| Chlorine is the most commonly used disinfectant due to its effectiveness, stability, easy of use, and low cost. Chlorine disinfection process will be a selection process for chlorine-resistant bacteria. In drinking water distribution systems(DWDS), chlorine-resistant bacteria would endanger drinking water microbial safety. To solve this problem, the population and chlorine resistance mechanisms of chlorine-resistant bacteria in the DWDS were investigated. It is critical to understand the distribution and resistance mechanisms of chlorine-resistant bacteria in DWDS for the assessment, control, and prevention of public health risks associated with drinking water.From the drinking water treatment plant to the terminal of DWDS in Northeast China, chlorine-resistant bacteria counts of bulk water and biofilm in different sampling points were investigated, and the chlorine-resistant bacterial diversity in biofilm was analyzed by Illumina Mi Seq high-throughput sequencing technology. Results showed that chlorine-resistant bacteria survived pervasively in the studied DWDS. And the chlorine-resistant bacteria counts in the biofilm were higher than in the bulk water with one orders of magnitude at least. The bacterial diversity in the clearwell was very low. But in the DWDS, the bacterial diversity presented gradual increase with the extension of the pipe network. In the terminal of branch pipe network, the bacterial diversity also significantly increased. Pseudomonas, Bacillus, Acinetobacter, Sphingobium, Sphingomonas, Acidovorax and Rhodopseudomonas were predominant in the front and middle of DWDS, and were all relatively resistant to chlorine. Chlorine-nonresistant bacteria were predominant in the terminal of DWDS where there were a certain proportion of the nitrifying bacteria(Nitrosomonadaceae and Nitrospira).The pipe materials and ?ow velocities may be variable in DWDS. The effects of pipe materials and ?ow velocities on the chlorine-resistant bacteria in biofilm and bulk water under more than 0.97 mg/L free chlorine were investigated by heterotrophic plate counts, flow cytometric and denaturing gradient gel electrophoresis. Results showed that both chlorine-resistant bacteria counts and diversity were varied in pipe materials, while flow velocities only affect chlorine-resistant bacteria counts in biofilm. In the essentially mature biofilm, the chlorine-resistant bacteria counts in the biofilms formed on the five pipe materials were ranked in the order: cast iron > concrete > copper > polyethylene > stainless steel 304. In the biofilm community structure, Moraxella osloensis(member of Gammaproteobacteria) and Sphingomonas sp.(member of Alphaproteobacteria) were observed in all the pipe materials, while Bacillus sp.(member of Firmicutes) was detected except in the concrete pipe. Other bacteria were only found from one to three pipe materials. It is noteworthy that there are eleven opportunistic pathogens in the seventeen classified bacterial strains.The purpose of the present study was to investigate the effects of chlorine-resistant bacterial diversities and interactions on biofilm formation and chlorine resistance. Five chlorine-resistant bacteria, viz. Acinetobacter sp., Acidovorax defluvii, Bacillus cereus, Microbacterium laevaniformans and Sphingomonas sp. isolated from a model DWDS were used to form single, dual and multispecies biofilms by 96-well microtiter-plate assays, and these 72 h cultured biofilms were used to assess their resistance to chlorine. Biofilms were characterized in terms of mass, metabolic activity and plate counts. Results showed that all of single-, dual- and multi- species were able to formed biofilms within 72 h. The largest single biofilm amounts was found for Acinetobacter sp. and the least was Acidovorax defluvii. Evidences of microbial interactions in dual-species biofilm formation were found for the following cases: synergy/cooperation was found for Acidovorax+Acinetobacter, Sphingomonas+Bacillus, Sphingomonas+Acidovorax and Acidovorax+Microbacterium, and antagonism/competition was found for Acinetobacter+Microbacterium, Sphingomonas+Microbacterium and Sphingomonas+Acinetobacter. A neutral interaction was found for Acidovorax+ Bacillus, Bacillus+Acinetobacter and Bacillus+Microbacterium. Multispecies of bacteria produced strong biofilms for the three sampling times. Five mixed multispecies biofilm formation was weakly inhibited in the present of Microbacterium laevaniformans, and was weakly increased in the present of any of the three species(Acinetobacter sp., Acidovorax defluvii and Bacillus cereus).While five mixed multispecies biofilm formation was weakly increased in the first, and then was weakly inhibited in the present of Sphingomonas sp.The chlorine resistance of bacteria in single-species biofilm ranked from high to low: Bacillus cereus > Sphingomonas sp. > Acidovorax defluvii > Microbacterium laevaniformans > Acinetobacter sp.. Bacillus cereus had the highest chlorine resistance, as this species could produce spores, and the optimal spores producing concentrations of chlorine were 0.6 mg/L and 1 mg/L. In the dual species biofilm, due to Microbacterium laevaniformans competition or neutrality with the other four species, chlorine resistance of bacteria in dual species biofilm composed of Microbacterium laevaniformans were inhibited to some extent. Multispecies biofilm bacteria were more resistant to chlorine than single- and dual- species biofilm bacteria. The chlorine resistance of five mixed multispecies biofilm bacteria was strongly improved in the present of Bacillus cereus, while the chlorine resistance was inhibited in the present of Microbacterium laevaniformans. So there are possible approaches to change chlorine resistance of multispecies biofilm by the control of these two species(Bacillus cereus and Microbacterium laevaniformans) in real DWDS. |
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