Disinfection of bacteria attached to granular activated carbon.

Heterotrophic plate count bacteria, coliform organisms, and pathogenic microorganisms attached to granular activated carbon particles were examined for their susceptibility to chlorine disinfection. When these bacteria were grown on carbon particles and then disinfected with 2.0 mg of chlorine per liter (1.4 to 1.6 mg of free chlorine residual per liter after 1 h) for 1 h, no significant decrease in viable counts was observed. Washed cells attached to the surface of granular activated carbon particles showed similar resistance to chlorine, but a progressive increase in sublethal injury was found. Observations made by scanning electron microscope indicated that granular activated carbon was colonized by bacteria which grow in cracks and crevices and are coated by an extracellular slime layer. These data suggest a possible mechanism by which treatment and disinfection barriers can be penetrated and pathogenic bacteria may enter drinking water supplies.     

A pilot study of bacteriological population changes through potable water treatment and distribution

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.     

A Pilot Study of Bacteriological Population Changes through Potable Water Treatment and Distribution

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.     

Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 x 10(5) bacteria ml(-1). The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 x 10(3) active AOB ml(-1) were detected in the membrane-intact fraction, and 1.40 x 10(4) active AOB ml(-1) were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml(-1) detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.     

A review of drinking-water-associated endotoxin,including potential routes of human exposure

In the past decade efforts have been made to reduce the formation of harmful disinfection byproducts during the treatment and distribution of drinking water. This has been accomplished in part by the introduction of processes that involve the deliberate encouragement of indigenous biofilm growth in filters. In a controlled environment, such as a filter, these biofilms remove compounds that would otherwise be available as disinfection byproduct precursors or support uncontrolled biological activity in distribution systems. In the absence of exposure to chlorinated water, most biofilm bacteria are gram negative and have an outer layer that contains endotoxin. To date, outbreaks of waterborne endotoxin-related illness attributable to contamination of water used in hemodialysis procedures have been only infrequently documented, and occurrences linked to ingestion or through dermal abrasions could not be located. However, a less obvious conduit, that of inhalation, has been described in association with aerosolized water droplets. This review summarizes documented drinking-water-associated incidents of endotoxin exposure attributable to hemodialysis and inhalation. Typical endotoxin levels in water and conditions under which substantial quantities can enter drinking water distribution systems are identified. It would appear that endotoxin originating in tap water can be inhaled but at present there is insufficient information available to quantify potential health risks.     

Colonization and Disinfection of Biofilms Hosting Coliform-Colonized Carbon Fines

The documented release of carbon fines from granular activated carbon filters is a concern for drinking water utilities, since these particles may carry coliform and even pathogenic bacteria through the disinfection barrier. Such a breakthrough could have an impact on distribution system biofilms. Using total cell counts, specific monoclonal antibody staining, and computerized image analysis, we monitored the colonization of introduced Klebsiella pneumoniae associated with carbon fines in mixed-population biofilms. The particles transported the coliforms to the biofilms and allowed successful colonization. Chlorine (0.5 mg/liter) was then applied as a disinfectant. Most K. pneumoniae along with the carbon fines left the biofilm under these conditions. The impact of chlorine was greater on the coliform bacteria and carbon fines than on the general fixed bacterial population. However, 10% of the introduced coliforms and 20% of the fines remained in the biofilm. The possibility that this represents a mechanism for bacteria of public health concern to be involved in regrowth events is discussed.     

Annual cancer risks from chemicals in North Carolina community water systems

Motivated partly by concerns about cancer, the U.S. Congress in 1986 amended the Safe Drinking Water Act (SDWA) by requiring that community water systems monitor 81 chemicals and remove those detected at concentrations above health-based standards. No prior research has used the resulting 30 years of monitoring data to analyze cancer risks from chemicals in US drinking water. To fill this gap, this paper uses chemical monitoring data from North Carolina's (NC's) 2,120 community water systems along with a risk assessment approach commonly applied in global burden of disease studies to quantify cancer risks of regulated chemicals in drinking water. The results indicate that 0.30% of NC cancer deaths are attributable to regulated drinking water contaminants and that the average annual individual risk is 7.2 × 10^?6. More than 99% of this risk arises from disinfection by-products, with the remaining risk mostly attributable to arsenic and alpha particle radiation. In no water system does the combined risk from chemicals other than disinfection by-products, arsenic, or alpha particles exceed 10^?4. The results suggest that regulated chemicals pose very low cancer risks and that risks from chemicals other than disinfection by-products, arsenic, and alpha particles are negligible in NC community water systems.     

Comparison of the mutagenic activity of XAD4 and blue rayon extracts of surface water and related drinking water samples

The combination of mutagenicity tests and selective extraction methodologies can be useful to indicate the possible classes of genotoxic organic contaminants in water samples. Treated and source water samples from two sites were analyzed: a river under the influence of an azo dye-processing plant discharge and a reservoir not directly impacted with industrial discharges, but contaminated with untreated domestic sewage. Organic extraction was performed in columns packed with XAD4 resin, that adsorbs a broad class of mutagenic compounds like polycyclic aromatic hydrocarbons (PAHs), arylamines, nitrocompounds, quinolines, antraquinones, etc., including the halogenated disinfection by-products; and with blue rayon that selectively adsorbs polycyclic planar structures. The organic extracts were tested for mutagenicity with the Salmonella assay using TA98 and TA100 strains and the potencies were compared. A protocol for cleaning the blue rayon fibers was developed and the efficiency of the reused fibers was analyzed with spiked samples. For the river water samples under the influence of the azo-type dye-processing plant, the mutagenicity was much higher for both blue rayon and XAD4 extracts when compared to the water from the reservoir not directly impacted with industrial discharges. For the drinking water samples, although both sites showed mutagenic responses with XAD4, only samples from the site under the influence of the industrial discharge showed mutagenic activity with the blue rayon extraction, suggesting the presence of polycyclic compounds in those samples. As expected, negative results were found with the blue rayon extracts of the drinking water collected from the reservoir not contaminated with industrial discharges. In this case, it appears that using the blue rayon to extract drinking water samples and comparing the results with the XAD resin extracts we were able to distinguish the mutagenicity caused by industrial contaminants from the halogenated disinfection by-products generated during water treatment.     

Formation of natural biofilms during chlorine dioxide and u.v. disinfection in a public drinking water distribution system

AIMS: The influence of two disinfection techniques on natural biofilm development during drinking water treatment and subsequent distribution is compared with regard to the supply of a high-quality drinking water. METHODS AND RESULTS: The growth of biofilms was studied using the biofilm device technique in a real public technical drinking water asset. Different pipe materials which are commonly used in drinking water facilities (hardened polyethylene, polyvinyl chloride, steel and copper) were used as substrates for biofilm formation. Apart from young biofilms, several months old biofilms were compared in terms of material dependence, biomass and physiological state. Vital staining of biofilms with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and the DNA-specific 4',6-diamidino-2-phenylindole (DAPI) staining resulted in a significant difference in physiological behaviour of biofilm populations depending on the disinfection technique. Compared with chlorine dioxide disinfection (0.12-0.16 mg l-1), the respiratory activities of the micro-organisms were increased on all materials during u.v. disinfection (u.v.254; 400 J m-2). The biofilm biocoenosis was analysed by in situ hybridization with labelled oligonucleotides specific for some subclasses of Proteobacteria. Using PCR and additional hybridization techniques, the biofilms were also tested for the presence of Legionella spp., atypical mycobacteria and enterococci. The results of the molecular-biological experiments in combination with cultivation tests showed that enterococci were able to pass the u.v. disinfection barrier and persist in biofilms of the distribution system, but not after chlorine dioxide disinfection. CONCLUSIONS: The results indicated that bacteria are able to regenerate and proliferate more effectively after u.v. irradiation at the waterworks, and chlorine dioxide disinfection appears to be more applicative to maintain a biological stable drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as the application of u.v. disinfection is used for conditioning of critical water sources for drinking water, the efficiency of u.v. irradiation in natural systems should reach a high standard to avoid adverse impacts on human health.     

Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 10⁵ bacteria ml⁻¹. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 × 10³ active AOB ml⁻¹ were detected in the membrane-intact fraction, and 1.40 × 10⁴ active AOB ml⁻¹ were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml⁻¹ detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.     

Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli

A cDNA of fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30-40% and 60-75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard (Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid beta-oxidation mutant (fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way.     

Bacteriology of drinking water distribution systems: an integral and multidimensional review

A drinking water distribution system (DWDS) is the final and essential step to supply safe and high-quality drinking water to customers. Biological processes, such as biofilm formation and detachment, microbial growth in bulk water, and the formation of loose deposits, may occur. These processes will lead to deterioration of the water quality during distribution. In extreme conditions, pathogens and opportunistic pathogens may proliferate and pose a health risk to consumers. It is, therefore, necessary to understand the bacteriology of DWDSs to develop effective strategies that can ensure the water quality at consumers' taps. The bacteriology of DWDSs, both the quantitative growth and the qualitative bacterial community, has attracted considerable research attention. However, the researchers have focused mainly on the pipe wall biofilm. In this review, DWDS bacteriology has been reviewed multidimensionally, including both the bacterial quantification and identification. For the first time, the available literature was reviewed with an emphasis on the subdivision of DWDS into four phases: bulk water, suspended solids, loose deposits, and pipe wall biofilm. Special concentration has been given to potential contribution of particulate matter: suspended particles and loose deposits. Two highlighted questions were reviewed and discussed: (1) where does most of the growth occur? And (2) what is the contribution of particle-associated bacteria to DWDS bacteriology and ecology? At the end of this review, recommendations were given based on the conclusion of this review to better understand the integral DWDS bacteriology.     

In vivo labeling and specific magnetic bead separation of RNA for biofilm characterization and stress-induced gene expression analysis in bacteria

The method of in vivo labeling and separation of bacterial RNA was developed as an approach to elucidating the stress response of natural bacterial populations. This technique is based on the incorporation of digoxigenin-11-uridine-5′-triphosphate (DIG-11-UTP) in the RNA of active bacteria. The digoxigenin fulfills a dual role as a label of de novo synthesized RNA and a target for magnetic bead separation from a total RNA extract.Depending on the growth conditions and the population's composition, the assembly rate of DIG-11-UTP ranged from 1.2% to 12.5% of the total RNA in gram-positive and gram-negative reference bacteria as well as in natural biofilms from drinking water, surface water, and lake sediment. Separation of DIG-RNA from total RNA extracts was performed with a biotinylated anti-digoxigenin antibody and streptavidin-functionalized magnetic particles. The average separation yield from total RNA extracts was about 95% of labeled RNA. The unspecific bindings of non-labeled nucleic acids were smaller than 0.2%, as was evaluated by spiking experiments with an unmarked DNA amplicon. Applicability of the method developed was demonstrated by rRNA-directed PCR-DGGE population analysis of natural biofilms and expression profiling of two stress-induced genes (vanA and rpoS) in reference bacteria.     

Bacteria associated with granular activated carbon particles in drinking water

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Microbial ecology of drinking water distribution systems

The supply of clean drinking water is a major, and relatively recent, public health milestone. Control of microbial growth in drinking water distribution systems, often achieved through the addition of disinfectants, is essential to limiting waterborne illness, particularly in immunocompromised subpopulations. Recent inquiries into the microbial ecology of distribution systems have found that pathogen resistance to chlorination is affected by microbial community diversity and interspecies relationships. Research indicates that multispecies biofilms are generally more resistant to disinfection than single-species biofilms. Other recent findings are the increased survival of the bacterial pathogen Legionella pneumophila when present inside its protozoan host Hartmannella vermiformis and the depletion of chloramine disinfectant residuals by nitrifying bacteria, leading to increased overall microbial growth. Interactions such as these are unaccounted for in current disinfection models. An understanding of the microbial ecology of distribution systems is necessary to design innovative and effective control strategies that will ensure safe and high-quality drinking water.     

Shift in the Microbial Ecology of a Hospital Hot Water System following the Introduction of an On-Site Monochloramine Disinfection System

Drinking water distribution systems, including premise plumbing, contain a diverse microbiological community that may include opportunistic pathogens. On-site supplemental disinfection systems have been proposed as a control method for opportunistic pathogens in premise plumbing. The majority of on-site disinfection systems to date have been installed in hospitals due to the high concentration of opportunistic pathogen susceptible occupants. The installation of on-site supplemental disinfection systems in hospitals allows for evaluation of the impact of on-site disinfection systems on drinking water system microbial ecology prior to widespread application. This study evaluated the impact of supplemental monochloramine on the microbial ecology of a hospital’s hot water system. Samples were taken three months and immediately prior to monochloramine treatment and monthly for the first six months of treatment, and all samples were subjected to high throughput Illumina 16S rRNA region sequencing. The microbial community composition of monochloramine treated samples was dramatically different than the baseline months. There was an immediate shift towards decreased relative abundance of Betaproteobacteria, and increased relative abundance of Firmicutes, Alphaproteobacteria, Gammaproteobacteria, Cyanobacteria and Actinobacteria. Following treatment, microbial populations grouped by sampling location rather than sampling time. Over the course of treatment the relative abundance of certain genera containing opportunistic pathogens and genera containing denitrifying bacteria increased. The results demonstrate the driving influence of supplemental disinfection on premise plumbing microbial ecology and suggest the value of further investigation into the overall effects of premise plumbing disinfection strategies on microbial ecology and not solely specific target microorganisms.     

Evaluation of a fluorescent antibody technique for the rapid enumeration of Bacteroides fragilis group of organisms in water

The Bacteroides fragilis group has been evaluated as a prospective rapid indicator of faecal contamination of water. Fluorescent antibody (FA) stained B. fragilis group bacteria were enumerated microscopically and compared with faecal coliform or Escherichia coli counts as indicators of faecal contamination. Environmental samples included surface waters (raw drinking water and known contaminated water). Laboratory disinfection experiments with ozone, chlorine and u.v. radiation were also performed. Bacteroides FA counts specifically detected recent human faecal contamination in field samples in 2-3 h. Samples with a high content of particulates or debris limited the sensitivity to about 10 FA counts/ml. Viable counts showed that the sensitivity to all three disinfection agents was essentially the same for Bacteroides and E. coli. Fluorescent antibody counts of Bacteroides, conversely, were not altered by any of the agents. Therefore, the Bacteroides FA method is not recommended for routine monitoring but may be useful for cases where extensive human faecal contamination is suspected (e.g. pipeline rupture or pollution of recreational water) and where rapid remedial action must be taken to protect the public health.