data is positively associated with that in metatranscriptomic data. Synchronized infection patterns were observed after disturbance where virus-host ratio (VHR) peaked in around November and was followed by a rapid decrease with a trough near June. The results of this study provide new insights into diversity, ecological roles, host connections and functional potential of prokaryotic viruses in WWTPs and provide a genomic reference for future design and timing of phage treatment to tackle sludge bulking and foaming problems.