Sphingopyxis Species Isolated from Sand Filter Biofilm at an Australian Drinking Water Treatment Works.

Three strains isolated by geosmin enrichment from a sand filter in an Australian drinking water treatment works were genome sequenced to identify their taxonomic placement, and a bench-scale batch experiment confirmed their geosmin-degrading capability. Using the average nucleotide identity based on the MUMmer algorithm (ANIm), pairwise digital DNA-DNA hybridization (dDDH), and phylogenomic analyses, the strains were identified as Sphingopyxis species.

Determination of rate constants and half-lives for the simultaneous biodegradation of several cyanobacterial metabolites in Australian source waters.

The fate of five cyanobacterial metabolites was assessed in water sourced from Lake Burragorang (Warragamba Dam) in New South Wales, Australia. All of the studied metabolites were shown to be biodegradable in this water source. For some metabolites, biodegradation was influenced by factors, including temperature, location (within the water body) and seasonal variations. The biodegradation of the metabolites was shown to follow pseudo-first-order kinetics with rate constants ranging from 8.0 × 10(-4) to 1.3 × 10(-2) h(-1). Half-lives of the metabolites were also estimated and ranged from 2.2 to 36.1 d. The order of ease of biodegradability in this water source followed the trend: microcystin-LR ≥ cylindrospermopsin > saxitoxins > geosmin ≥ 2-methylisoborneol. The lack of detection of the mlrA gene during microcystin biodegradation suggests that these toxins may be degraded via a different pathway. While no metabolite-degrading organisms were isolated in this study, the inoculation of previously isolated geosmin- and microcystin-degrading bacteria into Lake Burragorang water resulted in efficient biodegradation of the respective metabolites. For example, microcystin-degrading isolate TT25 was able to degrade three microcystin variants to concentrations below analytical detection within 24 h, suggesting that inoculation of such bacteria has the potential to enhance biodegradation in Lake Burragorang.

Comparison of drinking water treatment process streams for optimal bacteriological water quality.

Four pilot-scale treatment process streams (Stream 1 - Conventional treatment (coagulation/flocculation/dual media filtration); Stream 2 - Magnetic ion exchange (MIEX)/Conventional treatment; Stream 3 - MIEX/Conventional treatment/granular activated carbon (GAC) filtration; Stream 4 - Microfiltration/nanofiltration) were commissioned to compare their effectiveness in producing high quality potable water prior to disinfection. Despite receiving highly variable source water quality throughout the investigation, each stream consistently reduced colour and turbidity to below Australian Drinking Water Guideline levels, with the exception of Stream 1 which was difficult to manage due to the reactive nature of coagulation control. Of particular interest was the bacteriological quality of the treated waters where flow cytometry was shown to be the superior monitoring tool in comparison to the traditional heterotrophic plate count method. Based on removal of total and active bacteria, the treatment process streams were ranked in the order: Stream 4 (average log removal of 2.7) > Stream 2 (average log removal of 2.3) > Stream 3 (average log removal of 1.5) > Stream 1 (average log removal of 1.0). The lower removals in Stream 3 were attributed to bacteria detaching from the GAC filter. Bacterial community analysis revealed that the treatments affected the bacteria present, with the communities in streams incorporating conventional treatment clustering with each other, while the community composition of Stream 4 was very different to those of Streams 1, 2 and 3. MIEX treatment was shown to enhance removal of bacteria due to more efficient flocculation which was validated through the novel application of the photometric dispersion analyser.

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.

Removal of cyanobacterial metabolites through wastewater treatment plant filters.

Wastewaters have the potential to proliferate excessive numbers of cyanobacteria due to high nutrient levels. This could translate to the production of metabolites, such as the saxitoxins, geosmin and 2-methylisoborneol (MIB), which can impair the quality of wastewater destined for re-use. Biological sand filtration was assessed for its ability to remove these metabolites from a wastewater. Results indicated that the sand filter was incapable of effectively removing the saxitoxins and in some instances, the effluent of the sand filter displayed greater toxicity than the influent. Conversely, the sand filter was able to effectively remove geosmin and MIB, with removal attributed to biodegradation. Granular activated carbon was employed as an alternative filter medium to remove the saxitoxins. Results showed similar removals to previous drinking water studies, where efficient removals were initially observed, followed by a decrease in the removal; a consequence of the presence of competing organics which reduced adsorption of the saxitoxins.

Biodegradation of multiple cyanobacterial metabolites in drinking water supplies.

The fate of multiple cyanobacterial metabolites was assessed in two Australian source waters. The saxitoxins were the only metabolites shown to be non-biodegradable in Myponga Reservoir water, while microcystin-LR (MCLR) and geosmin were biodegradable in this water source. Likewise, cylindrospermopsin (CYN) was shown to be biodegradable in River Murray water. The order of ease of biodegradability followed the trend: MCLR>CYN>geosmin>saxitoxins. Biodegradation of the metabolites was affected by temperature and seasonal variations with more rapid degradation at 24°C and during autumn compared with 14°C and during winter. A microcystin-degrading bacterium was isolated and shown to degrade four microcystin variants within 4 h. This bacterium, designated as TT25, was shown to be 99% similar to a Sphingopyxis sp. based on a 16S rRNA gene fragment. Isolate TT25 was shown to contain a homologue of the mlrA gene; the sequence of which was 99% similar to that of a previously reported microcystin-degrader. Furthermore, isolate TT25 could degrade the microcystins in the presence of copper sulphate (0.5 mg L(-1) as Cu(2+)) which is advantageous for water authorities dosing such algicides into water bodies to control cyanobacterial blooms.

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.

Solar radiation induces non-nuclear perturbations and a false start to regulated exocytosis in Cryptosporidium parvum.

Stratospheric ozone depletion, climate warming and acidification of aquatic ecosystems have resulted in elevated levels of solar radiation reaching many aquatic environments with an increased deleterious impact on a wide range of living organisms. While detrimental effects on living organisms are thought to occur primarily through DNA damage, solar UV can also damage cellular proteins, lipids and signalling pathways. Cryptosporidium, a member of the eukaryotic phylum Apicomplexa, contain numerous vesicular secretory organelles and their discharge via regulated exocytosis is essential for the successful establishment of infection. Using flow cytometric techniques we demonstrate that solar UV rapidly induces sporozoite exocytosis resulting in a significant reduction in the ability of sporozoites to attach and invade host cells. We found that solar UV induced sporozoite membrane depolarization, resulting in reduced cellular ATP and increased cytosolic calcium. These changes were accompanied by a reduction in the internal granularity of sporozoites, indicative of apical organelle discharge, which was confirmed by analysis of sporozoites with an exocytosis-sensitive dye. The precise timing of apical organelle discharge in the presence of a compatible host cell is critical for sporozoite attachment and invasion. Our results demonstrate for the first time how solar UV radiation can interfere with exocytosis, a fundamental cellular process in all eukaryotic cells. We contend that not only may the forecast increases in solar radiation in both aquatic and terrestrial environments significantly affect members of the Apicomplexa, solar UV-induced membrane depolarizations resulting in cytosolic calcium perturbation may affect a wider range of eukaryotic organisms through antagonistic effects on a myriad of calcium dependant cellular functions.

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.

Development of an mlrA gene-directed TaqMan PCR assay for quantitative assessment of microcystin-degrading bacteria within water treatment plant sand filter biofilms.

We report for the first time a quantitative mlrA gene-directed TaqMan PCR assay for the rapid detection of microcystin-degrading bacteria. This was applied, in combination with 16S ribosomal DNA-directed quantitative PCR and denaturing gradient gel electrophoresis, to study virgin sand filter column biofilm development and to correlate mlrA gene abundance with microcystin removal efficiency.

Biodegradation of geosmin by a novel Gram-negative bacterium; isolation, phylogenetic characterisation and degradation rate determination.

Biologically active sand filters within water treatment plants (WTPs) are now recognised as an effective barrier for the removal of geosmin. However, little is known regarding the actual microbiological processes occurring or the bacteria capable of degrading geosmin. This study reports the enrichment and isolation of a Gram-negative bacterium, Geo48, from the biofilm of a WTP sand filter where the isolate was shown to effectively degrade geosmin individually. Experiments revealed that Geo48 degraded geosmin in a planktonic state by a pseudo-first-order mechanism. Initial geosmin concentrations ranging from 100 to 1000ng/l were shown to directly influence geosmin degradation in reservoir water by Geo48, with rate constants increasing from 0.010h(-1) (R(2)=0.93) to 0.029h(-1) (R(2)=0.97) respectively. Water temperature also influenced degradation of geosmin by Geo48 where temperatures of 11, 22 and 30 degrees C resulted in rate constants of 0.017h(-1) (R(2)=0.98), 0.023h(-1) (R(2)=0.91) and 0.019h(-1) (R(2)=0.85) respectively. Phylogenetic analysis using the 16S rRNA gene of Geo48 revealed it was a member of the Alphaproteobacteria and clustered with 99% bootstrap support with an isolate designated Geo24, a Sphingopyxis sp. previously described as degrading geosmin but only as a member of a bacterial consortium. Of the previously described bacteria, Geo48 was most similar to Sphingopyxis alaskensis (97.2% sequence similarity to a 1454bp fragment of the 16S rRNA gene). To date, this is the only study to report the isolation and characterisation of a Gram-negative bacterium from a biologically active sand filter capable of the sole degradation of geosmin.

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.

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.

FACS enrichment and identification of floc-associated alphaproteobacterial tetrad-forming organisms in an activated sludge community.

A precise phylogenetic identity of the Defluviicoccus-related glycogen-accumulating organisms (GAO) observed after FISH probing in a novel activated sludge process removing phosphorus was sought with the aim of exploring the phylogenetic diversity of this important group. These organisms, whose sequences were not revealed in previously generated community wide 16S rRNA gene clone libraries, were identified using flow cytometry cell sorting of FISH-positive cells. Sequencing of a 16S rRNA gene clone library created from this sorted population identified the Defluviicoccus-related GAO as being highly related to previous identified GAO from enhanced biological phosphorus removal systems, despite a marked environmental difference between the two systems.

Assessing the impact of the biological control agent Bacillus thuringiensis on the indigenous microbial community within the pepper plant phyllosphere.

Although biological control agents (BCAs) have been used extensively for controlling insects and pathogens of plants, little is known regarding the effects of such agents on the indigenous microbial communities within the plant phyllosphere. We assessed the effect of the BCA Bacillus thuringiensis (Bt) on the microbial communities within the pepper plant phyllosphere using culture-independent methodologies. Phospholipid fatty acid (PLFA) analysis suggested that the bacterial and fungal biomass were not significantly affected following Bt application. However, principal component analysis of PLFA data indicated that Bt did change the phyllosphere microbial community structure significantly. 16S rRNA gene-directed PCR with denaturing gradient gel electrophoresis (DGGE) also suggested a significant change in the phyllosphere bacterial community structure following Bt inoculation. Phylogenetic analysis of excised DGGE bands suggested a change in bacterial phyla; bands from untreated samples predominantly belonged to the Firmicutes, while Gammaproteobacteria abounded in the treated samples.

Isolation and identification of a novel microcystin-degrading bacterium from a biological sand filter.

A novel bacterium capable of degrading two microcystin analogues, microcystin-LR and -LA (MCLR and MCLA), was isolated from a biological sand filter which was previously shown to effectively remove these toxins from source waters. Based on phylogenetic analysis of the 16S rRNA gene sequence, the isolated organism, LH21, most likely belonged to the genus Sphingopyxis and of the previously cultured species clustered with Sphingopyxis witflariensis. Using polymerase chain reaction (PCR), isolate LH21 was shown to contain homologues to each of the four genes, mlrA, mlrB, mlrC and mlrD previously associated with the degradation of MCLR by Sphingomonas sp. ACM-3962. Isolate LH21 was able to effectively degrade MCLR and MCLA in batch experiments under environmentally relevant conditions, with complete removal observed within 5h after re-exposure of the toxins.

Biodegradation rates of 2-methylisoborneol (MIB) and geosmin through sand filters and in bioreactors.

Taste and odour (T&O) causing compounds, in particular, 2-methylisoborneol (MIB) and geosmin, are a problem for water authorities as they are recalcitrant to conventional water treatment. In this study, biological sand filtration was shown to be an effective process for the complete removal of MIB and geosmin, with removal shown to be predominantly through biodegradation. In addition, MIB and geosmin were also effectively degraded in batch bioreactor experiments using biofilm sourced from one of the sand filters as the microbial inoculum. The biodegradation of MIB and geosmin was determined to be a pseudo-first-order reaction with rate constants ranging between 0.10 and 0.58 d(-1) in the bioreactor experiments. Rate constants were shown to be dependent upon the initial concentration of the microbial inoculum but not the initial concentration of MIB and geosmin when target concentrations of 200 and 50 ng l(-1) were used. Furthermore, rate constants were shown to increase upon re-exposure of the biofilm to both T&O compounds. Enrichment cultures with subsequent community profile analysis using 16S rRNA-directed PCR-DGGE identified four bacteria most likely involved in the biodegradation of geosmin within the sand filters and bioreactors. These included a Pseudomonas sp., Alphaproteobacterium, Sphingomonas sp. and an Acidobacteriaceae member.

Bacterial degradation of microcystin toxins within a biologically active sand filter.

Microcystin toxins are a problem for water authorities as they are recalcitrant to conventional water treatment. In this study, biological sand filtration was assessed in laboratory column experiments for its ability to remove two microcystin analogues, microcystin-LR and microcystin-LA. A lag period of 3 days was evident prior to the commencement of degradation. Contact times were varied during the experiment; however, no microcystin was detected in the effluent after 4 days, even under conditions similar to those of a rapid sand filter. Removals of microcystin through the sand filters were shown to be primarily through biological degradation processes. Using polymerase chain reaction (PCR), biofilm, extracted from one of the sand filters that had effectively removed the microcystins, was shown to contain bacteria with the mlrA gene. Detection of this gene provided additional evidence that biological degradation of microcystin was the primary removal mechanism.

Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system.

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 x 10(5) 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 x 10(3) active AOB ml(-1) were detected in the membrane-intact fraction, and 1.40 x 10(4) 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.

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.