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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., (© 2017 The Society for Applied Microbiology.)

The use of carbon nanotubes (CNTs) as building blocks in the design of electrochemical biosensors has been attracting attention over the last few years, mainly due to their high electrical conductivity and large surface area. Here, we present two approaches based on tailored single-walled CNTs (SWCNTs) architectures to develop immunosensors for the bacteriophage MS2, a virus often detected in sewage-impacted water supplies. In the first approach, SWCNTs were used in the bottom-up design of sensors as antibody immobilization support. Carboxy-functionalised SWCNTs were covalently tethered onto gold electrodes via carbodiimide coupling to cysteamine-modified gold electrodes. These SWCNTs were hydrazide functionalized by electrochemical grafting of diazonium salts. Site-oriented immobilization of antibodies was then carried out through hydrazone bond formation. Results showed microarray electrode behavior, greatly improving the signal-to-noise ratio. Excellent sensitivity and limit of detection (9.3 pfu/mL and 9.8 pfu/mL in buffer and in river water, respectively) were achieved, due to the combination of the SWCNTs' ability to promote electron transfer reactions with electroactive species at low overpotentials and their high surface-to-volume ratio providing a favorable environment to immobilize biomolecules. In the second approach, SWCNTs were decorated with iron oxide nanoparticles. Diazonium salts were electrochemically grafted on iron-oxide-nanoparticle-decorated SWCNTs to functionalize them with hydrazide groups that facilitate site-directed immobilization of antibodies via hydrazone coupling. These magnetic immunocarriers facilitated MS2 separation and concentration on an electrode surface. This approach minimized non-specific adsorptions and matrix effects and allowed low limits of detection (12 pfu/mL and 39 pfu/mL in buffer and in river water, respectively) that could be further decreased by incubating the magnetic immunocarriers with larger volumes of sample. Significantly, both approaches permitted the detection of MS2 to levels regularly encountered in sewage-impacted environments., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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.

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.