River toxicity assessment using molecular biosensors: Heavy metal contamination in the Turag-Balu-Buriganga river systems, Dhaka, Bangladesh.

Pollution in rapidly urbanising cities and in delta systems is a serious problem that blights the lives and livelihoods of millions of people, damaging and restricting potable water supply and supplies to industry (Whitehead et al, 2015, 2018). Employing new technology based on luminescent molecular biosensors, the toxicity in the rivers around Dhaka in Bangladesh, namely the Turag, Tongi, Balu and Buriganga, has been assessed. Samples taken at 36 sites during medium and low flow conditions and during the Bishwa Ijtema Festival revealed high levels of cell toxicity, as well as high concentrations of metals, particularly aluminium, cadmium, chromium, iron, zinc, lithium, selenium and nickel. Chemical analysis also revealed low dissolved oxygen levels and anoxic conditions in the rivers at certain sites. The bacterial molecular biosensors were demonstrated to be fast, with results in 30 min, robust and a highly sensitive method for the assessment of water toxicity in the field. Furthermore, the biosensor toxicity analysis correlated with the metals data, and a multivariate regression relationship was developed relating toxicity to key metals, such a selenium, zinc and chromium. The resulting model has been validated against split samples and the Bishwa Ijtema Festival data. The combination of modelling and the molecular biosensor technology provides a new approach to detecting and managing pollution in urban river systems.

Comparative study of chemical and physical methods for distinguishing between passive and metabolically active mechanisms of water contaminant removal by biofilms.

In this study, physical and chemical approaches were employed to distinguish between passive and active mechanisms in biofilms removing contaminants in waste waters and their relative merits were assessed. Respiration, post-exposure recovery and scanning electron microscopic analysis demonstrated that both ultraviolet (UV) treatment (300 mJ/cm(2)) and sodium azide (10 mM) completely inhibited metabolic activity at 5 and 24 h exposure, respectively, whilst not damaging the integrity of the biofilms. Amongst the commonly used chemical inhibitors, only sodium azide showed complete inhibition after 24 h incubation with only about 10% (±4%) of biofilm carbon released into the bulk solution, compared to 33-41% (±8%) when exposed to 5 mM and 10 mM 2,4-dinitrophenol (DNP) and 69-80% (±5%) when exposed to 2% and 5% w/v formalin, respectively. Biofilm inhibition with UV and sodium azide was found to be equally effective at inhibiting biofilms for treatment of triethanolamine (TEA) and benzotriazole (BTA): the results confirming that the dominant removal mechanism was biodegradation. However, the rates of glucose removal by sodium azide-inhibited biofilms were similar to controls, suggesting that chemical inhibitors were not effective for distinguishing the removal mechanisms of simple sugars. Statistically similar amounts of metal were removed by biofilms treated with UV and sodium azide in zinc, copper and cadmium single-systems: the results indicated that the removal mechanism is predominantly a passive biosorption process.

Enhanced biotransformation of TCE using plant terpenoids in contaminated groundwater.

AIMS: To examine plant terpenoids as inducers of TCE (trichloroethylene) biotransformation by an indigenous microbial community originating from a plume of TCE-contaminated groundwater. METHODS AND RESULTS: One-litre microcosms of groundwater were spiked with 100 micromol 1(-1) of TCE and amended weekly for 16 weeks with 20 microl 1(-1) of the following plant monoterpenes: linalool, pulegone, R-(+) carvone, S-(-) carvone, farnesol, cumene. Yeast extract-amended and unamended control treatments were also prepared. The addition of R-carvone and S-carvone, linalool and cumene resulted in the biotransformation of upwards of 88% of the TCE, significantly more than the unamendment control (61%). The aforementioned group of terpenes also significantly (P < 0.05) allowed more TCE to be degraded than the remaining two terpenes (farnesol and pulegone), and the yeast extract treatment which biotransformed 74-75% of the TCE. The microbial community profile was monitored by denaturing gradient gel electrophoresis and demonstrated much greater similarities between the microbial communities in terpene-amended treatments than in the yeast extract or unamended controls. CONCLUSIONS: TCE biotransformation can be significantly enhanced through the addition of selected plant terpenoids. SIGNIFICANCE AND IMPACT OF THE STUDY: Plant terpenoid and nutrient supplementation to groundwater might provide an environmentally benign means of enhancing the rate of in situ TCE bioremediation.

Microbial analysis of soil and groundwater from a gasworks site and comparison with a sequenced biological reactive barrier remediation process.

AIMS: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater. METHODS AND RESULTS: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal. CONCLUSION: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident. SIGNIFICANCE AND IMPACT OF THE STUDY: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process.

Impact of electrokinetic remediation on microbial communities within PCP contaminated soil.

Electrokinetic techniques have been used to stimulate the removal of organic pollutants within soil, by directing contaminant migration to where remediation may be more easily achieved. The effect of this and other physical remediation techniques on the health of soil microbial communities has been poorly studied and indeed, largely ignored. This study reports the impact on soil microbial communities during the application of an electric field within ex situ laboratory soil microcosms contaminated with pentachlorophenol (PCP; 100mg kg(-1) oven dry soil). Electrokinetics reduced counts of culturable bacteria and fungi, soil microbial respiration and carbon substrate utilisation, especially close to the acidic anode where PCP accumulated (36d), perhaps exacerbated by the greater toxicity of PCP at lower soil pH. There is little doubt that a better awareness of the interactions between soil electrokinetic processes and microbial communities is key to improving the efficacy and sustainability of this remediation strategy.

Selection of microbial consortia for treating metal-working fluids.

The aim of this research was to determine the effectiveness of a strategy for constructing microbial consortia for treating chemically mixed industrial effluent, based on a more thorough understanding of communities within waste metal-working fluids (MWFs). Complementary phenotypic and genotypic methods revealed that the microbial communities in spent MWFs had low diversity and were very similar in species composition in samples originating from different locations and uses. Of 65 bacterial isolates studied, only 9 species were identified using fatty acid methyl ester (FAME) analysis. The results of genotypic analysis by denaturing gradient gel electrophoresis (DGGE) were congruent with observations made using FAME analysis. The metabolic potential of the isolates was assessed in terms of assimilation ability and tolerance of co-contaminants. The three isolates, selected (Clavibacter michiganensis, Methylobacterium mesophilicum, and Rhodococcus erythropolis) to form a consortium, were representative of three of the four most abundant populations and when combined could utilise or tolerate all of the individual MWF components, including the biocide and the recalcitrant compound benzotriazole.

Substrate-regulated cyanide hydratase (chy) gene expression in Fusarium solani: the potential of a transcription-based assay for monitoring the biotransformation of cyanide complexes.

The fungus Fusarium solani detoxifies cyanide through induction of the cyanide hydratase gene activity (chy) in the presence of either KCN or the metal-complexed cyanides, K2Ni(CN)4 or K4Fe(CN)6, at pH 7.0 and 4.0 respectively. Sequence analysis of the chy gene identified primers for reverse transcriptase-polymerase chain reaction (RT-PCR)-directed analysis of mRNA transcripts, which demonstrated that activity correlated to the substrate-specific induction of gene expression. chy transcription was initiated 30-60 min after exposure of F. solani cultures to cyanide complexes. Maximum expression was detected within 4.5 h, after which chy mRNA synthesis declined below the limits of detection within 26 h. A lag period of approximately 2 h, following initial transcription, was recorded before cyanide complexes were converted to formamide. mRNA transcripts of chy were not detected in the absence of cyanide or cyanide complexes. The presence of introns within the gene resulted in a difference in size of 100 bp for DNA compared with mRNA of the corresponding 5' region. This size difference facilitated PCR detection of gene and transcript respectively. Comparisons of the predicted amino acid sequence of the F. solani chy gene and those of Gloeocerospora sorghi, Fusarium lateritium and Leptosphaeria maculans demonstrate that cyanide hydratase genes are highly conserved and of a similar evolutionary origin. These data predict that the functional assay described here to monitor the induction of chy gene expression and, potentially, cyanide degradation would be applicable to a variety of polluted environments.

Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot).

The community composition and structure of planktonic heterotrophic bacteria (903 isolates) sampled from a small eutrophic lake in northern England (Priest Pot) was studied with respect to season (four samples) and depth (to 3.1 m). Bacteria (887) were isolated on tryptic soy broth agar and identified to 48 genera using fatty acid methyl ester analysis. The two most abundant genera isolated were Aeromonas and Pseudomonas which, respectively, dominated the middle to bottom depths in August and all depths in February. The structure of the sampled community was described using: species richness, Simpson's index and the Shannon-Wiener index. All three indices detected a number of significant differences with depth demonstrating stratification. The greatest stratification of the bacterial community was observed in August when bacterial counts correlated strongly and negatively with diversity. Using structural measures was found to be preferable to the use of species frequencies in the analysis of perturbation and succession in community structure. Insensitivity to one or more of eight antibiotics was observed in 71% (61/86) of the isolates tested particularly in Gram-negative genera. Bacteriocinogeny and lysogeny was observed in 36% (32/90) of isolates. Using sensitive indicator strains, two of 10 producing strains produced virus, while the others produced bacteriocins.

Biodegradation of diethyl phthalate in soil by a novel pathway.

Biodegradation of diethyl phthalate (DEP) has been shown to occur as a series of sequential steps common to the degradation of all phthalates. Primary degradation of DEP to phthalic acid (PA) has been reported to involve the hydrolysis of each of the two diethyl chains of the phthalate to produce the monoester monoethyl phthalate (MEP) and then PA. However, in soil co-contaminated with DEP and MeOH, biodegradation of the phthalate to PA resulted in the formation of three compounds, in addition to MEP. These were characterised by gas chromatography-electron ionisation mass spectrometry and nuclear magnetic resonance as ethyl methyl phthalate, dimethyl phthalate and monomethyl phthalate, and indicated the existence of an alternative pathway for the degradation of DEP in soil co-contaminated with MeOH. Transesterification or demethylation were proposed as the mechanisms for the formation of the three compounds, although the 7:1 ratio of H(2)O to MeOH means that transesterification is unlikely.

Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer.

A plasmid-free, non-pathogenic, ribosomal RNA group 1 fluorescent pseudomonad, Pseudomonas fluorescens SBW25, was selected from the microflora of sugar beet (Beta vulgaris) and modified to contain constitutively expressed marker genes. By site directed homologous recombination a KX cassette [kanamycin resistance (kanr) and catechol 2,3 dioxygenase (xylE)] and a ZY cassette [lactose utilization (lacZY, beta-galactosidase, lactose permease)] were introduced at least 1 Mbp apart on the 6.6 Mbp bacterial chromosome. Separate sites were selected to provide sensitive detection methods and allow assessments of marker gene stability of the genetically modified micro-organism (GMM), SBW25EeZY6KX, when it colonized the leaves and roots of sugar beet plants following seed inoculation.

Comparison of Borrelia isolated from UK foci of Lyme disease.

Restriction endonuclease digestion of linear borrelial chromosomal DNA showed that three isolates of UK Lyme disease spirochaetes differed markedly from each other and from published data for other isolates from North America and continental Europe. Analysis of linear plasmid bands revealed that UK isolates differed from each other in the number and sizes of the plasmids in isolates from different foci of UK Lyme disease. Fatty acid analysis (of fatty acid methyl ester (FAME) profiles) showed the UK isolates clustering together with the relapsing fever spirochaetes, Borrelia turicatae and Borrelia parkeri. These data are discussed in respect of current knowledge of Lyme borreliosis in the UK.

Fatty acid profiles of invertebrate iridescent viruses.

Eight invertebrate iridescent viruses (IVs) from diverse host taxa were grown in a common lepidopteran host, Galleria mellonella. The lipid composition of purified virus was assessed by fatty acid methyl esterase (FAME) analysis using a gas-liquid chromatograph. IV fatty acid profiles were markedly different from those of the host tissues. The interrelationships among the IVs did not follow previous serological and genetic findings. We conclude that FAME analysis is not a useful technique for revealing phylogenetic relationships among these viruses.

Phenotypic and genotypic diversity of fluorescent pseudomonads isolated from field-grown sugar beet.

A sample of 30 fluorescent pseudomonads isolated from the phyllosphere of sugar beet throughout a single growing season and shown to be closely related on the basis of fatty acid methyl ester (FAME) analysis was subjected to detailed phenotypic and genotypic characterization. Phenotypic traits were assessed on the basis of biochemical properties, assimilation of sole carbon sources, FAME analysis, organic pyrolysate content (MS-pyrolysis), and total cellular protein profiles. With the exception of total cellular protein profiles, numerical analysis of the data revealed two main clusters, each of which was divided into several subclusters. Numerical analysis of total cellular protein data failed to differentiate isolates into two main clusters, but nevertheless grouped isolates into six subclusters. On the basis of biochemical and carbon source assimilation profiles, 19 isolates were identified as Pseudomonas fluorescens biovar V, eight isolates as P. fluorescens biovar III and three isolates as P. syringae pathovar syringae. In general, all methods of phenotypic analysis grouped isolates according to time of sampling and leaf type. Genome analysis was undertaken by pulsed-field gel electrophoresis (PFGE) of PacI, SpeI, SwaI and XbaI macrorestriction fragments and revealed the presence of eight distinct genomic (clonal) groups. These groups correlated closely with the clusters generated by numerical analysis of phenotypic data, but there was no correlation between macrorestriction fragment profile and isolate identification; in fact the variation in macrorestriction fragment patterns within P. fluorescens biovars was as great as the variation detected between biovars, and between P. fluorescens and P. syringae. Statistical evaluation of macrorestriction fragment patterns revealed two examples of recent strain divergence: one was due to the presence of a 400 kbp plasmid within one isolate of a collection of nine otherwise genomically identical isolates, and the other was observed between two phenotypically similar isolates sampled 220 d apart. Genetic variation was expressed in terms of nucleotide diversity (pi) and pairwise comparisons yielded values ranging from 0.0029 to 0.1517. The mean intrapopulation genetic variation was high (0.0993), but limited genetic variation was detected among isolates sampled on each occasion. Taken together this suggests a population comprised of a variety of apparently distantly related clones (genomic groups), each adapted to local conditions. Genome sizes were estimated from the sum of SpeI restriction fragments and ranged from 4.2 to 5.5 Mbp. Examination of the distribution of XbaI, SpeI, SwaI and PacI restriction endonuclease sites showed that the distribution of SpeI sites differed significantly from the expected (random) distribution.

Genome and fatty acid analysis of Pseudomonas stutzeri.

A genome and fatty acid analysis of 16 Pseudomonas stutzeri reference strains having DNA compositions ranging from 62.2 to 65.5 mol% G+C was performed by pulsed-field gel electrophoresis of XbaI and SpeI macrorestriction fragments and gas chromatography of total cellular fatty acids. Macrorestriction fragment patterns were evaluated by using previously described algorithms (D. Grothues and B. Tümmler, Mol. Microbiol. 5:2763-2776, 1991), and the results allowed us to subdivide the species into two groups which correlated with G+C content. Two examples of recent strain divergence were observed among clinical isolates, but in general a marked degree of heterogeneity was observed in the macrorestriction fragment patterns, and even phenotypically similar strains produced divergent patterns. While the differences were not sufficiently great to exclude any strain from P. stutzeri, they suggest that recombination and niche-specific selection may be significant factors responsible for generating and maintaining the heterogeneity inherent in the species. Genome sizes were estimated from the sums of SpeI restriction fragment sizes and ranged from 3.4 to 4.3 Mbp; the genome sizes of the low-G+C-content strains (G+C contents, approximately 62 mol%) were confined to a narrow range between 3.9 and 4.1 Mbp. An examination of the distributions of macrorestriction fragments resulting from digestion with XbaI and SpeI showed that both distributions differed significantly from the expected (random) distribution, suggesting that there is a supragenic level of chromosomal organization. An analysis of fatty acid methyl ester data by using Microbial Identification System software revealed a similar correlation between phenotype and G+C content, indicating that division of the species is possible by the method used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of intra-specific variation in the fatty acid profiles of Borrelia burgdorferi.

Analysis of the fatty acid methyl esters (FAMEs) of bacteria is a commonly used chemotaxonomic technique. Application of this methodology to spirochaetes associated with Lyme borreliosis revealed distinct clusters corresponding to three genetically distinguished groups: Borrelia burgdorferi sensu stricto, B. garinii, and the VS461 group. However, B. garinii formed a common group with B. hermsii, a relapsing fever spirochaete, and VS461 grouped with B. turicatae and B. parkeri, two other relapsing fever spirochaetes. The diversity in fatty acid profiles of Lyme disease spirochaetes has implications for the protean clinical manifestations of the disease.

Comparison of the fatty acid profiles of Borrelia, Serpulina and Leptospira species.

Fatty acid methyl ester (FAME) derivatives were examined as a means of characterizing Borrelia burgdorferi isolates and distinguishing them from other spirochaetes. Analysis was performed using a gas liquid chromatography column in conjunction with Microbial Identification System (MIS) software. Reproducible FAME profiles were produced which distinguished Borrelia species, Serpulina hyodysenteriae and Leptospira icterohaemorrhagiae. Furthermore, the FAME profiles of four recognized Borrelia species (including two American isolates of Borrelia burgdorferi, B31 and JD1) were distinct from one another and from the BSK II medium in which they were grown. The results confirm previous reports that FAME profiles of bacteria represent a diagnostic phenotypic property and suggest that they may have applications in the chemotaxonomic classification of Borrelia species.

The effect of polluted and leached snow melt waters on the soil bacterial community-quantitative response.

The effect of polluted snow melt waters on the number of soil bacteria was determined using soil cores extracted from an upland catchment in the Cairngorm Mountains, Scotland. Total numbers of viable heterotrophic bacteria and bacterial denitrifiers were determined using plate and MPN counts. Separate soil cores were treated with simulated melt waters representative of either the composition of the first melt fraction from polluted or leached snowpacks. The number of bacteria in the Ah soil horizon (Hodgson, 1974) treated with polluted snow melt (PSM) water decreased significantly by 28-fold, but increased by 11-fold in the BC horizon. Denitrifier numbers decreased by 8-fold in the Ah horizon, but increased by over 2-fold lower down the profile. Overall the bacterial community exposed to simulated leached snow melt (LSM) waters showed little change in the Ah horizon. In the BC horizon (Hodgson, 1974), total viable bacterial numbers decreased by 20-fold, but denitrifiers numbers were unaffected.

Learned taste aversion to saccharin produced by orally consumed d-amphetamine.

Rats were presented with solutions containing both saccharin and d-amphetamine and the development of taste aversions to solutions of either or both of these substances was studied. In Experiment 1 it was found that taste aversions developed to solutions of saccharin (1 mg/ml) which contained amphetamine at concentrations of 0.01, 0.03 and 0.1 mg/ml. Experiment 2 showed that a taste aversion conditioned to a solution of saccharin (2 mg/ml) and amphetamine (0.2 mg/ml) generalised to solutions containing saccharin at concentrations between 0.625 and 20 mg/ml but not to a solution containing only amphetamine. In the third experiment it was found that the degree of generalisation of a taste aversion to lower saccharin concentrations depended upon the concentration used during conditioning trials. When the conditioning concentration was 0.625 mg/ml the aversion generalised to concentrations as low as 0.075 mg/ml but when a 10 mg/ml solution was used for conditioning the aversion did not generalise to concentrations below 2 mg/ml. The characteristics of taste aversions conditioned with orally consumed amphetamine are similar to those of conditioning involving injections of the drug.