ABSTRACTA metagenome-based approach was used to assess the taxonomic affiliation and function potential of microbial populations in free-chlorine-treated (CHL) and monochloramine-treated (CHM) drinking water (DW). In all, 362,640 (averaging 544 bp) and 155,593 (averaging 554 bp) pyrosequencing reads were analyzed for the CHL and CHM samples, respectively. Most annotated proteins were found to be of bacterial origin, although eukaryotic, archaeal, and viral proteins were also identified. Differences in community structure and function were noted. Most notably,Legionella-like genes were more abundant in the CHL samples while mycobacterial genes were more abundant in CHM samples. Genes associated with multiple disinfectant mechanisms were identified in both communities. Moreover, sequences linked to virulence factors, such as antibiotic resistance mechanisms, were observed in both microbial communities. This study provides new insights into the genetic network and potential biological processes associated with the molecular microbial ecology of DW microbial communities.

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The microbial composition of concrete biofilms within wastewater collection systems was studied using molecular assays. SSU rDNA clone libraries were generated from 16 concrete surfaces of manholes, a combined sewer overflow, and sections of a corroded sewer pipe. Of the 2457 sequences analyzed, α-, β-, γ-, and δ-Proteobacteria represented 15%, 22%, 11%, and 4% of the clones, respectively. β-Proteobacteria (47%) sequences were more abundant in the pipe crown than any of the other concrete surfaces. While 178 to 493 Operational Taxonomic Units (OTUs) were associated with the different concrete samples, only four sequences were shared among the different clone libraries. Bacteria implicated in concrete corrosion were found in the clone libraries while archaea, fungi, and several bacterial groups were also detected using group-specific assays. The results showed that concrete sewer biofilms are more diverse than previously reported. A more comprehensive molecular database will be needed to better study the dynamics of concrete biofilms.

BackgroundConcrete corrosion of wastewater collection systems is a significant cause of deterioration and premature collapse. Failure to adequately address the deteriorating infrastructure networks threatens our environment, public health, and safety. Analysis of whole-metagenome pyrosequencing data and 16S rRNA gene clone libraries was used to determine microbial composition and functional genes associated with biomass harvested from crown (top) and invert (bottom) sections of a corroded wastewater pipe.ResultsTaxonomic and functional analysis demonstrated that approximately 90% of the total diversity was associated with the phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The top (TP) and bottom pipe (BP) communities were different in composition, with some of the differences attributed to the abundance of sulfide-oxidizing and sulfate-reducing bacteria. Additionally, human fecal bacteria were more abundant in the BP communities. Among the functional categories, proteins involved in sulfur and nitrogen metabolism showed the most significant differences between biofilms. There was also an enrichment of genes associated with heavy metal resistance, virulence (protein secretion systems) and stress response in the TP biofilm, while a higher number of genes related to motility and chemotaxis were identified in the BP biofilm. Both biofilms contain a high number of genes associated with resistance to antibiotics and toxic compounds subsystems.ConclusionsThe function potential of wastewater biofilms was highly diverse with level of COG diversity similar to that described for soil. On the basis of the metagenomic data, some factors that may contribute to niche differentiation were pH, aerobic conditions and availability of substrate, such as nitrogen and sulfur. The results from this study will help us better understand the genetic network and functional capability of microbial members of wastewater concrete biofilms.

Monochloramine is an increasingly used drinking water disinfectant and has been shown to increase nitrifying bacteria and mycobacteria in drinking waters. The potential successions and development of these bacteria were examined by 16S rRNA gene clone libraries generated from various biofilms within a water distribution system simulator. Biofilms were obtained from in-line and off-line devices using borosilicate glass beads, along with polycarbonate coupons from annular reactors incubated for up to 8 months in monochloramine-treated drinking water. No significant difference in community structures was observed between biofilm devices and coupon material; however, all biofilm communities that developed on different devices underwent similar successions over time. Early stages of biofilm formation were dominated by Serratia (29%), Cloacibacterium (23%), Diaphorobacter (16%), and Pseudomonas (7%), while Mycobacterium-like phylotypes were the most predominant populations (> 27%) in subsequent months. The development of members of the nontuberculous mycobacteria (NTM) after 3 months may impact individuals with predisposing conditions, while nitrifiers (related to Nitrospira moscoviensis and Nitrosospira multiformis) could impact water quality. Overall, 90% of the diversity in all the clone library samples was associated with the phyla Proteobacteria, Actinobacteria, and Bacteroidetes. These results provide an ecological insight into biofilm bacterial successions in monochloramine-treated drinking water.

CIRCOS plots representing the pan-genome and resistome of waterborne resistant bacteria.

We investigated the bacterial composition of water samples from two service areas within a drinking water distribution system (DWDS), each associated with a different primary source of water (groundwater, GW; surface water, SW) and different treatment process. Community analysis based on 16S rRNA gene clone libraries indicated that Actinobacteria (Mycobacterium spp.) and α-Proteobacteria represented nearly 43 and 38% of the total sequences, respectively. Sequences closely related to Legionella, Pseudomonas, and Vibrio spp. were also identified. In spite of the high number of sequences (71%) shared in both areas, multivariable analysis revealed significant differences between the GW and SW areas. While the dominant phylotypes where not significantly contributing in the ordination of samples, the populations associated with the core of phylotypes (1-10% in each sample) significantly contributed to the differences between both service areas. Diversity indices indicate that the microbial community inhabiting the SW area is more diverse and contains more distantly related species coexisting with local assemblages as compared with the GW area. The bacterial community structure of SW and GW service areas were dissimilar, suggesting that their respective source water and/or water quality parameters shaped by the treatment processes may contribute to the differences in community structure observed.

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly 62% of all DNA sequences analyzed. Other phylogenetic groups identified included Proteobacteria (20%), Actinobacteria (9%), Cyanobacteria (4%), and Bacteroidetes (2%). The composition of RNA-based libraries (1122 sequences) was similar to the DNA-based libraries with a few notable exceptions: Proteobacteria were more dominant in the RNA clone libraries (i.e., 35% RNA; 20% DNA). Differences in the Proteobacteria composition were also observed; alpha-Proteobacteria was 22 times more abundant in the RNA-based clones while beta-Proteobacteria was eight times more abundant in the DNA libraries. Nearly twice as many DNA operational taxonomic units (OTUs) than RNA OTUs were observed at distance 0.03 (101 DNA; 53 RNA). Twenty-four OTUs were shared between all RNA- and DNA-based libraries (OTU0.03) representing only 18% of the total OTUs, but 81% (1527/1883) of all sequences. Such differences between clone libraries demonstrate the necessity of generating both RNA- and DNA-derived clone libraries to compare these two different molecular approaches for community analyses.