Bacterial community radial-spatial distribution in biofilms along pipe wall in chlorinated drinking water distribution system of East China

Applied Microbiology and Biotechnology - Biofilms in the pipe wall may lead to water quality deterioration and biological instability in drinking water distribution systems (DWDSs). In this study,...     

Phylogenetic diversity of drinking water bacteria in a distribution system simulator

AIMS: To characterize the composition of microbial populations in a distribution system simulator (DSS) by direct sequence analysis of 16S rDNA clone libraries. METHODS AND RESULTS: Bacterial populations were examined in chlorinated distribution water and chloraminated DSS feed and discharge water. Bacterial strains isolated from DSS discharge water on R2A medium were identified using 16S rDNA sequence analysis. The majority of the bacteria identified were alpha-proteobacteria, ranging from approx. 34% in the DSS discharge water to 94% of the DSS isolates. Species richness estimators Chao1 and ACE (abundance-based coverage estimators) indicated that the chlorinated distribution water sample was representative of the total population diversity, while the chloraminated DSS feed water sample was dominated by Hyphomicrobium sp. sequences. The DSS discharge water contained the greatest diversity of alpha-, beta-, gamma-proteobacteria, with 36% of the sequences being operational taxonomic units (OTUs, sequences with >97.0% homology). CONCLUSIONS: This work demonstrated the dominance of alpha-proteobacteria in distribution system water under two different disinfectant residuals. The shift from chlorine to monochloramine residual may have played a role in bacterial population dynamics. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of bacteria present in treated drinking water is needed in order to better determine the risk of regrowth of potentially pathogenic organisms within distribution systems.     

Genotoxic effects of various chlorinated butenoic acids identified in chlorinated drinking water

The mutagenic activities of the chlorinated butenoic acids recently identified in chlorinated drinking waters were determined by the Salmonella microsome assay and by the SOS chromotest. The Salmonella typhimurium tester strains TA97, TA98 and TA100 were used without and with S9 mix. In the SOS chromotest Escherichia coli PQ37 was used as an indicator organism with and without metabolic activation. In addition, the extremely potent Ames test mutagen (Z)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid (MX, in the open form), was studied by the micronucleus test with mice using intraperitoneal treatment. The results of the Salmonella assay and the SOS chromotest showed that MX was by far the most potent mutagen of the compounds tested. Mutations were also induced by the reduced form of MX, (Z)-2-chloro-3-(dichloromethyl)-4-hydroxybut-2-enoic acid (red-MX), and by the geometric isomer of MX, (E)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid (EMX). However, since the solution of EMX contained approximately 5% MX, most of its activity might be attributable to MX. The oxidised form of EMX, (E)-2-chloro-3-(dichloromethyl)-butenedioic acid (ox-EMX), was marginally active in the SOS chromotest only. All these compounds were directly acting mutagens and in the presence of metabolic activation (S9 mix) they did not generate mutagenicity. The oxidised form of MX, (Z)-2-chloro-3-(dichloromethyl)-butenedioic acid (ox-MX), was not mutagenic at the dose levels tested. MX did not induce micronuclei in the bone marrow of mice.     

Identification of culturable bacteria present in haemodialysis water and fluid

Water used to prepare haemodialysis fluid is not sterile, and its microbiological control is important for the prevention of haemodialysis-associated illness. Bacterial populations inhabiting a distribution system for haemodialysis water were studied over an 18-month period. 203 planktonic bacteria isolated on R2A medium were identified by restriction analysis and sequencing of 16S rRNA gene. A diverse bacterial community was detected, containing predominantly Gram-negative members of the Alphaproteobacteria and Betaproteobacteria, as well as representatives of the genus Mycobacterium. Ecological and clinical consequences are discussed: bacteria from the genera Novosphingobium, Pseudomonas and Sphingomonas have been described in the build-up of biofilms, and others like Acinetobacter, Mycobacterium or Brevibacterium may represent a health risk to patients under haemodialysis treatment.     

Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Inhibition of growth ofLegionella species by heterotrophic plate count bacteria isolated from chlorinated drinking water

The ability of heterotrophic plate count bacterial strains isolated from chlorinated drinking water on low-nutrient media to inhibit the growth ofLegionella species was examined. Between 16% and 32% of these strains were able to inhibit the growth ofLegionella species when tested on buffered charcoal yeast extract agar. The exact proportion of inhibiting strains varied with the individualLegionella species. Two strains that inhibited the growth of severalLegionella species could also stimulate the growth of the same species when both the test strain and theLegionella species were grown on buffered charcoal yeast extract agar that lacked the essential amino acidl-cysteine.     

Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters.

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Influence of phosphorus on biofilm formation in model drinking water distribution systems

Aims:  This study investigated the effects of phosphorus on biofilm formation via annular reactor systems in terms of biofilm cell growth, exopolysaccharide (EPS) production, biofilm structure and cell metabolic potential.
Methods and Results:  Drinking water biofilms were developed in annular reactors with supplement of carbon and different levels of phosphorus. The biofilm formation was monitored over a period of 30 days. Biofilm related parameters were examined by various methods, which included heterotrophic plate count, total carbohydrate content, confocal laser scanning microscopy and GN2 microplate assay. Our results showed that phosphorus addition can promote the biofilm cell growth (cell count increased about 1 log with addition of 30 and 300 μg l⁻¹ of phosphorus). However, the addition of 30 and 300 μg l⁻¹ of phosphorus caused 81% and 77% decrease in EPS production, respectively. The results of biofilm structure analysis showed that the addition of 30 and 300 μg l⁻¹ of phosphorus can induce thicker and less homogeneous biofilms with more biomass. Furthermore, the addition of 30 and 300 μg l⁻¹ of phosphorus dramatically increased the biofilm cell metabolic potential. The addition of 3 μg l⁻¹ of phosphorus was found to have minor effects on the parameters examined.
Conclusions:  The results indicate phosphorus addition to drinking water distribution system (DWDS) has a complicated effect on the biofilm formation.
Significance and Impact of the Study:  As the addition of phosphorus at certain levels can affect the biofilm growth in DWDS, care should be taken when phosphate-based corrosion inhibitors are used in the DWDS.     

Factors promoting survival of bacteria in chlorinated water supplies

Results of our experiments showed that the attachment of bacteria to surfaces provided the greatest increase in disinfection resistance. Attachment of unencapsulated Klebsiella pneumoniae grown in medium with high levels of nutrients to glass microscope slides afforded the microorganisms as much as a 150-fold increase in disinfection resistance. Other mechanisms which increased disinfection resistance included the age of the biofilm, bacterial encapsulation, and previous growth conditions (e.g., growth medium and growth temperature). These factors increased resistance to chlorine from 2- to 10-fold. The choice of disinfectant residual was shown to influence the type of resistance mechanism observed. Disinfection by free chlorine was affected by surfaces, age of the biofilm, encapsulation, and nutrient effects. Disinfection by monochloramine, however, was only affected by surfaces. Importantly, results showed that these resistance mechanisms were multiplicative (i.e., the resistance provided by one mechanism could be multiplied by the resistance provided by a second mechanism).     

High levels of mutagenic activity in chlorinated drinking water in Finland

The organic extract from the drinking water of Kuopio, Eastern Finland, turned out to be highly mutagenic in the Ames test. The direct mutagenic activities of the acid/neutral fractions of 48 drinking water samples were on an average 1700 net revertants/l in strain TA100. The highest activities were more than 6000 net revertants/l, and one drinking water sample exceeded 10 000 net revertants/l. The drinking water of Kuopio is produced from lake water which has a relatively high content of humic substances. Water processing involved two chlorination phases. The organic constituents of the water samples were isolated either by continuous liquid-liquid extraction or by absorption on XAD 8 resin.     

Identification of bacteria in biofilm and bulk water samples from a nonchlorinated model drinking water distribution system: detection of a large nitrite-oxidizing population associated with Nitrospira spp

In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 microg/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P = 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 x 10(5) to 2 x 10(5) nitrifying cells/ml in the bulk water and 3 x 10(5) cells/cm(2) on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.     

Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant

The complex ecology of free-living amoebae (FLA) and their role in spreading pathogenic microorganisms through water systems have recently raised considerable interest. In this study, we investigated the presence of FLA and amoebae-resisting bacteria (ARB) at various stages of a drinking water plant fed with river water. We isolated various amoebal species from the river and from several points within the plant, mostly at early steps of water treatment. Echinamoeba- and Hartmannella-related amoebae were mainly recovered in the drinking water plant whereas Acanthamoeba- and Naegleria-related amoebae were recovered from the river water and the sand filtration units. Some FLA isolates were recovered immediately after the ozonation step, thus suggesting resistance of these microorganisms to this disinfection procedure. A bacterial isolate related to Mycobacterium mucogenicum was recovered from an Echinamoeba-related amoeba isolated from ozone-treated water. Various other ARB were recovered using co-culture with axenic Acanthamoeba castellanii, including mycobacteria, legionella, Chlamydia-like organisms and various proteobacteria. Noteworthy, a new Parachlamydia acanthamoebae strain was recovered from river water and from granular activated carbon (GAC) biofilm. As amoebae mainly multiply in sand and GAC filters, optimization of filter backwash procedures probably offers a possibility to better control these protists and the risk associated with their intracellular hosts.     

Factors promoting survival of bacteria in chlorinated water supplies.

Results of our experiments showed that the attachment of bacteria to surfaces provided the greatest increase in disinfection resistance. Attachment of unencapsulated Klebsiella pneumoniae grown in medium with high levels of nutrients to glass microscope slides afforded the microorganisms as much as a 150-fold increase in disinfection resistance. Other mechanisms which increased disinfection resistance included the age of the biofilm, bacterial encapsulation, and previous growth conditions (e.g., growth medium and growth temperature). These factors increased resistance to chlorine from 2- to 10-fold. The choice of disinfectant residual was shown to influence the type of resistance mechanism observed. Disinfection by free chlorine was affected by surfaces, age of the biofilm, encapsulation, and nutrient effects. Disinfection by monochloramine, however, was only affected by surfaces. Importantly, results showed that these resistance mechanisms were multiplicative (i.e., the resistance provided by one mechanism could be multiplied by the resistance provided by a second mechanism).     

Phylogenetic analysis of culturable dimethyl sulfide-producing bacteria from a spartina-dominated salt marsh and estuarine water

Dimethylsulfoniopropionate (DMSP), an abundant osmoprotectant found in marine algae and salt marsh cordgrass, can be metabolized to dimethyl sulfide (DMS) and acrylate by microbes having the enzyme DMSP lyase. A suite of DMS-producing bacteria isolated from a salt marsh and adjacent estuarine water on DMSP agar plates differed markedly from the pelagic strains currently in culture. While many of the salt marsh and estuarine isolates produced DMS and methanethiol from methionine and dimethyl sulfoxide, none appeared to be capable of producing both methanethiol and DMS from DMSP. DMSP, and its degradation products acrylate and beta-hydroxypropionate but not methyl-3-mecaptopropionate or 3-mercaptopropionate, served as a carbon source for the growth of all the alpha- and beta- but only some of the gamma-proteobacterium isolates. Phylogenetic analysis of 16S rRNA gene sequences showed that all of the isolates were in the group Proteobacteria, with most of them belonging to the alpha and gamma subclasses. Only one isolate was identified as a beta-proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species of Alcaligenes faecalis. Although bacterial population analysis based on culturability has its limitations, bacteria from the alpha and gamma subclasses of the Proteobacteria were the dominant DMS producers isolated from salt marsh sediments and estuaries, with the gamma subclass representing 80% of the isolates. The alpha-proteobacterium isolates were all in the Roseobacter subgroup, while many of the gamma-proteobacteria were closely related to the pseudomonads; others were phylogenetically related to Marinomonas, Psychrobacter, or Vibrio species. These data suggest that DMSP cleavage to DMS and acrylate is a characteristic widely distributed among different phylotypes in the salt marsh-estuarine ecosystem.     

Long-term succession of structure and diversity of a biofilm formed in a model drinking water distribution system

In this study, we examined the long-term development of the overall structural morphology and community composition of a biofilm formed in a model drinking water distribution system with biofilms from 1 day to 3 years old. Visualization and subsequent quantification showed how the biofilm developed from an initial attachment of single cells through the formation of independent microcolonies reaching 30 micro m in thickness to a final looser structure with an average thickness of 14.1 micro m and covering 76% of the surface. An analysis of the community composition by use of terminal restriction fragment length polymorphisms showed a correlation between the population profile and the age of the sample, separating the samples into young (1 to 94 days) and old (571 to 1,093 days) biofilms, whereas a limited spatial variation in the biofilm was observed. A more detailed analysis with cloning and sequencing of 16S rRNA fragments illustrated how a wide variety of cells recruited from the bulk water initially attached and resulted in a species richness comparable to that in the water phase. This step was followed by the growth of a bacterium which was related to Nitrospira, which constituted 78% of the community by day 256, and which resulted in a reduction in the overall richness. After 500 days, the biofilm entered a stable population state, which was characterized by a greater richness of bacteria, including Nitrospira, Planctomyces, Acidobacterium, and Pseudomonas. The combination of different techniques illustrated the successional formation of a biofilm during a 3-year period in this model drinking water distribution system.