Global diversity and biogeography of DNA viral communities in activated sludge systems

Abstract

Background: Activated sludge (AS) systems in wastewater treatment plants (WWTPs) harbor enormous viruses that regulate microbial metabolism and nutrient cycling, significantly influencing the stability of AS systems. However, our knowledge about the diversity of viral taxonomic groups and functional traits in global AS systems is still limited. To address this gap, we investigated the global diversity and biogeography of DNA viral communities in AS systems using 85,114 viral operational taxonomic units (vOTUs) recovered from 144 AS samples collected across 54 WWTPs from 13 different countries.

Results: AS viral communities and their functional traits exhibited distance-decay relationship (DDR) at the global scale and latitudinal diversity gradient (LDG) from equator to mid-latitude. Furthermore, it was observed that AS viral community and functional gene structures were largely driven by the geographic factors and wastewater types, of which the geographic factors were more important. Carrying and disseminating auxiliary metabolic genes (AMGs) associated with the degradation of polysaccharides, sulfate reduction, denitrification, and organic phosphoester hydrolysis, as well as the lysis of crucial functional microbes that govern biogeochemical cycles were two major ways by which viruses could regulate AS functions. It was worth noting that our study revealed a high abundance of antibiotic resistance genes (ARGs) in viral genomes, suggesting that viruses were key reservoirs of ARGs in AS systems.

Conclusions: Our results demonstrated the highly diverse taxonomic groups and functional traits of viruses in AS systems. Viral lysis of host microbes and virus-mediated HGT can regulate the biogeochemical and nutrient cycles, thus affecting the performance of AS systems. These findings provide important insights into the viral diversity, function, and ecology in AS systems on a global scale. Video Abstract.

Keywords: Activated sludge; Auxiliary metabolic genes; Biogeography; Viral community; Virus-microbe interaction.

Fig. 1

Overview of activated sludge (AS) viruses. A Geographic distribution of collected AS samples. The location sites of AS are presented as orange circles, with circle size representing the number of wastewater treatment plants (WWTPs). B UpSet plot showing the number of viral operational taxonomic units (vOTUs) and their sharedness between AS with different wastewater types at the species level. C Pie chart showing the number of vOTUs clustered in AS from different countries at the species level. D Accumulation curve of vOTUs (orange) and viral protein clusters (vPCs, gray). Dots represent the average number of vOTUs and PCs, and error bars represent the range. The numbers of vPCs are divided by five for better visualization. E Histogram showing the distribution of viral genome size (log₁₀ scale) and quality

Fig. 2

Biogeographic diversity patterns of activated sludge (AS) viruses. A, B Distance-decay relationships (DDRs) based on Bray–Curtis dissimilarity of viral community and functional gene structures. C Latitudinal diversity patterns of viral community richness. First- and second-order polynomial fits are shown in blue and black, respectively. The best polynomial fit was determined (as underlined) based on the corrected Akaike Information Criterion (AICc)

Fig. 3

Alpha and beta diversity analysis of AS viral communities. A Shannon index of viral communities across different datasets. The significant difference test is determined using Student’s t-test, ns indicates no significant difference, *** indicates P < 0.001, **** indicates P < 0.0001. The significant differences in viral diversity in AS with different countries are shown in the heatmap. B NMDS analysis of viral communities based on the Bray–Curtis dissimilarity calculated by the normalized mean coverage of viral operational taxonomic units (vOTUs). ANOSIM is applied to detect the differences in viral communities in AS between different wastewater types or countries

Fig. 4

Taxonomy and clustering of activated sludge (AS) viruses with known viruses. A The number of shared viral clusters (VCs) in AS with different wastewater types and NCBI viral RefSeq. B The number of shared VCs in AS from different countries. C The relative abundance of viral taxa (n = 22,673) across different AS samples based on the normalized mean coverage. AS samples are separated by wastewater types (black lines) and countries (different colors). Viral taxa with relative abundance of less than 0.1% are classified as others

Fig. 5

Analysis of antibiotic resistance genes (ARGs) carried by viruses in activated sludge (AS). A The relative abundance of ARGs carried by viruses across different AS samples. The significant difference test was determined by using Student’s t-test, ns indicates no significant difference, ** indicates P < 0.01. B Correlation analysis of the relative abundance of virus-associated ARGs with the relative abundance of total ARGs. C Correlation analysis of the relative abundance of virus-associated ARGs with the relative abundance of lysogenic viruses. D The number and relative abundance distribution of different ARGs classes across AS with the three wastewater types. Bubble size represents the number of ARGs, and the different colors represent the relative abundance of ARGs. The bars represent the total relative abundance of each ARG class

Fig. 6

Potential contribution of viruses to biogeochemical cycle in activated sludge (AS). A The glycoside hydrolases (GHs) encoded by AS viruses. The schematic shows the degradation of complex polysaccharides by different GHs encoded by AS viruses. The blue font indicates the classes of GHs. B Contribution of AS viruses to the sulfur (S) cycle. The schematic shows three pathways of the S cycle, including sulfur oxidation, assimilatory sulfate reduction, and dissimilatory sulfate reduction. Key genes in each pathway are indicated on the arrows with black font. AMGs carried by viruses are colored in brown. C Contribution of AS viruses to the nitrogen (N) cycle. The schematic shows the major pathways of the N cycle. The different arrow colors represent different pathways. Key genes in each pathway are indicated on the arrows with black font. AMGs carried by viruses are colored in green. D Contribution of AS viruses to the phosphorus (P) cycle. The schematic shows the pathways of organic phosphoester hydrolysis. Key genes in this pathway are indicated on the arrows in black font. AMGs carried by viruses are colored in gold

Fig. 7

Virus-host interactions in activated sludge (AS). A Relative abundances of vOTUs and their hosts grouped by the host taxonomy in each AS ample. AS samples are separated by wastewater types (black lines) and countries (different colors). B, C Linear regression model analysis is performed based on the virus-host abundance correlations for the specific lineage or functional group in each dataset (domestic wastewater: n = 51, industrial wastewater: n = 49, and mixed wastewater: n = 44). Based on linear regression analysis, the R² values and P values of each dataset in linear regression models are presented in different colors. Two-way ANOVA P values (black font) indicate the significant differences between the three datasets