Bio-analytical applications of microbial fuel cell-based biosensors for onsite water quality monitoring.

Globally, sustainable provision of high-quality safe water is a major challenge of the 21st century. Various chemical and biological monitoring analytics are presently utilized to guarantee the availability of high-quality water. However, these techniques still face some challenges including high costs, complex design and onsite and online limitations. The recent technology of using microbial fuel cell (MFC)-based biosensors holds outstanding potential for the rapid and real-time monitoring of water source quality. MFCs have the advantages of simplicity in design and efficiency for onsite sensing. Even though some sensing applications of MFCs were previously studied, e.g. biochemical oxygen demand sensor, recently numerous research groups around the world have presented new practical applications of this technique, which combine multidisciplinary scientific knowledge in materials science, microbiology and electrochemistry fields. This review presents the most updated research on the utilization of MFCs as potential biosensors for monitoring water quality and considers the range of potentially toxic analytes that have so far been detected using this methodology. The advantages of MFCs over established technology are also considered as well as future work required to establish their routine use.

Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

Development and field testing of a real-time PCR assay for cylindrospermopsin-producing cyanobacteria.

AIMS: To develop and test a real-time PCR assay to detect and quantify genes specific to Cylindrospermopsis sp. and cylindrospermopsin-producing cyanobacteria. METHOD AND RESULTS: A duplex real-time PCR assay was developed that targets a cylindrospermopsin-specific and Cylindrospermopsis raciborskii-specific DNA sequence. The C. raciborskii-specific sequence was based on the rpoC1 DNA-dependent RNA polymerase gene, whilst the cylindrospermopsin-specific sequence was selected by surveying an extensive number of potential cylindrospermopsin-producing cyanobacterial strains for genes implicated in toxin production, aoaA, aoaB and aoaC. In toxic strains, sequences of each of these three genes were always present; whilst in nontoxic strains the distribution of these sequences was patchy, resulting in what are likely to be natural deletion mutants. The real-time assay was optimized on a fixed and portable device, with results indicating that the reliable limit of detection for the assay was 100 copies per reaction or 1000 cells ml(-1) for both target sequences on both devices. In routine environmental samples enumerated by microscopy, the assay results were positive for all samples where C. raciborskii cells were observed at >1000 cells ml(-1) and negative in 15 samples where no C. raciborskii cells were observed. In field samples, the number of copies of the rpoC1 sequence more closely approximated the number of cells enumerated by microscopy, the number of copies of the pks sequence and detection of the toxin-specific sequence matched the results of toxin testing. CONCLUSIONS: The duplex real-time PCR assay was a sensitive and rapid method for detecting potential cylindrospermopsin-producing cyanobacteria in the laboratory or in the field. The observation of probable natural deletion mutants provides further evidence that the aoaA, aoaB and aoaC genes are involved in toxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay provides a new monitoring capability for tracking cylindrospermopsin-producing cyanobacteria that are an emerging threat to water quality.

Bathroom greywater recycling using polyelectrolyte-complex bilayer membrane: Advanced study of membrane structure and treatment efficiency.

Polyelectrolyte-complex bilayer membrane (PCBM) was fabricated using biodegradable chitosan and alginate polymers for subsequent application in the treatment of bathroom greywater. In this study, the properties of PCBMs were studied and it was found that the formation of polyelectrolyte network reduced the molecular weight cut-off (MWCO) from 242kDa in chitosan membrane to 2.71kDa in PCBM. The decrease in MWCO of PCBM results in better greywater treatment efficiency, subsequently demonstrated in a greywater filtration study where treated greywater effluent met the household reclaimed water standard of <2 NTU turbidity and <30ppm total suspended solids (TSS). In addition, a further 20% improvement in chemical oxygen demand (COD) removal was achieved as compared to a single layer chitosan membrane. Results from this study show that the biodegradable PCBM is a potential membrane material in producing clean treated greywater for non-potable applications.

A catabolic plasmid involved in 4-methyl-o-phthalate and 4-hydroxy-iso-phthalate degradation in Pseudomonas cepacia.

Genes involved in 4-methyl-o-phthalate and 4-hydroxy-iso-phthalate catabolism reside on a 226-232 kbp catabolic plasmid termed MOP. This was confirmed by transformation and conjugation into an isogenic heat-cured (MOP-) derivative of the wild-type isolate, identified and termed Pseudomonas cepacia Pc701. Transformation confirmed the presence of Tn1 in MOP derived from Pc704, a mutant deficient in 4-methyl-o-phthalate catabolism. pCS1, a recombinant plasmid bearing MOP DNA, complemented MOP::Tn1 restoring the ability of Pc704 to grow on 4-methyl-o-phthalate. DNA-DNA hybridization using pCS1 as probe confirmed that loss of 4-methyl-o-phthalate catabolism by Pc704 was the result of Tn1 insertion into a 2.1 kbp HindIII fragment of MOP.

A PCR test for the identification and discrimination of Legionella longbeachae serogroups 1 and 2.

A PCR test has been developed for the specific identification of Legionella longbeachae. The test targeted sequence unique to both L. longbeachae serogroups 1 and 2 within the mip gene and permitted both species and serogroup identification. The test was trialed on a range of closely related species and 20 clinical isolates originating from Australia, the USA and Israel. Results were consistent with previous identification analyses. From 20 water samples known to contain Legionella spp. one sample yielded isolates which consistently tested positive by L. longbeachae serogroup 1 PCR. DNA sequencing of the PCR product, 5S rRNA gene sequence and hybridisation analysis with a specific oligonucleotide probe definitively identified one isolate as L. longbeachae serogroup 1. PCR testing was demonstrated as a superior method of identification to traditional seroagglutination reactions, which were ambiguous and could explain the previous failure to identify the presence of this microorganism in water.

Development of glucosidase agar for the confirmation of water-borne Enterococcus.

Analysis of 56 river water samples by the Enterolert defined substrate technique, and standard m-Enterococcus agar isolation followed by confirmation, indicated that after 24 h incubation. Enterolert significantly underestimated the true numbers of enterococci. Extending Enterolert incubatioin to 36 h improved detection but also revealed false positives. These findings prompted the development of a novel confirmation medium we have termed glucosidase agar, which was prepared by dissolving Enterolert substrate in 2% (w/v) bacteriological agar. Analysis of 1,043 colonies arising on m-Enterococcus agar from 280 freshwater, marine and sewage effluent samples, demonstrated that 2-4 h incubation on glucosidase agar was a rapid and accurate means of confirming presumptive enterococci, when compared to standard confirmation procedures that take 48 h. The combination of primary isolation on m-Enterococcus agar followed by confirmation on glucosidase agar permits maximum recovery of Enteroccus whilst effectively eliminating false positives/negatives and provides a reliable alternative use of the Enterolert defined substrate technology.

Development of a nested-PCR assay for the detection of Cryptosporidium parvum in finished water.

A nested-PCR assay, incorporating an internal positive control, was developed for Cryptosporidium monitoring in finished water. This assay was capable of reproducibly detecting 8 oocysts in spiked-filtered water samples collected from 5 South Australian water treatment plants. The RT-PCR assay of Kaucner and Stinear (Appl. Environ. Microbiol. 64(5) (1998) 1743) was also evaluated for the detection of Cryptosporidium parvum. Initially, under our experimental conditions, a detection level of 27 oocysts was achieved for spiked reagent water samples. This level was improved to 5 oocysts by modification of the method. Untreated South Australian source waters concentrated by calcium carbonate flocculation were found to be highly inhibitory to the RT-PCR assay. Concentration of similar samples using Envirochek filters appeared to eliminate PCR inhibition. While both methods possessed similar sensitivities the nested-PCR assay was more reproducible, more cost effective, simpler to perform and could detect both viable and non-viable intact Cryptosporidium parvum oocysts, which is an important consideration for plant operators. These factors make the nested-PCR assay the method of choice for screening large numbers of potable water samples, where a reliable low level of detection is essential.

The "Nostocoida limicola" story: resolving the phylogeny of this morphotype responsible for bulking in activated sludge.

On the basis of 16S rRNA sequence analyses of several isolates of "Nostocoida limicola" from activated sludge plants in Australia and other countries, it is clear that "N. limicola" I, II and III are not three morphological variants of a single bacterium but at least three phylogenetically different bacteria. Data show that "N. limicola" I are members of at least two genera in the low mol% G+C gram-positive bacteria, while some isolates of "N. limicola" II belong to the high mol% G+C gram positive bacteria, and "N. limicola" III is a member of the Planctomycetales. Design and application of 16S rRNA targeted probes for each to biomass samples suggests that their phylogeny is more diverse than pure culture studies would suggest.

Rapid preparation of cyanobacterial DNA for real-time PCR analysis.

AIMS: To develop a rapid preparation method for real-time PCR analysis of cyanobacteria from cultures or field samples. METHODS AND RESULTS: Field samples and cultures containing Anabaena circinalis, Cylindrospermopsis raciborskii or Microcystis aeruginosa were subjected to three cell disruption treatments: (i) heating during thermocycling, (ii) microwave irradiation in the presence of detergent and (iii) probe sonication. Treated samples were directly added to the PCR reaction and analysed on two different real-time devices. A statistically significant difference was evident in the cycle thresholds for each of the treatments in all but one culture and one environmental sample, sonication and microwave treatments performing better than direct addition. The microwave treatment was also compared to the Qiagen DNA Mini kit and performance was equivalent when treated samples were analysed as above. CONCLUSIONS: Whilst microwave treatment was slightly less effective than probe sonication across all samples, it was more amenable to processing multiple samples and significantly better than heat treating the sample during thermocycling. SIGNIFICANCE AND IMPACT OF THE STUDY: The microwave method described here is a simple, rapid and effective preparation method for cyanobacterial DNA that can be easily deployed in the field, making the most of the speed and flexibility offered by fixed and portable real-time PCR devices.

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. METHODS AND RESULTS: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. CONCLUSION: Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.

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. METHODS AND RESULTS: Geosmin-degrading bacteria from a biologically active sand filter column were enriched by their growth in a minimal medium supplemented with geosmin as the sole carbon source. By day 51, 21.7 mg l(-1) of geosmin had been degraded as determined by solid-phase microextraction gas chromatography/mass spectrometry, and was accompanied by a 2.12 log(10) increase in active bacterial numbers as measured using the BacLight(TM) bacterial viability kit and flow cytometric enumeration. During the onset of geosmin degradation, the predominance of three bacteria, most similar to previously cultured species of Sphingopyxis alaskensis, Novosphingobium stygiae and Pseudomonas veronii based on 16S rRNA gene sequences was detected by denaturing gradient gel electrophoresis. Subsequent isolation of these organisms revealed that degradation of geosmin, when present as either the sole carbon source (ranging from 40 ng l(-1) to 20 mg l(-1)) or when spiked into sterile reservoir water (37 and 131 ng l(-1)), occurred only when all three isolates were present. None of the isolates was shown to be capable of degrading geosmin either individually or in any combination of two. CONCLUSIONS: This study has reported, for the first time, the cooperative degradation of geosmin by a consortium comprising three gram-negative bacteria isolated from a biologically active sand filter column. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are important for researchers currently employing molecular-based approaches to further understand the biodegradation of geosmin by bacteria, as such studies may be complicated by the discovery of geosmin degradation occurring by a consortium. This study also advances the knowledge surrounding the types of bacteria capable of degrading the taste and odour compound, as investigations to date regarding this are limited.

Isolates of 'Candidatus Nostocoida limicola' Blackall et al. 2000 should be described as three novel species of the genus Tetrasphaera, as Tetrasphaera jenkinsii sp. nov., Tetrasphaera vanveenii sp. nov. and Tetrasphaera veronensis sp. nov.

Despite differences in their morphologies, comparative analyses of 16S rRNA gene sequences revealed high levels of similarity (>94 %) between strains of the filamentous bacterium 'Candidatus Nostocoida limicola' and the cocci Tetrasphaera australiensis and Tetrasphaera japonica and the rod Tetrasphaera elongata, all isolated from activated sludge. These sequence data and their chemotaxonomic characters, including cell wall, menaquinone and lipid compositions and fingerprints of their 16S-23S rRNA intergenic regions, support the proposition that these isolates should be combined into a single genus containing six species, in the family Intrasporangiaceae in the Actinobacteria. This suggestion receives additional support from DNA-DNA hybridization data and when partial sequences of the rpoC1 gene are compared between these strains. Even though few phenotypic characterization data were obtained for these slowly growing isolates, it is proposed, on the basis of the extensive chemotaxonomic and molecular evidence presented here, that 'Candidatus N. limicola' strains Ben 17, Ben 18, Ben 67, Ben 68 and Ben 74 all be placed into the species Tetrasphaera jenkinsii sp. nov. (type strain Ben 74(T)=DSM 17519(T)=NCIMB 14128(T)), 'Candidatus N. limicola' strain Ben 70 into Tetrasphaera vanveenii sp. nov. (type strain Ben 70(T)=DSM 17518(T)=NCIMB 14127(T)) and 'Candidatus N. limicola' strains Ver 1 and Ver 2 into Tetrasphaera veronensis sp. nov. (type strain Ver 1(T)=DSM 17520(T)=NCIMB 14129(T)).

Profiling bacterial survival through a water treatment process and subsequent distribution system.

AIMS: To profile fractions of active bacteria and of bacteria culturable with routine heterotrophic plate count (HPC) methods through a typical water treatment process and subsequent distribution system. In doing so, investigate how water treatment affects both bacterial abundance and diversity, and reveal the identities of active bacteria not detected by traditional HPC culture. METHODS AND RESULTS: Profiling active fractions was performed by flow cytometric cell sorting of either membrane-intact (BacLight kit) or enzymatically active (carboxyfluorescein diacetate, CFDA) bacteria, followed by eubacterial 16S rDNA-directed PCR and denaturing gradient gel electrophoresis (DGGE). Water treatment significantly reduced active bacterial numbers detected by the BacLight kit and CFDA assay by 2.89 and 2.81 log respectively. Bacterial diversity was also reduced from > 20 DGGE bands in the active fractions of reservoir water to only two bands in the active fractions of finished water. These two bands represented Stenotrophomonas maltophila, initially culturable by HPC, and a Burkholderia-related species. Both species maintained measurable traits of physiological activity in distribution system bulk water but were undetected by HPC. CONCLUSIONS: Flow cytometric cell sorting with PCR-DGGE, to assess water treatment efficacy, identified active bacteria from a variety of major phylogenetic groups undetected by routine HPC. Following treatment S. maltophila and a Burkholderia-related species retained activity and entered distribution undetected by HPC. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods used here demonstrate how water treatment operators can better monitor water treatment plant efficacy and assess distribution system instability by the detection and identification of active bacteria recalcitrant to routine HPC culture.

A colony based confirmation assay for Legionella and Legionella pneumophila employing the EnviroAmp Legionella system and seroagglutination.

Modifications to the EnviroAmp Legionella detection system are described which permit the rapid analysis of bacterial colonies taken from Legionella selective media. Capillary PCR permitted twice the number of samples to be analysed with a single kit. When PCR was positive for Leg. pneumophila, this result was confirmed by seroagglutination. The reverse dot blot hybridization assay was only used where PCR indicated a Legionella sp. other than Leg. pneumophila, permitting further savings on detection system components. This technique and standard confirmation procedures were applied to 133 isolates arising from 63 water samples plated to Legionella isolation media. Results agreed except for two isolates which gave a positive result for Legionella spp. by PCR and hybridization but were negative using standard procedures. Raising the annealing/extension temperature of the PCR by 2 degrees C eliminated the false positive result with these two isolates but did not adversely effect the sensitivity of the assay, as determined by re-testing of 68 environmental isolates and testing of 69 new environmental isolates and 12 Legionella reference species. The modified technique provides a convenient and cost effective alternative to standard confirmation procedures.

Molecular phylogeny of Anabaena circinalis and its identification in environmental samples by PCR.

Although the cyanobacterium Anabaena circinalis occurs worldwide, Australian isolates are believed to exclusively possess the saxitoxin group neurotoxins (paralytic shellfish poisons). Identification of A. circinalis in a mixed population is complicated due to limited morphological differences between Anabaena species. Sequence analysis of the DNA-dependent RNA polymerase (rpoC1) gene from 24 Anabaena isolates, including 12 designated A. circinalis, permitted a phylogenetic analysis to be performed. In addition, an A. circinalis-specific PCR was developed and tested successfully on environmental samples.

4-Methylphthalate catabolism in Burkholderia (Pseudomonas) cepacia Pc701: a gene encoding a phthalate-specific permease forms part of a novel gene cluster.

We have determined the entire nucleotide sequence of a 4.4 kbp fragment of pMOP, a plasmid involved in 4-methylphthalate catabolism in Burkholderia cepacia (formerly Pseudomonas cepacia) Pc701. Two complete ORFs were identified and termed mopA and mopB. mopB encodes a 4-methylphthalate permease which is a member of a superfamily of symport proteins found in both prokaryotes and eukaryotes. Functionality was assigned to MopB by detailed analysis of the predicted amino acid sequence, resulting in the identification of 12 hydrophobic membrane-spanning domains and motifs associated with this class of protein. An assay was developed to demonstrate MopB function in substrate uptake. Of 4-methylphthalate, 4-hydroxyisophthalate, benzoate, p-toluate and phthalate, only uptake of 4-methylphthalate and phthalate was demonstrated, suggesting that two carboxyl groups in the ortho position are essential for substrate recognition. The predicted protein MopA showed significant levels of homology to reductase proteins implicated in aromatic and aliphatic catabolism, and contained motifs recognized as binding the ADP and flavin moieties of FAD/NAD. Northern hybridization experiments determined that mopA and mopB are contranscribed, but expression was only seen in cells grown on 4-methylphthalate and not in cells grown on closely related structural analogues, including phthalate. mopA and mopB may be situated at the 3' terminus of a cistron about 10 kbp in size. The isolation and characterization of a 4-methylphthalate permease gene may lead to the identification of other permeases involved in bacterial biodegradation processes and possibly the construction of strains with enhanced degradative abilities.

Nucleotide sequences and regulational analysis of genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22(pTDN1).

A 9,233-bp HindIII fragment of the aromatic amine catabolic plasmid pTDN1, isolated from a derivative of Pseudomonas putida mt-2 (UCC22), confers the ability to degrade aniline on P. putida KT2442. The fragment encodes six open reading frames which are arranged in the same direction. Their 5' upstream region is part of the direct-repeat sequence of pTDN1. Nucleotide sequence of 1.8 kb of the repeat sequence revealed only a single base pair change compared to the known sequence of IS1071 which is involved in the transposition of the chlorobenzoate genes (C. Nakatsu, J. Ng, R. Singh, N. Straus, and C. Wyndham, Proc. Natl. Acad. Sci. USA 88:8312-8316, 1991). Four open reading frames encode proteins with considerable homology to proteins found in other aromatic-compound degradation pathways. On the basis of sequence similarity, these genes are proposed to encode the large and small subunits of aniline oxygenase (tdnA1 and tdnA2, respectively), a reductase (tdnB), and a LysR-type regulatory gene (tdnR). The putative large subunit has a conserved [2Fe-2S]R Rieske-type ligand center. Two genes, tdnQ and tdnT, which may be involved in amino group transfer, are localized upstream of the putative oxygenase genes. The tdnQ gene product shares about 30% similarity with glutamine synthetases; however, a pUC-based plasmid carrying tdnQ did not support the growth of an Escherichia coli glnA strain in the absence of glutamine. TdnT possesses domains that are conserved among amidotransferases. The tdnQ, tdnA1, tdnA2, tdnB, and tdnR genes are essential for the conversion of aniline to catechol.

Rapid confirmation of Clostridium perfringens by using chromogenic and fluorogenic substrates.

The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for C. perfringens confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life.

Loss of Tdn catabolic genes by deletion from and curing of plasmid pTDN1 in Pseudomonas putida: rate and mode of loss are substrate and pH dependent.

The ability to degrade aromatic amines and m-toluate (Tdn+ phenotype), encoded by plasmid pTDN1, was lost from Pseudomonas putida hosts after subculture in benzoate, succinate, acetate and glucose minimal medium, the fastest rate of loss occurring where benzoate was the substrate. Tdn- cells had either lost the entire pTDN1 plasmid or suffered a recombinational deletion of a specific 26 kbp region. Proportional increase of Tdn- cells resulted from their growth-rate advantage, and additionally, where benzoate was the substrate, from its metabolism via the chromosomal ortho-cleavage pathway incorporating a short lag phase. The ratio of whole plasmid loss to deletion was substrate and pH dependent. Deletion of catabolic genes was not required for loss of pTDN1 but by comparison was a prerequisite for loss of TOL plasmid pWW0. It appeared that m-toluate and benzoate were channelled via chromosomally encoded benzoate oxygenase and dihydroxycyclohexadiene carboxylate dehydrogenase prior to pTDN1 encoded meta-cleavage.