Daniel Hoefel

Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques.

Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of physiologically active bacteria from raw and potable waters taken from various locations around South Australia. Results were compared to the direct viable count (DVC) and quantitative DVC (qDVC), in addition to the culture-based methods of the heterotrophic plate count (HPC) and a commercial SimPlate technique. Raw and potable water analysis revealed that DVC and culture-based techniques reported significantly fewer viable bacteria compared to the number of physiologically active bacteria detected using the rapid FCM assays, where this difference appeared to be nonlinear across different samples. Inconclusive results were obtained using qDVC as a viability assay. In particular, HPC results were 2-4 log orders of magnitude below that reported by the FCM assays for raw waters. Few bacteria in potable waters examined were culturable by HPC, even though FCM assays reported between 5.56 x 10(2) and 3.94 x 10(4) active bacteria ml(-1). These differences may be attributed to the presence of nonheterotrophic bacteria, sublethal injury or the adoption of an active but nonculturable (ABNC) state.

Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

ABSTRACT Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 105 bacteria ml−1. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 × 103 active AOB ml−1 were detected in the membrane-intact fraction, and 1.40 × 104 active AOB ml−1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml−1 detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.

Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters

Aims:  To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters.

The impacts of cypermethrin pesticide application on the non-target microbial community of the pepper plant phyllosphere.

Although pesticides have been extensively used for controlling insects and disease pathogens of plants, little is known regarding the impacts of applying these pesticides on the microbial community in the plant phyllosphere. Here, we report the effects of cypermethrin pesticide application upon the microbial community of the pepper plant phyllosphere. Assessments were made using culture-independent techniques including phospholipid fatty acid analysis (PLFA) and 16S rRNA gene directed Polymerase Chain Reaction with Denaturing Gradient Gel Electrophoresis (PCR-DGGE). During the 21 day greenhouse study, PLFA results indicated that both total and bacterial biomass increased after application of the pesticide. PLFA profiles also indicated that Gram-negative bacteria became predominant. DGGE analysis confirmed a significant change in bacterial community structure within the phyllosphere following the pesticide application where different dendrogram clusters were observed between control and treated samples. Phylogenetic analysis also suggested a change in bacterial phyla following treatment, where bands sequenced within control cultures were predominantly of the Firmicutes phylum, but those bands sequenced in the treated samples were predominantly members of the Bacteroidetes and gamma-Proteobacteria phyla. In conclusion, this study revealed an increase in bacterial abundance and a shift in community composition within the pepper plant phyllosphere following the pesticide application, and highlighted the effective use of PLFA and PCR-DGGE for studying the effect of pesticides upon indigenous phyllosphere microbes.

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

Aims:  To profile the fractions of bacteria in heat‐treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen.

Cooperative biodegradation of geosmin by a consortium comprising three gram-negative bacteria isolated from the biofilm of a sand filter column

Aims:  To isolate and identify bacteria from a sand filter column capable of degrading the taste and odour compound, geosmin. In doing so, to investigate if these organisms degrade geosmin either individually or if an alternative mechanism is utilized.

Investigations into the biodegradation of microcystin-LR in wastewaters.

Microcystins are potent hepatotoxins that can be produced by cyanobacteria. These organisms can proliferate in wastewaters due to a number of factors including high concentrations of nutrients for growth. As treated wastewaters are now being considered as supplementary drinking water sources, in addition to their frequent use for irrigated agriculture, it is imperative that these wastewaters are free of toxins such as microcystins. This study investigated the potential for biodegradation of microcystin-LR (MCLR) in wastewaters through a biological sand filtration experiment and in static batch reactor experiments. MCLR was effectively removed at a range of concentrations and at various temperatures, with degradation attributed to the action of microorganisms indigenous to the wastewaters. No hepatotoxic by-products were detected following the degradation of MCLR as determined by a protein phosphatase inhibition assay. Using TaqMan polymerase chain reaction, the first gene involved in bacterial degradation of MCLR (mlrA) was detected and the responsible bacteria shown to increase with the amount of MCLR being degraded. This finding suggested that the degradation of MCLR was dependent upon the abundance of MCLR-degrading organisms present within the wastewater, and that MCLR may provide bacteria with a significant carbon source for proliferation; in turn increasing MCLR removal.

Assessing granular media filtration for the removal of chemical contaminants from wastewater

Granular media filtration was evaluated for the removal of a suite of chemical contaminants that can be found in wastewater. Laboratory- and pilot-scale sand and granular activated carbon (GAC) filters were trialled for their ability to remove atrazine, estrone (E1), 17α-ethynylestradiol (EE2), N-nitrosodimethylamine (NDMA), N-nitrosomorpholine (NMOR) and N-nitrosodiethylamine (NDEA). In general, sand filtration was ineffective in removing the contaminants from a tertiary treated wastewater, with the exception of E1 and EE2, where efficient removals were observed after approximately 150 d. Batch degradation experiments confirmed that the removal of E1 was through biological activity, with a pseudo-first-order degradation rate constant of 7.4 × 10(-3) h(-1). GAC filtration was initially able to effectively remove all contaminants; although removals decreased over time due to competition with other organics present in the water. The only exception was atrazine where removal remained consistently high throughout the experiment. Previously unreported differences were observed in the adsorption of the three nitrosamines, with the ease of removal following the trend, NDEA > NMOR > NDMA, consistent with their hydrophobic character. In most instances the removals from the pilot-scale filters were generally in agreement with the laboratory-scale filter, suggesting that there is potential in using laboratory-scale filters as monitoring tools to evaluate the performance of pilot- and possibly full-scale sand and GAC filters at wastewater treatment plants.

Enhancing the biofiltration of geosmin by seeding sand filter columns with a consortium of geosmin-degrading bacteria.

Geosmin is a secondary metabolite that can be produced by many species of cyanobacteria and Actinomycetes. It imparts a musty/earthy taste and odour to drinking water which can result in consumer complaints and a general perception that there is a problem with the water quality. As geosmin is recalcitrant to conventional water treatment, processes are sought to ensure effective removal of this compound from potable water. Biological filtration (biofiltration) is an attractive option for geosmin removal as this compound has been shown to be biodegradable. However, effective biofiltration of geosmin can be site specific as it is highly dependent upon the types of organism present and there is often an extended acclimation period before efficient removals are achieved. We report here, a novel approach to enhance the biofiltration of geosmin by seeding sand filter columns with a bacterial consortium previously shown to be capable of effectively degrading geosmin. Geosmin removals of up to 75% were evident through sand columns which had been inoculated with the geosmin-degrading bacteria, when compared with non-inoculated sand columns where geosmin removals were as low as 25%. These low geosmin removals through the non-inoculated sand columns are consistent with previous studies and were attributed to physical/abiotic losses. The presence of an existing biofilm was shown to influence geosmin removal, as the biofilm allowed for greater attachment of the geosmin-degrading consortium (as determined by an ATP assay), and enhanced removals of geosmin. Minimal difference in geosmin removal was observed when the geosmin-degrading bacteria were inoculated into the sand columns containing either an active or inactive biofilm.

Biodegradation of di-n-butyl phthalate by an isolated Gordonia sp. strain QH-11: Genetic identification and degradation kinetics

Di-n-butyl phthalate (DBP) is one of the most widely used phthalic acid esters (PAEs), which have shown increasing environmental concerns worldwide. A bacterial strain designated as QH-11, was isolated from activated sludge and found to be capable of utilizing DBP as carbon and energy sources for growth. 16S rRNA and gyrb gene sequence analysis revealed that strain QH-11 was most closely related to Gordonia sp. Kinetics studies of DBP degradation by the strain QH-11 revealed that DBP depletion curves fit with the modified Gompertz model (R(2)>0.98). Meanwhile, substrate utilization tests showed that strain QH-11 could utilize other common PAEs and also the main intermediate product phthalic acid (PA). A gene encoding the large subunit of the phthalate dioxygenase, which is responsible for PA degradation, was successfully detected in strain QH-11. Furthermore, the results of reverse transcription quantitative PCR demonstrate that mRNA expression level of phthalate dioxygenase increased significantly after strain QH-11 was induced by DBP and PA.