RESUMEN
Harmful cyanobacteria have been an important concern for drinking water quality for quite some time, as they may produce cyanotoxins and odorants. Microcystis and Cylindrospermopsis are two common harmful cyanobacterial genera detected in freshwater lakes and reservoirs, with microcystins (MCs) and cylindrospermopsin (CYN) as their important metabolites, respectively. In this study, two sets of duplex qPCR systems were developed, one for quantifying potentially-toxigenic Microcystis and Microcystis, and the other one for cylindrospermopsin-producing cyanobacteria and Cylindrospermopsis. The duplex qPCR systems were developed and validated in the laboratory by using 338 samples collected from 29 reservoirs in Taiwan and her offshore islands. Results show that cell numbers of Microcystis and Cylindorspermopsis enumerated with microscopy, and MCs and CYN concentrations measured with the enzyme-linked immuno-sorbent assay method, correlated well with their corresponding gene copies determined with the qPCR systems (range of coefficients of determination R² = 0.392-0.740). The developed qPCR approach may serve as a useful tool for the water industry to diagnose the presence of harmful cyanobacteria and the potential presence of cyanotoxins in source waters.
Asunto(s)
Toxinas Bacterianas/análisis , Cianobacterias/aislamiento & purificación , Agua Potable/análisis , Microcistinas/análisis , Uracilo/análogos & derivados , Contaminantes del Agua/análisis , Alcaloides , Cianobacterias/genética , Toxinas de Cianobacterias , ADN Bacteriano/análisis , Agua Potable/microbiología , Monitoreo del Ambiente , Agua Dulce/análisis , Agua Dulce/microbiología , Lagos/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Taiwán , Uracilo/análisisRESUMEN
2-Methylisoborneol (2-MIB) is a commonly detected cyanobacterial odorant in drinking water sources in many countries. To provide safe and high-quality water, development of a monitoring method for the detection of 2-MIB-synthesis (mibC) genes is very important. In this study, new primers MIBS02F/R intended specifically for the mibC gene were developed and tested. Experimental results show that the MIBS02F/R primer set was able to capture 13 2-MIB producing cyanobacterial strains grown in the laboratory, and to effectively amplify the targeted DNA region from 17 2-MIB-producing cyanobacterial strains listed in the literature. The primers were further coupled with a TaqMan probe to detect 2-MIB producers in 29 drinking water reservoirs (DWRs). The results showed statistically significant correlations between mibC genes and 2-MIB concentrations for the data from each reservoir (R2=0.413-0.998; p<0.05), from all reservoirs in each of the three islands (R2=0.302-0.796; p<0.01), and from all data of the three islands (R2=0.473-0.479; p<0.01). The results demonstrate that the real-time PCR can be an alternative method to provide information to managers of reservoirs and water utilities facing 2-MIB-related incidents.
Asunto(s)
Canfanos/análisis , Cianobacterias/crecimiento & desarrollo , Agua Potable , Genes Bacterianos , Microbiología del Agua/normas , Recursos Hídricos/provisión & distribución , Canfanos/metabolismo , Cianobacterias/genética , Cianobacterias/metabolismo , Agua Potable/química , Agua Potable/microbiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Geosmin is one of the most commonly detected off-flavor chemicals present in reservoirs and drinking water systems. Quantitative real-time PCR (qPCR) is useful for quantifying geosmin-producers by focusing on the gene encoding geosmin synthase, which is responsible for geosmin synthesis. In this study, several primers and probes were designed and evaluated to detect the geosmin synthase gene in cyanobacteria. The specificity of primer and probe sets was tested using 21 strains of laboratory cultured cyanobacteria isolated from surface waters in Australia (18) and Taiwan (2), including 6 strains with geosmin producing ability. The results showed that the primers designed in this study could successfully detect all geosmin producing strains tested. The selected primers were used in a qPCR assay, and the calibration curves were linear from 5 × 10(1) to 5 × 10(5) copies mL(-1), with a high correlation coefficient (R(2) = 0.999). This method was then applied to analyze samples taken from Myponga Reservoir, South Australia, during a cyanobacterial bloom event. The results showed good correlations between qPCR techniques and traditional methods, including cell counts determined by microscopy and geosmin concentration measured using gas chromatography (GC) coupled with a mass selective detector (MSD). Results demonstrate that qPCR could be used for tracking geosmin-producing cyanobacteria in drinking water reservoirs. The qPCR assay may provide water utilities with the ability to properly characterize a taste and odor episode and choose appropriate management and treatment options.
Asunto(s)
Anabaena/genética , Monitoreo del Ambiente , Naftoles/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Australia , Genes Bacterianos , Geografía , Estándares de Referencia , Factores de TiempoRESUMEN
Two molecular methods, denaturing gradient gel electrophoresis (DGGE) and quantitative real-time polymerase chain reaction (qPCR) with the Universal ProbeLibrary (UPL) probe, were developed and used for the characterization and quantification of several microcystin producers in Moo-Tan Reservoir (MTR), Taiwan and its associated water treatment plant (Shih-Men Water Treatment Plant, SMWTP). Internal transcribed spacer (ITS) sequence, a highly diversified region between the 16S rRNA and 23S rRNA genes, was used to further identify the isolated strains from MTR and also used in DGGE for the detection of the specific DNA fragments and biomarkers for 11 strains observed in MTR. These ITS-DGGE biomarkers were successfully applied to monitor the community changes of potential toxigenic Microcystis sp. over a period of five years. Two highly specific primers were combined with UPL probes to measure microcystins synthesis gene (mcyB) and phycocyanin intergenic spacer region (cpcB) concentrations in water samples. The copy concentrations of UPL-mcyB and UPL-cpcB correlated well with MC-RR concentrations/water temperature and Microcystis sp. cell numbers in the water samples, respectively. For SMWTP, toxin concentrations were low, but the DGGE bands clearly demonstrated the presence of potential microcystin producers in both water treatment plants and finished water samples. It was demonstrated that toxigenic Microcystis sp. may penetrate through the treatment processes and pose a potential risk to human health in the drinking water systems.
Asunto(s)
Agua Dulce/microbiología , Microcystis/crecimiento & desarrollo , Microbiología del Agua , Secuencia de Bases , Electroforesis en Gel de Gradiente Desnaturalizante , Agua Potable/química , Agua Potable/microbiología , Agua Dulce/química , Humanos , Microcystis/clasificación , Microcystis/genética , Microcystis/aislamiento & purificación , Datos de Secuencia Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , Medición de Riesgo , Eliminación de Residuos Líquidos , Abastecimiento de Agua/estadística & datos numéricosRESUMEN
The effect of permanganate oxidation on the formation and degradation of wood odorant ß-cyclocitral in water was investigated. Oxidation experiments were conducted for ß-cyclocitral prepared from pure chemicals and extracted from Microcystis aeruginosa. The data were simulated with appropriate kinetic rate models. The formation and degradation of ß-cyclocitral during the oxidation of ß-carotene were also monitored and modeled. The degradation of ß-cyclocitral prepared from pure chemicals followed second-order kinetics with a rate constant of 91.7 ± 2.4M(-1)s(-1), and that of the ß-cyclocitral precursor, ß-carotene, followed first-order kinetics with a rate constant of 0.0054 ± 0.0004 min(-1). During the oxidation of ß-carotene, ß-cyclocitral was produced. The formation and degradation can both be simulated by first-order kinetics with respect to ß-carotene and ß-cyclocitral concentrations, respectively. The degradation rate of ß-cyclocitral produced from ß-carotene was found to be much slower than that for ß-cyclocitral obtained from pure chemicals, very likely due to a mass transfer limitation. The kinetic models were further employed to simulate the oxidation of ß-cyclocitral in the presence of ß-carotene and for ß-cyclocitral extracted from M. aeruginosa, respectively. The models well predict/fit the experimental data, with rate constants similar to other experiments, indicating that the models may be used for simulating the formation and degradation of ß-cyclocitral in water treatment systems.
Asunto(s)
Aldehídos/química , Diterpenos/química , Compuestos de Manganeso/química , Óxidos/química , Madera , Cinética , Microcystis/química , Oxidación-Reducción , beta Caroteno/químicaRESUMEN
A solid-phase extraction (SPE)-liquid chromatography (LC)-mass spectrometry (MS) method was developed to concentrate and detect nine cyanotoxins simultaneously, including six microcystins (MCs) congeners, nodularin (NOD), anatoxin-a (ATX) and cylindrospermopsin (CYN), in pure and natural waters. A dual cartridge SPE assembly was tested for the operating parameters of cyanotoxin extraction. A surrogate standard (SS), 1,9-diaminononane, was spiked in all the samples before the SPE extraction, and an internal standard (IS), 2,3,5-trimethylphenyl methyl carbamate, was spiked before LC/MS analysis. The method detection limit (MDL) was 2-100 ng/L for nine cyanotoxins in pure water and was increased by a factor of three to ten in a more complicated water matrix. The recoveries based on SS were between 83 and 104%, while those based on IS were 80-120%. The developed method was successfully employed in analyzing 33 water samples collected from eutrophic lakes, water treatment plants and distribution taps. MCs, NOD, and CYN were detected in the reservoir water, with concentrations as high as 36 µg/L. In addition, for the first time in Taiwan's tap water, CYN was detected at concentrations as high as 8.6 µg/L. Quality control data for the field samples shows that the analytical scheme developed is appropriate for monitoring cyanotoxins.