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.

Release and oxidation of cell-bound saxitoxins during chlorination of Anabaena circinalis cells.

Surface water sources are increasingly subject to proliferation of toxic cyanobacteria. Direct chlorination of source water containing toxic cyanobacterial cells for different treatment purposes might cause cell damage and toxin release. There is limited information available on chlorination of saxitoxins (STXs: saxitoxin, C-toxins, and gonyautoxins) produced by Anabaena circinalis. This work: (1) investigated the impact of chlorination on cell lysis and toxin/odor compound release in natural waters; (2) assessed the rates of chlorination of total STXs, and (3) estimated apparent rate constants for STX oxidation in ultrapure and natural waters. With a chlorine exposure (CT) value of 7.0 mg x min/L all cells lost viability causing toxin release. Cell-membrane damage occurred faster than released STXs oxidation. All saxitoxin and more than 95% of other STX analogues were subsequently oxidized. Kinetic analysis of the oxidation of STX analogues revealed significant differences in the susceptibility to chlorine, saxitoxin being the easiest to oxidize. Also, concentrations of trihalomethanes, haloacetic acids, and N-nitrosodimethylamine as chlorination byproducts were respectively <50 µg/L and 11 ng/L even at the highest CT value (50.3 mg x min/L).

Determining the fate of Microcystis aeruginosa cells and microcystin toxins following chloramination.

The cyanobacterium Microcystis aeruginosa can produce potent toxins known as microcystins. While many studies have focussed on the chlorination of microcystin toxins, little work has been conducted with respect to the chloramination of the microcystins. In addition, no studies have been reported on the effect of chloramination on intact Microcystis cells. This study was conducted to determine the fate of M. aeruginosa cells and microcystin toxins following chloramination of a drinking water source. Results indicate that monochloramine could effectively oxidise dissolved microcystin-LR (MCLR) provided high CT values were employed, typically greater than 30,000 mg min L(-1). The decay of MCLR was demonstrated to be a pseudo first-order reaction with rate constants ranging from 9.3x10(-7) to 1.1x10(-5) s(-1) at pH 8.5. However, in the presence of Microcystis cells, monochloramine was ineffective in oxidising microcystin toxins due to the cells exerting a demand on the oxidant. The doses of monochloramine applied (2.8 and 3.5 mg L(-1)) were shown to rapidly release intracellular microcystins into the dissolved state. Flow cytometric analysis of the cells determined that the lower monochloramine dose did not compromise the cell membrane integrity, even though microcystins were rapidly released from the cells. In contrast the higher monochloramine dose resulted in cell membrane disruption with up to 90% of the cells shown to be non-viable after the high dose was applied.

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.

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 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.

Optimising water treatment practices for the removal of Anabaena circinalis and its associated metabolites, geosmin and saxitoxins.

The cyanobacterium Anabaena circinalis has the ability to co-produce geosmin and saxitoxins, compounds which can compromise the quality of drinking water. This study provides pertinent information in optimising water treatment practices for the removal of geosmin and saxitoxins. In particular, it demonstrates that pre-oxidation using potassium permanganate could be applied at the head of water treatment plants without releasing intracellular geosmin and saxitoxins from A. circinalis. Furthermore, powdered activated carbon (PAC) was shown to be an effective treatment barrier for the removal of extracellular (dissolved) geosmin and saxitoxins, with similar adsorption trends of both compounds. The relative removal of the saxitoxins compared with geosmin was determined to be 0.84 +/- 0.27, which implies that saxitoxin removal with PAC can be estimated to be approximately 60 to 100% of the removal of geosmin under equivalent conditions. Chlorine was shown to be effective for the oxidation of the saxitoxins with CT values of approximately 30 mg min l(-1) required for greater than 90% destruction of the saxitoxins.

Investigating the fate of saxitoxins in biologically active water treatment plant filters.

The saxitoxins are potent neurotoxins, which can be produced by freshwater cyanobacteria. This study assessed the fate of five saxitoxins variants through biologically active laboratory filters containing media sourced from the filters beds of two water treatment plants (WTPs). Decreases in the concentration of the less toxic variants coincided with increases in the concentrations of the more toxic variants through the filters containing anthracite sourced from two different WTPs. No changes in toxin concentrations were evident through parallel filters containing sand. The results strongly suggest that organisms within the biofilm of the anthracite filters possessed the ability to biotransform the saxitoxins variants, which has important implications for drinking water treatment, particularly since this has the potential to increase the toxicity of the filtered water.

Bacterial degradation of microcystin toxins in drinking water eliminates their toxicity.

Microcystin-LR and -LA were readily biodegraded by a bacterium, Sphingpoyxis sp. LH21, in a treated reservoir water. Detection of the microcystins was conducted using high-performance liquid chromatography (HPLC), protein phosphatase 2A (PP2A) inhibition assay and a cell-based cytotoxicity assay. The HPLC results correlated well with the two assays. The decrease in cytotoxicity, coupled with the associated decrease in microcystin concentrations, indicated that no cytotoxic by-products were being generated, highlighting the applicability of biodegradation as a feasible treatment option for effective microcystin removal.

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.

Discriminating and assessing adsorption and biodegradation removal mechanisms during granular activated carbon filtration of microcystin toxins.

Microcystins are cyanobacterial toxins that are problematic for water authorities due to their resistance to conventional water treatment. Granular activated carbon (GAC) filtration has been shown to be effective in removing microcystin from water using both adsorption and biodegradation removal mechanisms; however, little is known regarding which removal mechanism predominates and to what extent. In this study, microcystin removal due to adsorption and biodegradation in GAC filtration were discriminated and assessed by commissioning three parallel laboratory columns, including a sterile GAC column, a conventional GAC column and a sand column. The results demonstrate that biodegradation is an efficient removal mechanism once it commences and that the rate of biodegradation was dependent upon temperature and initial bacterial concentration. Adsorption of microcystins was prevalent during the initial stages of the GAC columns and was modelled using the homogeneous surface diffusion model (HSDM). The HSDM provided evidence that an active biofilm present on the surface of the conventional GAC hindered adsorption of microcystin compared with the sterile GAC with no active biofilm. Up to 70% removal of microcystin-LR was still observed after 6 months of operation of the sterile GAC column, indicating that adsorption still played a vital role in the removal of this toxin.

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.

Differences in the chlorine reactivity of four microcystin analogues.

The presence of microcystin toxins in drinking water is highly undesirable as they have the potential to adversely affect human health. Consequently, effective removal of these toxins from water is a major goal for water authorities. In this study, four microcystin analogues were chlorinated in two treated waters, and two of the analogues were chlorinated in deionised water. The oxidation of the microcystins was related to the chlorine exposure (CT) of the sample waters with the ease of oxidation following the trend: microcystin-YR > microcystin-RR > microcystin-LR > or = microcystin-LA. This trend was in agreement with published data on model compounds and free amino acids. Values of CT of up to 25 mg min L(-1) were required for oxidation of all microcystin analogues to below the World Health Organization guideline value of 1.0 microg L(-1). Results from this study indicate that for some water resources it is important to determine the speciation of the microcystin analogues to optimise chlorination practices.

Effect of NOM, turbidity and floc size on the PAC adsorption of MIB during alum coagulation.

The effect of natural organic material (NOM) and turbidity on the powdered activated carbon (PAC) adsorption of the odour compound 2-methylisoborneol (MIB) was evaluated during alum coagulation. The character of the flocs, in terms of their size and fractal dimensions (Df), was used to interpret the observed adsorption behaviour of MIB during the coagulation process. As the alum dose was increased, the adsorption of MIB decreased. This was determined to be due to the size of the flocs, with larger flocs incorporating PAC into their structure, reducing the efficiency of mixing, and the bulk diffusion kinetics for the MIB molecule. The presence of turbidity also reduced MIB adsorption due to the formation of larger flocs. The character of NOM was found to have a greater influence on the adsorption of MIB than the floc structure.

Impact of UV-H2O2 Advanced Oxidation and Aging Processes on GAC Capacity for the Removal of Cyanobacterial Taste and Odor Compounds.

Cyanobacteria and their taste and odor (T&O) compounds are a growing concern in water sources globally. Geosmin and 2-methylisoborneol (MIB) are the most commonly detected T&O compounds associated with cyanobacterial presence in drinking water sources. The use of ultraviolet and hydrogen peroxide (H2O2) as an advanced oxidation treatment for T&O control is an emerging technology. However, residual H2O2 (>80% of the initial dose) has to be removed from water prior final disinfection. Recently, granular activated carbon (GAC) is used to remove H2O2 residual. The objective of this study is to assess the impact of H2O2 quenching and aging processes on GAC capacity for the removal of geosmin and MIB. Pilot columns with different types of GAC and presence/absence of H2O2 have been used for this study. H2O2 removal for the operational period of 6 months has no significant impact on GAC capacity to remove the geosmin and MIB from water.

Surface analysis of pilot distribution system pipe autopsies: The relationship of organic and inorganic deposits to input water quality.

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis was conducted to characterise deposits in polyethylene pipes used in a novel pilot water distribution system (PDS). The system consisted of four (4) parallel distribution systems receiving water from different treatment processes, ranging from conventional coagulation through to an advanced membrane filtration system. After two years of operation, the distribution system was shut down and samples of pipe were collected for autopsy analysis. Inlet and outlet samples from each PDS were collected for purpose of comparison. ToF-SIMS was used to assess chemical differences in surface biofilm accumulation and particulate deposition, which resulted as a consequence of the treatment method and operational mode of each system. These data supplemented previously collected bacteriological and chemical water quality data. Results from the inorganic analysis of the pipes were consistent with corrosion and contamination events that occurred upstream in the corresponding treatment systems. Principal component analysis of data on organic constituents showed oxygen and nitrogen containing fragments were associated with the treatment inlet and outlet samples. These types of signals can often be ascribed to biofilm polysaccharides and proteins. A trend was observed when comparing samples from the same PDS, showing an association of lower molecular weight (MW) organic fragments with the inlet and higher MW organic fragments with the outlet samples.

Fate of geosmin and 2-methylisoborneol in full-scale water treatment plants.

The increasing frequency and intensity of taste and odour (T&O) producing cyanobacteria in water sources is a growing global issue. Geosmin and 2-methylisoborneol (MIB) are the main cyanobacterial T&O compounds and can cause complaints from consumers at levels as low as 10 ng/L. However, literature concerning the performance of full-scale treatment processes for geosmin and MIB removal is rare. Hence, the objectives of this study were to: 1) estimate the accumulation and breakthrough of geosmin and MIB inside full-scale water treatment plants; 2) verify the potential impact of sludge recycling practice on performance of plants; and, 3) assess the effectiveness of aged GAC for the removal of these compounds. Sampling after full-scale treatment processes and GAC pilot assays were conducted to achieve these goals. Geosmin and MIB monitoring in full-scale plants provided the opportunity to rank the performance of studied treatment processes with filtration and granular activated carbon providing the best barriers for removal of total and extracellular compounds, correspondingly. Geosmin was removed to a greater extent than MIB using GAC. Geosmin and MIB residuals in water post GAC contactors after two years of operation was 20% and 40% of initial concentrations, correspondingly. Biological activity on the GAC surface enhanced the removal of T&O compounds. These observations demonstrated that a multi-barrier treatment approach is required to ensure cyanobacteria and their T&O compounds are effectively removed from drinking water.

Influence of the character of NOM on the ozonation of MIB and geosmin.

Tastes and odours (T&Os) are a major concern in drinking water as they are not efficiently removed by conventional water treatment. Ozonation has been effective for their destruction in some studies. However, the natural organic matter (NOM) in waters can affect the ozonation process and subsequently affect the destruction of T&Os. Five NOM fractions were isolated and ozonated in synthetic waters. The fraction containing the more highly coloured, higher molecular weight compounds exhibited the highest ozone (O3) demand, whereas the low aromatic fraction exhibited the lowest O3 demand. The character of the NOM fractions influenced the ozonation of MIB and geosmin. The destruction of MIB and geosmin was significantly higher in the fraction with the highest colour and UV/visible absorbance at all O3 doses. The destruction of the compounds in the other fractions showed the same trends, increasing MIB and geosmin destruction with increasing UV/visible absorbing character of the NOM. MIB was also ozonated in two real waters. with results showing a competing effect between NOM concentration and NOM character. The O3 reaction time was shown to be important for the destruction of both compounds.