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Legionella pneumophila in potable water installations poses a potential health risk, but quantitative information about its replication in biofilms in relation to water quality is scarce. Therefore, biofilm formation on the surfaces of glass and chlorinated polyvinyl chloride (CPVC) in contact with tap water at 34 to 39°C was investigated under controlled hydraulic conditions in a model system inoculated with biofilm-grown L. pneumophila. The biofilm on glass (average steady-state concentration, 23 Ϯ 9 pg ATP cm Ϫ2 ) exposed to treated aerobic groundwater (0.3 mg C liter Ϫ1 ; 1 g assimilable organic carbon [AOC] liter Ϫ1 ) did not support growth of the organism, which also disappeared from the biofilm on CPVC (49 Ϯ 9 pg ATP cm Ϫ2 ) after initial growth. L. pneumophila attained a level of 4.3 log CFU cm Ϫ2 in the biofilms on glass (1,055 Ϯ 225 pg ATP cm Ϫ2 ) and CPVC (2,755 Ϯ 460 pg ATP cm Ϫ2 ) exposed to treated anaerobic groundwater (7.9 mg C liter Ϫ1 ; 10 g AOC liter Ϫ1 ). An elevated biofilm concentration and growth of L. pneumophila were also observed with tap water from the laboratory. The Betaproteobacteria Piscinibacter and Methyloversatilis and amoeba-resisting Alphaproteobacteria predominated in the clones and isolates retrieved from the biofilms. In the biofilms, the Legionella colony count correlated significantly with the total cell count (TCC), heterotrophic plate count, ATP concentration, and presence of Vermamoeba vermiformis. This amoeba was rarely detected at biofilm concentrations of Ͻ100 pg ATP cm Ϫ2 . A threshold concentration of approximately 50 pg ATP cm Ϫ2 (TCC ϭ 1 ϫ 10 6 to 2 ϫ 10 6 cells cm Ϫ2 ) was derived for growth of L. pneumophila in biofilms.IMPORTANCE Legionella pneumophila is the etiologic agent in more than 10,000 cases of Legionnaires' disease that are reported annually worldwide and in most of the drinking water-associated disease outbreaks reported in the United States. The organism proliferates in biofilms on surfaces exposed to warm water in engineered freshwater installations. An investigation with a test system supplied with different types of warm drinking water without disinfectant under controlled hydraulic conditions showed that treated aerobic groundwater (0.3 mg liter Ϫ1 of organic carbon) induced a low biofilm concentration that supported no or very limited growth of L. pneumophila. Elevated biofilm concentrations and L. pneumophila colony counts were observed on surfaces exposed to two types of extensively treated groundwater, containing 1.8 and 7.9 mg C liter Ϫ1 and complying with the microbial water quality criteria during distribution. Control measures in warm tap water installations are therefore essential for preventing growth of L. pneumophila.

In the presence of bromide, ozonation as applied in water treatment results in the formation of bromate, an ion with carcinogenic properties. The reduction of bromate by mixed bacterial populations as well as pure cultures was studied under laboratory conditions. Bromate was reduced to bromide by a mixed bacterial population with and without a preceding nitrate reduction step in an anaerobically incubated medium with ethanol as the energy and carbon source at 20 and 25؇C. The predominating bacteria isolated from the batches showing bromate reduction were identified as Pseudomonas spp. Strains of Pseudomonas fluorescens reduced BrO 3 ؊ to Br ؊ but at a much lower rate than the mixed bacterial population did. Nitrate is a preferred electron acceptor for the bromate-reducing bacteria. Bromate reduction did not occur in the presence of NO 3 ؊ , and the rate of bromate reduction was at least 100 times lower than the rate of nitrate reduction. Bromate was completely converted to Br ؊ , indicating that intermediates, e.g., BrO 2 ؊ , did not accumulate during bromate reduction. on July 6, 2020 by guest http://aem.asm.org/ Downloaded from 240 HIJNEN ET AL. APPL. ENVIRON. MICROBIOL. on July 6, 2020 by guest http://aem.asm.org/ Downloaded from a This group included isolates Br1, Br4, Br5, Br14, Br16, Br22, and Br23. b This group included isolates Br15, Br17, Br18, Br19, Br21, Br24, and Br25. c This group included isolates Br2, Br3, Br6 to Br13, and Br20.