This paper describes a method for determining the potential of a water for supporting microbial regrowth in a drinking water distribution system. The method is based on the growth of fluorescent pseudomonads as a function of the concentration of easily assimilable organic carbon (AOC) in water. Using this method, the authors analyzed water samples from various stages of water treatment and water in which samples of nonmetallic piping materials had been placed. The concentration of AOC in water increased if the water was ozonated or placed in contact with plasticized polyvinyl‐chloride.

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The ammonia-oxidizing prokaryote (AOP) community in three groundwater treatment plants and connected distribution systems was analyzed by quantitative real-time PCR and sequence analysis targeting the amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Results demonstrated that AOB and AOA numbers increased during biological filtration of ammonia-rich anoxic groundwater, and AOP were responsible for ammonium removal during treatment. In one of the treatment trains at plant C, ammonia removal correlated significantly with AOA numbers but not with AOB numbers. Thus, AOA were responsible for ammonia removal in water treatment at one of the studied plants. Furthermore, an observed negative correlation between the dissolved organic carbon (DOC) concentration in the water and AOA numbers suggests that high DOC levels might reduce growth of AOA. AOP entered the distribution system in numbers ranging from 1.5 ؋ 10 3 to 6.5 ؋ 10 4 AOPs ml ؊1 . These numbers did not change during transport in the distribution system despite the absence of a disinfectant residual. Thus, inactive AOP biomass does not seem to be degraded by heterotrophic microorganisms in the distribution system. We conclude from our results that AOA can be commonly present in distribution systems and groundwater treatment, where they can be responsible for the removal of ammonia.Ammonia can be present in source water used for drinking water production or added to treated water with chlorine to form chloramines as a disinfectant. However, the presence of ammonia in drinking water is undesirable because nitrification might lead to toxic levels of nitrite (29) or adverse effects on water taste and odor (4) and might increase heterotrophic bacteria, including opportunistic pathogens (29). Two-thirds of the drinking water in The Netherlands is produced from groundwater. Most of the groundwater used for drinking water production is anoxic with relatively high concentrations of methane, iron, manganese, dissolved organic carbon (DOC), and ammonia. Treatment of anoxic groundwater aims at achieving biologically stable water, because drinking water in The Netherlands is distributed without a disinfectant residual. As a result, a highly efficient nitrification process during rapid medium filtration is required.Nitrification is the microbial oxidation of ammonia to nitrate and consists of two processes: the oxidation of ammonia to nitrite by ammonia-oxidizing prokaryotes (AOP) and the oxidation of nitrite to nitrate by nitrite-oxidizing bacteria (NOB). Recently it was shown that in addition to bacteria, archaea also are capable of ammonia oxidation (13). Since then, ammoniaoxidizing archaea (AOA) have been found in many different ecosystems, including wastewater treatment systems (10,20,24). However, it is currently unknown if AOA are present in drinking water treatment processes and distribution systems. Recent studies have focused on nitrification in drinking water treatment (16,28). In those studies, AOB and NOB were enumerated by traditional most-proba...

The need for protozoa for the proliferation of Legionella pneumophila in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of L. pneumophila in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous L. pneumophila originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-m pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable L. pneumophila, and for their concentrations of protozoa. L. pneumophila grew to high numbers (6.3 log CFU/cm 2 ) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of Hartmannella vermiformis in all flasks in which L. pneumophila multiplied and also when cycloheximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of L. pneumophila in trophozoites of H. vermiformis, with 25.9% ؎ 10.5% of the trophozoites containing L. pneumophila on day 10 and >90% containing L. pneumophila on day 14. Calculations confirmed that intracellular growth was most likely the only way for L. pneumophila to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher L. pneumophila concentrations, and therefore the growth of L. pneumophila within engineered water systems can be limited by controlling biofilm formation.Legionella pneumophila is widespread in natural freshwater environments, despite its fastidious nature (16). The bacterium has also frequently been observed in engineered water systems such as warm water distributing systems, cooling towers, humidifiers, and fountains (25,43,50). The multiplication of the organism in water systems poses a potential human health risk when aerosolization can occur (12).Legionellae grow in vitro only in complex media with supplements of cysteine and iron salts (11). For their multiplication in vivo, other microorganisms are required, but different species of heterotrophic bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae, and Flavobacterium sp.) alone do not support the growth of L. pneumophila (31,45). In vitro studies using cocultures have repeatedly demonstrated the intracellular multiplication of L. pneumophila in amoebae (Acanthamoeba, Echinamoeba, Hartmannella, Naegleria, Vahlkampfia, and Dictyostelium) and in a ciliated protozoon (Tetrahymena pyriformis) (12,15,21,39). Amoebae have been observed in water systems associated with Legionnaires' disease (5, 7), and L. pneumophila can recolonize water distributing systems within a few weeks after disinfection (26,27). Batch experiments with tap wate...

Representatives of the genus Legionella were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies. Legionella concentrations ranged from 1.1 ؋ 10 3 to 7.8 ؋ 10 5 cells liter ؊1 and were significantly higher in SW treated with multiple barriers at 4°C than in GW treated at 9 to 12°C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter ؊1 ) were detected in treated water by the culture method. Legionella was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the Legionella concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with Legionella sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described Legionella species and types, including L. bozemanii, L. worsleiensis, Legionella-like amoebal pathogen types, L. quateirensis, L. waltersii, and L. pneumophila. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of Legionella. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15°C.

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