Evaluating online data of water quality changes in a pilot drinking water distribution system with multivariate data exploration methods.

The distribution of drinking water generates soft deposits and biofilms in the pipelines of distribution systems. Disturbances in water distribution can detach these deposits and biofilms and thus deteriorate the water quality. We studied the effects of simulated pressure shocks on the water quality with online analysers. The study was conducted with copper and composite plastic pipelines in a pilot distribution system. The online data gathered during the study was evaluated with Self-Organising Map (SOM) and Sammon's mapping, which are useful methods in exploring large amounts of multivariate data. The objective was to test the usefulness of these methods in pinpointing the abnormal water quality changes in the online data. The pressure shocks increased temporarily the number of particles, turbidity and electrical conductivity. SOM and Sammon's mapping were able to separate these situations from the normal data and thus make those visible. Therefore these methods make it possible to detect abrupt changes in water quality and thus to react rapidly to any disturbances in the system. These methods are useful in developing alert systems and predictive applications connected to online monitoring.

Survival of Mycobacterium avium in drinking water biofilms as affected by water flow velocity, availability of phosphorus, and temperature.

Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4',6'-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 microg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20 degrees C versus 7 degrees C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.

Survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and caliciviruses in drinking water-associated biofilms grown under high-shear turbulent flow.

Most of the bacteria in drinking water distribution systems are associated with biofilms. In biofilms, their nutrient supply is better than in water, and biofilms can provide shelter against disinfection. We used a Propella biofilm reactor for studying the survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and canine calicivirus (CaCV) (as a surrogate for human norovirus) in drinking water biofilms grown under high-shear turbulent-flow conditions. The numbers of M. avium and L. pneumophila were analyzed with both culture methods and with peptide nucleic acid fluorescence in situ hybridization (FISH) methods. Even though the numbers of pathogens in biofilms decreased during the experiments, M. avium and L. pneumophila survived in biofilms for more than 2 to 4 weeks in culturable forms. CaCV was detectable with a reverse transcription-PCR method in biofilms for more than 3 weeks. E. coli was detectable by culture for only 4 days in biofilms and 8 days in water, suggesting that it is a poor indicator of the presence of certain waterborne pathogens. With L. pneumophila and M. avium, culture methods underestimated the numbers of bacteria present compared to the FISH results. This study clearly proved that pathogenic bacteria entering water distribution systems can survive in biofilms for at least several weeks, even under conditions of high-shear turbulent flow, and may be a risk to water consumers. Also, considering the low number of virus particles needed to result in an infection, their extended survival in biofilms must be taken into account as a risk for the consumer.

Estimates of microbial quality and concentration of copper in distributed drinking water are highly dependent on sampling strategy.

The numbers of bacteria generally increase in distributed water. Often household pipelines or water fittings (e.g., taps) represent the most critical location for microbial growth in water distribution systems. According to the European Union drinking water directive, there should not be abnormal changes in the colony counts in water. We used a pilot distribution system to study the effects of water stagnation on drinking water microbial quality, concentration of copper and formation of biofilms with two commonly used pipeline materials in households; copper and plastic (polyethylene). Water stagnation for more than 4h significantly increased both the copper concentration and the number of bacteria in water. Heterotrophic plate counts were six times higher in PE pipes and ten times higher in copper pipes after 16 h of stagnation than after only 40 min stagnation. The increase in the heterotrophic plate counts was linear with time in both copper and plastic pipelines. In the distribution system, bacteria originated mainly from biofilms, because in laboratory tests with water, there was only minor growth of bacteria after 16 h stagnation. Our study indicates that water stagnation in the distribution system clearly affects microbial numbers and the concentration of copper in water, and should be considered when planning the sampling strategy for drinking water quality control in distribution systems.

The effects of changing water flow velocity on the formation of biofilms and water quality in pilot distribution system consisting of copper or polyethylene pipes.

We studied the effects of flow velocity on the formation of biofilms and the concentration of bacteria in water in copper and plastic (polyethylene, PE) pipes. The formation of biofilms increased with the flow velocity of water. The increase in microbial numbers and contents of ATP was clearer in the PE pipes than in the copper pipes. This was also seen as increased consumption of microbial nutrients in the pipeline system. This indicates that the mass transfer of nutrients is in major role in the growth of biofilms. However, the increased biomass of biofilms did not affect microbial numbers in the water. Rapid changes in water flow rate resuspended biofilms and sediments which increased the concentrations of bacteria and copper in water. The disturbance caused by the changing water flow was also seen as an increase in the particle counts and water turbidity recorded with online instrumentation.

Fluorescence in situ hybridization using peptide nucleic acid probes for rapid detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in potable-water biofilms.

Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.

Advantages of peptide nucleic acid oligonucleotides for sensitive site directed 16S rRNA fluorescence in situ hybridization (FISH) detection of Campylobacter jejuni, Campylobacter coli and Campylobacter lari.

Traditionally fluorescence in situ hybridization (FISH) has been performed with labeled DNA oligonucleotide probes. Here we present for the first time a high affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting thermotolerant Campylobacter spp. using FISH. Thermotolerant Campylobacter spp, including the species Campylobacter coli, Campylobacter jejuni and Campylobacter lari, are important food and water borne pathogens. The designed PNA probe (CJE195) bound with higher affinity to a previously reported low affinity site on the 16S rRNA than the corresponding DNA probe. PNA also overcame the problem of the lack of affinity due to the location of the binding site and the variation of the target sequence within species. The PNA probe specificity was tested with several bacterial species, including other Campylobacter spp. and their close relatives. All tested C. coli, C. jejuni and C. lari strains were hybridized successfully. Aging of the Campylobacter cultures caused the formation of coccoid forms, which did not hybridize as well as bacteria in the active growth phase, indicating that the probe could be used to assess the physiological status of targeted cells. The PNA FISH methodology detected C. coli by membrane filtration method from C. coli spiked drinking water samples.

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.

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.

Rapid enumeration of virus-like particles in drinking water samples using SYBR green I-staining.

We studied the suitability of SYBR green I-staining for determining total counts of virus-like particles and bacteria in drinking water. Low background fluorescence and lack of unspecific staining made drinking water samples an excellent matrix for SYBR green I-staining. Direct microscopic count method is a rapid and economical tool for assessing the total number of virus-like particles in aquatic samples, compared to culture-dependent or molecular biology methods. We applied this method to show the efficiency of a large-scale drinking water purification process in the removal of virus-like particles and bacteria from lake water.

Mycobacteria in water and loose deposits of drinking water distribution systems in Finland.

Drinking water distribution systems were analyzed for viable counts of mycobacteria by sampling water from waterworks and in different parts of the systems. In addition, loose deposits collected during mechanical cleaning of the main pipelines were similarly analyzed. The study covered 16 systems at eight localities in Finland. In an experimental study, mycobacterial colonization of biofilms on polyvinyl chloride tubes in a system was studied. The isolation frequency of mycobacteria increased from 35% at the waterworks to 80% in the system, and the number of mycobacteria in the positive samples increased from 15 to 140 CFU/liter, respectively. Mycobacteria were isolated from all 11 deposits with an accumulation time of tens of years and from all 4 deposits which had accumulated during a 1-year follow-up time. The numbers of mycobacteria were high in both old and young deposits (medians, 1.8 x 10(5) and 3.9 x 10(5) CFU/g [dry weight], respectively). Both water and deposit samples yielded the highest numbers of mycobacteria in the systems using surface water and applying ozonation as an intermediate treatment or posttreatment. The number and growth of mycobacteria in system waters correlated strongly with the concentration of assimilable organic carbon in the water leaving the waterworks. The densities of mycobacteria in the developing biofilms were highest at the distal sites of the systems. Over 90% of the mycobacteria isolated from water and deposits belonged to Mycobacterium lentiflavum, M. tusciae, M. gordonae, and a previously unclassified group of mycobacteria. Our results indicate that drinking water systems may be a source for recently discovered new mycobacterial species.

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.

Formation of biofilms in drinking water distribution networks, a case study in two cities in Finland and Latvia.

The formation of biofilms in drinking water distribution networks is a significant technical, aesthetic and hygienic problem. In this study, the effects of assimilable organic carbon, microbially available phosphorus (MAP), residual chlorine, temperature and corrosion products on the formation of biofilms were studied in two full-scale water supply systems in Finland and Latvia. Biofilm collectors consisting of polyvinyl chloride pipes were installed in several waterworks and distribution networks, which were supplied with chemically precipitated surface waters and groundwater from different sources. During a 1-year study, the biofilm density was measured by heterotrophic plate counts on R2A-agar, acridine orange direct counting and ATP-analyses. A moderate level of residual chlorine decreased biofilm density, whereas an increase of MAP in water and accumulated cast iron corrosion products significantly increased biofilm density. This work confirms, in a full-scale distribution system in Finland and Latvia, our earlier in vitro finding that biofilm formation is affected by the availability of phosphorus in drinking water.

Impact of UV disinfection on microbially available phosphorus, organic carbon, and microbial growth in drinking water.

UV irradiation at a wavelength of 253.7 nm (UV(254)) is commonly used for drinking water disinfection. UV radiation is known to convert organically combined phosphorus to orthophosphate and to degrade natural organic matter. We studied if UV disinfection increases the amount of microbially available forms of organic carbon and phosphorus in drinking waters with different characteristics, and if these changes in water chemical quality could enhance the microbial growth in drinking water. The UV(254) dose (15-50 mWs/cm(2)) used in waterworks reduced the concentration of assimilable organic carbon and the sum of the molecular size fractions. The release of microbially available phosphorus needed higher doses (204 mWs/cm(2)) of UV(254) radiation. Of bacteria in drinking water, 90% were inactivated with UV(254)-irradiation doses below 50 mWs/cm(2). A high dose (501 mWs/cm(2)) of UV(254) radiation inhibited the microbial growth in water.

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.

Biofilm formation in drinking water affected by low concentrations of phosphorus.

Abstract: There are geographical regions where microbial growth in drinking waters is limited by phosphorus instead of organic carbon. In these drinking waters even a low amount of phosphorus can strongly enhance microbial growth. The formation of biofilm can be limited by low availability of phosphorus in drinking waters with low content of phosphorus. The formation of biofilms on polyvinyl chloride plates was studied in laboratory experiments with water containing 48 microg/L assimilable organic carbon and 0.19 microg/L microbially available phosphorus. We found that low additions of phosphate (1-5 microg/L PO4(3-)-P) to water increased microbial growth in the water and in the biofilm. The effect of phosphorus on microbial growth could be detected by determining either the microbial cell production or the content of ATP in biofilms. Also, in steady-state biofilms, microbial concentrations were higher with phosphorus addition as enumerated by heterotrophic plate counts on R2A-agar and acridine orange direct counting. This work confirms the earlier findings of the importance of phosphorus for microbial growth in humic-rich drinking waters.

The microbial community structure of drinking water biofilms can be affected by phosphorus availability.

Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 microg of phosphorus liter(-1) and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs). Phosphate increased the proportion of PLFAs 16:1 omega 7c and 18:1 omega 7c and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure. Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.