Markku J. Lehtola

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.

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

ABSTRACT 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 × 105 and 3.9 × 105 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.

Bacterial biofilm formation on polyvinyl chloride, polyethylene and stainless steel exposed to ozonated water

The formation of biofilm on polyvinyl chloride (PVC), polyethylene (PE) and stainless steel surfaces was studied in bank infiltrated and chemically purified waters without (control water) and with ozonation. Ozonation increased the concentration of assimilable organic carbon (AOC) in water (78 vs 450 μgC/l). Ozonation of water increased the viable numbers and cell volume of heterotrophic bacteria on the surfaces. The highest bacterial count, 7.7×105 cfu/cm2, was detected on PVC surface after three weeks’ exposure to ozonated water. The numbers of heterotrophic bacteria on surfaces reflected the bacterial numbers in water. There was no difference in total cell count on different surfaces and in control and ozonated waters. Direct staining of microbes on the surfaces produced higher cell count per cm2 than enumerating microbes detached from surfaces by sonication. In general, the accumulation of biofilm on different surface materials was quite similar. Only the cell volume was slightly higher on PE than on PVC surface.

Survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and Caliciviruses in Drinking Water-Associated Biofilms Grown under High-Shear Turbulent Flow

ABSTRACT 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.

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.

Microbially available organic carbon, phosphorus, and microbial growth in ozonated drinking water

Ozonation is a disinfection technique commonly used in the treatment of drinking water. It destroys harmful microbes, but it also degrades organic matter in water, increasing the bioavailability of organic matter. Recently, it was found that not only organic carbon but also phosphorus can limit the microbial growth in drinking water, which contains high amount of organic matter. We used a bioassay to analyze whether ozone could also increase the microbially available phosphorus (MAP) in drinking water, and whether MAP in ozone-treated water was associated with the growth of heterotrophic microbes. We found that both assimilable organic carbon and MAP concentrations were increased by ozone treatment. In ozonated water, microbial growth was mainly limited by phosphorus, and even minor changes in MAP concentration dramatically increased the growth potential of heterotrophic microbes. In this study, ozonation increased the MAP by 0.08-0.73 microgram P/l, resulting in an increase of 80,000-730,000 CFU/ml in water samples. In contrast to MAP, the content of assimilable organic carbon (AOCpotential) did not correlate with microbial growth. The results show that in water treatment not only AOCpotential but also MAP should be considered as an important factor that can limit microbial growth in drinking water.

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

ABSTRACT Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 μg 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 16:1ω7c and 18:1ω7c 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.

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 chorine 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.

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

ABSTRACT 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.

Survival of Mycobacterium avium in Drinking Water Biofilms as Affected by Water Flow Velocity, Availability of Phosphorus, and Temperature

ABSTRACT 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 μg/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°C versus 7°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.