Microbiology, chemistry and biofilm development in a pilot drinking water distribution system with copper and plastic pipes

We studied the changes in water quality and formation of biofilms occurring in a pilot-scale water distribution system with two generally used pipe materials: copper and plastic (polyethylene, PE). The formation of biofilms with time was analysed as the number of total bacteria, heterotrophic plate counts and the concentration of ATP in biofilms. At the end of the experiment (after 308 days), microbial community structure, viable biomass and gram-negative bacterial biomass were analysed via lipid biomarkers (phospholipid fatty acids and lipopolysaccharide 3-hydroxy fatty acids), and the numbers of virus-like particles and total bacteria were enumerated by SYBR Green I staining. The formation of biofilm was slower in copper pipes than in the PE pipes, but after 200 days there was no difference in microbial numbers between the pipe materials. Copper ion led to lower microbial numbers in water during the first 200 days, but thereafter there were no differences between the two pipe materials. The number of virus-like particles was lower in biofilms and in outlet water from the copper pipes than PE pipes. Pipe material influenced also the microbial and gram-negative bacterial community structure in biofilms and water.     

Pipeline materials modify the effectiveness of disinfectants in drinking water distribution systems

We studied how pipe material can modify the effectiveness of UV- and chlorine disinfection in drinking water and biofilms. This study was done with two pipe materials: copper and composite plastic (polyethylene, PE) in a pilot scale water distribution network. UV-disinfection decreased viable bacterial numbers in the pilot waterworks and outlet water of pipes on average by 79%, but in biofilms its disinfecting effect was minor. Chlorine decreased effectively the microbial numbers in water and biofilms of PE pipes. In outlet water from copper pipes, the effect of chlorination was weaker; microbial numbers increased back to the level before chlorination within a few days. In the biofilms present in the copper pipes, chlorine decreased microbial numbers only in front of the pipeline. One reason for weaker efficiency of chlorine in copper pipes was that its concentration declined more rapidly in the copper pipes than in the PE pipes. These results means that copper pipes may require a higher chlorine dosage than plastic pipes to achieve effective disinfection of the pipes.     

Effects of fluid-flow velocity and water quality on planktonic and sessile microbial growth in water hydraulic system

Water hydraulic systems use water instead of oil as a pressure medium. Microbial growth in the system may restrict the applicability this technology. The effects of fluid-flow velocity and water quality on microbial growth and biofilm formation were studied with a pilot-scale water hydraulic system. The fluid-flow velocities were 1.5-5.2 m/s and the corresponding shear stresses 9.1-84 N/m2. The fluid-flow velocity had an insignificant effect on the total bacterial numbers and the numbers of viable heterotrophic bacteria in the pressure medium. Microbial attachment occurred under high shear stresses. The fluid-flow velocity did not affect the biofilm formation in the tank. Increase in the flow velocity decreased the bacterial densities on the pipe surfaces indicating preferable biofilm formation on areas with low flow velocity. Using ultrapure water as the pressure medium decreased the total cell numbers and resulted in slower growth of bacteria in the pressure medium. Lowering the nutrient concentration retarded biofilm formation but did not affect the final cell densities. The decreasing pressure medium nutrient concentration favoured microbial attachment in the tank instead of the pipelines. In conclusion, microbial growth and biofilm formation in water hydraulic systems cannot be controlled by the fluid-flow velocity or the quality of the pressure medium.     

Removal of soft deposits from the distribution system improves the drinking water quality

Deterioration in drinking water quality in distribution networks represents a problem in drinking water distribution. These can be an increase in microbial numbers, an elevated concentration of iron or increased turbidity, all of which affect taste, odor and color in the drinking water. We studied if pipe cleaning would improve the drinking water quality in pipelines. Cleaning was arranged by flushing the pipes with compressed air and water. The numbers of bacteria and the concentrations of iron and turbidity in drinking water were highest at 9 p.m., when the water consumption was highest. Soft deposits inside the pipeline were occasionally released to bulk water, increasing the concentrations of iron, bacteria, microbially available organic carbon and phosphorus in drinking water. The cleaning of the pipeline decreased the diurnal variation in drinking water quality. With respect to iron, only short-term positive effects were obtained. However, removing of the nutrient-rich soft deposits did decrease the microbial growth in the distribution system during summer when there were favorable warm temperatures for microbial growth. No Norwalk-like viruses or coliform bacteria were detected in the soft deposits, in contrast to the high numbers of heterotrophic bacteria.     

Occurrence of nitrifiers and diversity of ammonia-oxidizing bacteria in developing drinking water biofilms

We studied the population dynamics of nitrifying bacteria during the development of biofilms up to 233 or 280 days on polyvinylchloride pipes connected to two full-scale drinking water distribution networks supplying processed and chloraminated surface water. The numbers of nitrifiers in biofilms were enumerated at intervals of 10–64 days by the most probable number (MPN) method at waterworks and at several study sites in distribution network areas. The numbers of nitrifiers increased towards the distal sites. The highest detected MPN counts of ammonia-oxidizing bacteria (AOB) for study areas 1 and 7 were 500 MPN cm⁻² and 1.0×10⁶ MPN cm⁻², and those of nitrite-oxidizing bacteria (NOB) 96 MPN cm⁻² and 2.2×10³ MPN cm⁻², respectively. The diversity of AOB was determined by PCR amplifying, cloning and sequencing the partial ammonia monooxygenase (amoA) gene of selected biofilm samples presenting different biofilm ages. The PCR primers used, A189 and A682, also amplified a fragment of particulate methane monooxygenase (pmoA) gene of methane-oxidizing bacteria. The majority of biofilm clones (24 out of 30 studied) contained Nitrosomonas amoA-like sequences. There were only two pmoA-like sequences of Type I methanotrophs, and four sequences positioned in amoA/pmoA sequence groups of uncultured bacteria. From both study area very similar or even completely identical Nitrosomonas amoA-like sequences were obtained despite of high difference in AOB numbers. The results show that the conditions in newly formed biofilms in drinking water distribution systems favor the growth of Nitrosomonas-type AOB.     

Soft deposits, the key site for microbial growth in drinking water distribution networks

In this project we studied the microbiological quality of soft pipeline deposits removed from drinking water distribution networks during mechanical cleaning. Drinking water and deposit samples were collected from 16 drinking water distribution networks located at eight towns in different parts of Finland. Soft pipeline deposits were found to be the key site for microbial growth in the distribution networks. The microbial numbers in the soft deposits were significantly higher than numbers in running water. The highest microbial numbers were detected in the main deposit pushed ahead by the first swab. The deposits contained high numbers of heterotrophic bacteria, actinomycetes and fungi. Also coliform bacteria were often isolated from deposit samples. Manganese and copper in the deposits correlated negatively with the numbers of heterotrophic bacteria. After a year, the viable microbial numbers in the new deposits were almost as high as in the old deposits before the first mechanical cleaning. The bacterial biomass production was higher in the new than in the old deposits.     

Dynamics of drinking water biofilm in flow/non-flow conditions

Drinking water biofilm formation on polyvinyl chloride (PVC), cross-linked polyethylene (PEX), high density polyethylene (HDPE) and polypropylene (PP) was followed in three different reactors operating under stagnant or continuous flow regimes. After one week, a quasi-steady state was achieved where biofilm total cell numbers per unit surface area were not affected by fluctuations in the concentration of suspended cells. Metabolically active cells in biofilms were around 17-35% of the total cells and 6-18% were able to form colony units in R(2)A medium. Microbiological analysis showed that the adhesion material and reactor design did not affect significantly the biofilm growth. However, operating under continuous flow (0.8-1.9 Pa) or stagnant water had a significant effect on biofilm formation: in stagnant waters, biofilm grew to a less extent. By applying mass balances and an asymptotic biofilm formation model to data from biofilms grown on PVC and HDPE surfaces under turbulent flow, specific growth rates of bacteria in the biofilm were found to be similar for both materials (around 0.15 day(-1)) and much lower than the specific growth rates of suspended bacteria (around 1.8 day(-1)).     

Role of discontinuous chlorination on microbial production by drinking water biofilms

Microbial quality in water distribution systems is strongly affected by the development of microbial biofilms. Production and release of microbial cells by the biofilm affect microbial levels in the water column and in some cases this fact constitutes a public health concern. In this study, we attempt to analyze in which way the existence of different episodes of chlorine depletion affects both biofilm formation and microbial load of an artificial laboratory system. The work was carried out using two parallel packed bed reactors both supplied with running tap water. One of the reactors was used as a control and was permanently exposed to the action of chlorine. In the other reactor, chlorine was neutralized at selected times during the experiment and for periods of variable length. During the experiment the concentration of total and viable cells from the effluent was monitored at the exit of each of the reactors. The data obtained were used to estimate microbial production from the biofilms. As an average, release of microbial cells to the water phase increased tenfold in the absence of chlorine. The results also indicate that disinfectant efficiency against the biofilm was not recovered when chlorine returned to normal levels after each event of chlorine neutralization. Cell viability in the water phase in the presence of chlorine was low at the beginning of the experiment but increased 4 orders of magnitude after five neutralization periods. Therefore, subsequent episodes of chlorine depletion may accelerate the development of microbial communities with reduced susceptibility to disinfection in real drinking water systems.     

Investigation of natural biofilms formed during the production of drinking water from surface water embankment filtration

Populations of bacteria in biofilms from different sites of a drinking water production system were analysed. Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analyses revealed changing DNA band patterns, suggesting a population shift during bank filtration and processing at the waterworks. In addition, common DNA bands that were attributed to ubiquitous bacteria were found. Biofilms even developed directly after UV disinfection (1-2m distance). Their DNA band patterns only partly agreed with those of the biofilms from the downstream distribution system. Opportunistic pathogenic bacteria in biofilms were analysed using PCR and Southern blot hybridisation (SBH). Surface water appeared to have a direct influence on the composition of biofilms in the drinking water distribution system. In spite of preceding filtration and UV disinfection, opportunistic pathogens such as atypical mycobacteria and Legionella spp. were found in biofilms of drinking water, and Pseudomonas aeruginosa was detected sporadically. Enterococci were not found in any biofilm. Bacterial cell counts in the biofilms from surface water to drinking water dropped significantly, and esterase and alanine-aminopeptidase activity decreased. beta-glucosidase activity was not found in the biofilms. Contrary to the results for planktonic bacteria, inhibitory effects were not observed in biofilms. This suggested an increased tolerance of biofilm bacteria against toxic compounds.     

Microbiological investigation of an industrial ultra pure supply water plant using cultivation-based and cultivation-independent methods

Ultra pure waters (UPW), characterized by extremely low salt and nutrient concentrations, can suffer from microbial contamination which causes biofouling and biocorrosion, possibly leading to reduced lifetime and increased operational costs. Samples were taken from an ultra pure supply water producing plant of a power plant. Scanning electron microscopic examination was carried out on the biofilms formed in the system. Biofilm, ion exchange resin, and water samples were characterized by culture-based methods and molecular fingerprinting (terminal restriction fragment length polymorphism [T-RFLP] analysis and molecular cloning). Identification of bacteria was based on 16S rDNA sequence comparison. A complex microbial community structure was revealed. Nearly 46% of the clones were related to as yet uncultured bacteria. The community profiles of the water samples were the most diverse and most of bacteria were recruited from bacterial communities of tube surface and ion exchange resin biofilms. Microbiota of different layers of the mixed bed ion exchange resin showed the highest similarity. Most of the identified taxa (dominated by β-Proteobacteria) could take part in microbially influenced corrosion.     

Overnight stagnation of drinking water in household taps induces microbial growth and changes in community composition

Drinking water quality is routinely monitored in the distribution network but not inside households at the point of consumption. Fluctuating temperatures, residence times (stagnation), pipe materials and decreasing pipe diameters can promote bacterial growth in buildings. To test the influence of stagnation in households on the bacterial cell concentrations and composition, water was sampled from 10 separate households after overnight stagnation and after flushing the taps. Cell concentrations, measured by flow cytometry, increased (2-3-fold) in all water samples after stagnation. This increase was also observed in adenosine tri-phosphate (ATP) concentrations (2-18-fold) and heterotrophic plate counts (4-580-fold). An observed increase in cell biovolume and ATP-per-cell concentrations furthermore suggests that the increase in cell concentrations was due to microbial growth. After 5 min flushing of the taps, cell concentrations and water temperature decreased to the level generally found in the drinking water network. Denaturing gradient gel electrophoresis also showed a change in the microbial composition after stagnation. This study showed that water stagnation in household pipes results in considerable microbial changes. While hygienic risk was not directly assessed, it emphasizes the need for the development of good material validation methods, recommendations and spot tests for in-house water installations. However, a simple mitigation strategy would be a short flushing of taps prior to use.     

Biofilm formation and multiplication of Legionella in a model warm water system with pipes of copper, stainless steel and cross-linked polyethylene

Legionella pneumophila was grown in a model warm water system with pipes of copper (Cu), stainless steel (SS) and cross-linked polyethylene (PEX) during recirculation of tap water at 25--35 degrees C. Subsequently, domestic use of warm (37 degrees C) water was simulated using tap water with a low AOC concentration (<10 microg C/L). Two times each week the temperature of the water in the electric heaters (not in the pipes) was elevated to 70 degrees C for 30 min. ATP concentrations in the water sampled from the pipes over a 2-year period were significantly different for the pipe materials, with median values of 2.1 ng/l (Cu), 2.5 ng/l (SS) and 4.5 ng/l (PEX), respectively. Median values of the biofilm concentration were similar on Cu and SS (about 630 pg ATP/cm(2)) and 1870 pg ATP/cm(2) on PEX. Legionella multiplied in these biofilms and median values of Legionella concentrations in water were 1500 CFU/l (Cu) and about 4300 CFU/l for SS and PEX. Legionella to ATP ratios in water had median values of about 0.8 CFU/pg. Hot water flushing (70 degrees C) of the pipes on day 552, followed by 2 weeks of recirculation at 37 degrees C, caused strongly increased concentrations of ATP (up to 300 ng/l) and Legionella (>10(7)CFU/l), with about 100 CFU/pg ATP. Concentrations declined to original levels within 1 week of domestic water use, etc. Legionella concentrations in water and biofilms were at the same levels for all materials after 2 years. Hence, copper temporarily limited the growth of Legionella under the applied conditions and a rapid biomass development strongly increased the Legionella to ATP ratio.     

Factors affecting bulk to total bacteria ratio in drinking water distribution systems

Bacteria in drinking water systems can grow in bulk water and as biofilms attached to pipe walls, both causing regrowth problems in the distribution system. While studies have focused on evaluating the factors influencing the bacteria in bulk water and in biofilms separately, there is a need for understanding biofilm characteristics relative to the bulk water phase. The current study evaluated the effects of chlorine and residence time on the presence of culturable bacteria in biofilms relative to that in bulk water. The results showed that when no chlorine residual was present in the system, the median ratio of bulk to total bacteria was 0.81, indicating that 81% of the bacteria were present in bulk water, whereas only 19% were present in the biofilm. As chlorine concentration increased to 0.2, 0.5, and 0.7 mg/L, the median percentage of bacteria present in bulk water decreased to 37, 28, and 31, respectively. On the other hand, as the residence times increased to 8.2, 12, 24, and 48h, the median percentage of bacteria present in bulk water increased to 7, 37, 58, and 88, respectively, in the presence of a 0.2mg/L chlorine residual. The common notion that biofilms dominate the distribution system is not true under all conditions. These findings suggest that bulk water bacteria may dominate in portions of a distribution system that have a low chlorine residual.     

Drinking water and biofilm disinfection by Fenton-like reaction

A Fenton-like disinfection process was conducted with Fenton's reagent (H₂O₂) at pH 3 or 5 on autochthonous drinking water biofilms grown on corroded or non-corroded pipe material. The biofilm disinfection by Fenton-like oxidation was limited by the low content of iron and copper in the biomass grown on non-corroded plumbing. It was slightly improved by spiking the distribution system with some additional iron source (soluble iron II or ferrihydrite particles appeared as interesting candidates). However successful in situ disinfection of biofilms was only achieved in fully corroded cast iron pipes using H₂O₂ and adjusting the pH to 5. These new results provide additional support for the use of Fenton's processes for cleaning drinking water distribution systems contaminated with biological agents or organics.     

限制基质条件下生物膜特性的研究

在氮作为限制基质的情况下,采用库爱特-泰勒反应器分别培养异养菌生物膜和异养/自养硝化菌混合生物膜。当生物膜系统达到稳定后,通过提高水力剪切力使生物膜发生脱落,以研究生物膜内层基本特性及活性变化。结果表明,随着水力强度的增加则生物膜发生逐步脱落,脱落前后两种生物膜的特性及微生物活性均发生了较大变化。在两种生物膜内,靠近载膜片的生物膜比靠近液相的生物膜具有更强的粘结力,能抵抗高达10 Pa的水力剪切力,且生物活性较高。对于自养硝化菌生物膜,在生物膜发生脱落后,残余生物膜对氨氮的表面去除速率几乎保持不变,甚至对氨氮的比去除速率还略有增加,说明自养硝化菌可能主要分布在生物膜的内层。     

The hazards of incorporating charcoal filters into domestic water systems

Charcoal filters used in domestic and commercial applications for the removal of objectionable taste and odor support the growth of bacteria to alarmingly high counts. The charcoal beds concentrate both bacteria and organic nutrients that are present in water at low concentration. This results in marked growth of bacteria during the overnight period when the water is stagnant and in their subsequent release into the morning flow.     

Changes in content of microbially available phosphorus,assimilable organic carbon and microbial growth potential during drinking water treatment processes

There are regions where microbial growth in drinking water is limited by phosphorus instead of organic carbon. In phosphorus limited waters small changes in phosphorus concentration significantly affect microbial growth. We studied how water treatment processes in waterworks affect the availability of microbial nutrients and microbial growth potential in drinking water. The nutrients studied were assimilable organic carbon (AOCpotential) and microbially available phosphorus (MAP) which both were quantified by bioassays. Chemical coagulation, commonly used in surfacewater works, effectively removed AOCpotential and MAP. In contrast to activated carbon filtration, ozonation increased the concentrations of AOCpotential and MAP, and also microbial growth potential. In most of the drinking waters, microbial growth was limited by phosphorus, and microbial growth potential correlated with the MAP concentration. Microbial growth potential was lowest in drinking waters produced from surface waters with efficient treatment technique and highest in less treated ground waters.     

Association and decontamination of Bacillus spores in a simulated drinking water system

The objective of this work was to elucidate the disinfectant susceptibility of Bacillus anthracis Sterne (BA) and a commercial preparation of Bacillus thuringiensis (BT) spores associated with a simulated drinking water system. Biofilms composed of indigenous water system bacteria were accumulated on copper and polyvinyl chloride (PVC) pipe material surfaces in a low-flow pipe loop and uniformly mixed tank reactor (CDC biofilm reactor). Application of a distributed shear during spore contact resulted in approximately a 1.0 and 1.6 log₁₀ increase in the number of spores associated with copper and PVC surfaces, respectively. Decontamination of spores in both free suspension and after association with biofilm-conditioned pipe materials was attempted using free chlorine and monochloramine. Associated spores required 5- to 10-fold higher disinfectant concentrations to observe the same reduction of viable spores as in suspension. High disinfectant concentrations (103 mg/L free chlorine and 49 mg/L monochloramine) yielded less than a 2-log₁₀ reduction in viable associated spores after 60 min. Spores associated with biofilms on copper surfaces consistently yielded higher Ct values than PVC.     

Implications of nutrient release from iron metal for microbial regrowth in water distribution systems

Control of microbial regrowth in iron pipes is a major challenge for water utilities. This work examines the inter-relationship between iron corrosion and bacterial regrowth, with a special focus on the potential of iron pipe to serve as a source of phosphorus. Under some circumstances, corroding iron and steel may serve as a source for all macronutrients necessary for bacterial regrowth including fixed carbon, fixed nitrogen and phosphorus. Conceptual models and experimental data illustrate that levels of phosphorus released from corroding iron are significant relative to that necessary to sustain high levels of biofilm bacteria. Consequently, it may not be possible to control regrowth on iron surfaces by limiting phosphorus in the bulk water.     

Removal of soluble COD by a biofilm formed on a membrane in a jet loop type membrane bioreactor

The soluble chemical oxygen demand (COD) removal efficiency through a cake layer (biofilm) deposited on the surfaces of a membrane was investigated as a function of biofilm thickness in a jet loop type membrane bioreactor (JL-MBR). The mechanisms for the removal were investigated based on the microbial characteristics of the biofilm. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to identify the microbial community and a Specific oxygen uptake rate (SOUR) analysis was applied to determine the activities of microbial communities in a biofilm. Both the activities and community of microbial communities in the biofilm were similar to those found in a mixed liquor since, in JL-MBR all the substrates, dissolved oxygen, and nutrients are forced to flow through the biofilm, which differs from the biofilms grown on a non-permeable substratum in conventional biofilm process. The removal efficiency of soluble COD in a reactor through the active biofilm increased, reaching a constant value of approximately 92% despite the continuous increase in the thickness of active biofilm with the operation time. This might be attributed to (i) the presence of soluble COD that is not readily biodegraded, (ii) the presence of small and non-biodegradable organic molecules that could easily pass through the biofilm as well as the membrane, and (iii) too short a contact time of soluble solutes with the active microorganisms in the biofilm.