Detection and enumeration of haloacetic acid-degrading bacteria in drinking water distribution systems using dehalogenase genes.
ABSTRACT To develop a PCR-based tracking method for the detection of a subset of bacteria in drinking water distribution systems capable of degrading haloacetic acids (HAAs).
Published degenerate PCR primers were used to determine that 54% of tap water samples (7/13) were positive for a deh gene, indicating that drinking water distribution systems may harbour bacteria capable of HAA degradation. As the published primer sets were not sufficiently specific for quantitative PCR, new primers were designed to amplify dehII genes from selected indicator strains. The developed primer sets were effective in directly amplifying dehII genes from enriched consortia samples, and the DNA extracted from tap water provided that an additional nested PCR step for detection of the dehII gene was used.
This study demonstrates that drinking water distribution systems harbour microbes capable of degrading HAAs. In addition, a quantitative PCR method was developed to detect and quantify dehII genes in drinking water systems.
The development of a technique to rapidly screen for the presence of dehalogenase genes in drinking water distribution systems could help water utilities determine if HAA biodegradation is occurring in the distribution system.
- SourceAvailable from: Anne K Camper[show abstract] [hide abstract]
ABSTRACT: One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.Applied and Environmental Microbiology 09/2007; 73(16):5111-7. · 3.68 Impact Factor
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ABSTRACT: Haloacetic acids (HAAs) are toxic organic chemicals that are frequently detected in surface waters and in drinking water distribution systems. The aerobic biodegradation of HAAs was investigated in serum bottles containing a single HAA and inoculated with washed microorganisms obtained from enrichment cultures maintained on either monochloroacetic acid (MCAA) or trichloroacetic acid (TCAA) as the sole carbon and energy source. Biodegradation was observed for each of the HAAs tested at concentrations similar to those found in surface waters and in drinking water distribution systems. The MCAA culture was able to degrade both MCAA and monobromoacetic acid (MBAA) with pseudo-first order rate constants of 1.06 x 10(-2) and 1.13 x 10(-2) l(mg protein)(-1) d(-1), respectively, for concentrations ranging from 10(-5) to 2 mM. The pseudo-first order rate constant for TCAA degradation by the TCAA culture was 6.52 x 10(-3) l(mg protein)(-1) d(-1) for concentrations ranging from 5.33 x 10(-5) to 0.72 mM. The TCAA culture was also able to degrade MCAA with the rate accelerating as incubation time increased. Experiments with radiolabeled HAAs indicated that the 14C was primarily converted to 14CO2 with minor incorporation into cell biomass. The community structure of the enrichment cultures was analyzed by both cultivation-dependent and cultivation-independent approaches. Denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene fragments showed that each of the two enrichment cultures had multiple bacterial populations, none of which corresponded to HAA-degrading bacteria cultivated on HAA-supplemented agar plates. This research indicates that biodegradation is a potential loss mechanism for HAAs in surface waters and in drinking water distribution systems.Chemosphere 06/2004; 55(6):915-25. · 3.14 Impact Factor
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ABSTRACT: The decomposition of trihaloacetic acids [bromodichloroacetic acid (BDCAA), dibromochloroacetic acid (DBCAA), tribromoacetic acid (TBAA)], and the formation of the corresponding trihalomethanes [bromodichloromethane (BDCM), dibromochloromethane (DBCM), tribromomethane (TBM)] were studied. Like TBAA, the two mixed chlorobromo-species, BDCAA and DBCAA, were found to decompose to form BDCM and DBCM, respectively, via a decarboxylation pathway. The decomposition of BDCAA, DBCAA and TBAA in water at neutral pH follows a first-order reaction, with rate constants of 0.0011, 0.0062 and 0.040 day(-1) at 23 degrees C, respectively; and 0.000028, 0.00014 and 0.0016 day(-1) at 4 degrees C, respectively. The activation energies for the decomposition reaction of BDCAA, DBCAA and TBAA in water at neutral pH were found to be 35.0, 34.5 and 29.2 kcal/mol, respectively. The effect of pH in the range of 6-9 and the effect of a drinking water matrix on the decomposition of BDCAA, DBCAA, and TBAA in water were found to be insignificant. Measurement and health implications due to decomposition of trihaloacetic acids and formation of the corresponding trihalomethanes were discussed. By applying the technique of quantitative structure-activity relationships (QSAR), the decomposition rate constants of six iodinated trihaloacetic acids were estimated.Water Research 09/2002; 36(14):3665-73. · 4.66 Impact Factor