Effect of arsenic contaminated irrigation water on Lens culinaris L. and toxicity assessment using lux marked biosensor.

Contamination of irrigation water represents a major constraint to Bangladesh agriculture, resulting in elevated levels in the terrestrial systems. Lux bacterial biosensor technology has previously been used to measure the toxicity of metals in various environmental matrices. While arbuscular mycorrhizal fungi have their most significant effect on phosphorus uptake, but showed alleviated metal toxicity to the host plant. The study examined the effects of arsenic and inoculation with an arbuscular mycorrhizal fungus, Glomus mosseae, on lentil (Lens culinaris L. cv. Titore). Plants were grown with and without arbuscular mycorrhizal inoculum for 9 weeks in a sand and terra-green mixture (50:50, V/V) and watered with five levels of arsenic (0, 1, 2, 5, 10 mg As/L arsenate). The results showed that arsenic addition above 1 mg/L significantly reduced percentage of mycorrhizal root infection. On further analysis a close relationship was established with the vegetative and reproductive properties of lentil (L. culinaris) plants compared to the percentage bioluminescence of the soil leachate. However, arbuscular mycorrhizal fungal inoculation reduced arsenic concentration in roots and shoots. Higher concentrations of arsenic (5, 10 mg As/L arsenate) reduced the mycorrhizal efficiency to increase phosphorus content and nitrogen fixation. Therefore, this study showed that increased concentration of arsenic in irrigation water had direct implications to the lentil (L. culinaris) plants overall performance. Moreover the use of bioassay demonstrated that mycorrhiza and clay particle reduced arsenic bioavailability in soil.

Persistence and metabolic activity of Escherichia coli O157:H7 in farm animal faeces.

Ruminants, and to a lesser extent monogastric farm animals, are known to be natural reservoirs of Escherichia coli O157:H7, and contact with contaminated faeces has been linked to human infection. This study used a nontoxigenic, chromosomally marked, lux reporter strain to compare the persistence and activity (bioluminescence) of E. coli O157:H7 over 21 days in the faecal liquor of five farm animals: horse, sheep, cow, pig and piglet. Samples were inoculated with the lux E. coli O157:H7 (7.82 log CFU mL(-1)) and stored at 20 +/- 1 degrees C. The organism was recovered from all samples throughout the experimental period, although lower numbers were recovered from horse faecal liquor relative to all other types (P<0.001). The organisms' activity declined in all samples over time and no luminescence could be detected in any sample 21 days postinoculation. However, activity did increase greatly within pig and piglet faeces during initial stages of monitoring and overall luminescence was greater in piglet samples compared with all other samples (P<0.001). This is the first study to demonstrate how both the persistence and metabolic activity of E. coli O157:H7 notably varies within a range of ruminant and nonruminant animal faeces. Further research is needed to elucidate the factors that govern differential persistence and metabolic activity of E. coli O157:H7 within such matrices.

Bacteria associated with ectomycorrhizas of slash pine (Pinus elliottii) in south-eastern Queensland, Australia.

Bacterial communities associated with ectomycorrhizal and uncolonized roots of Pinus elliottii (slash pine) collected from a plantation in south-east Queensland, Australia, were investigated, using cultivation-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene PCR products obtained using a cultivation-independent approach revealed that bacterial communities associated with ectomycorrhizal root tips differed significantly from those associated with roots uncolonized by ectomycorrhizal fungi. DGGE analysis of cultivable bacterial communities revealed no significant difference between ectomycorrhizal and uncolonized roots. Neither analytical approach revealed significant differences between the bacterial communities associated with ectomycorrhizal roots colonized by a Suillus sp. or an Atheliaceae taxon. Cloned bacterial 16S rRNA genes revealed sequence types closely related with that of Burkholderia phenazinium, common in both ectomycorrhizal-colonized and -uncolonized roots, while sequence types most similar to the potentially phyopathogenic bacteria Burkholderia andropogonis and Pantoea ananatis were only detected in ectomycorrhizal roots. These results highlight the possibility of global movement of microorganisms, including putative pathogens, as a result of the introduction of exotic pine plantations.

PCR profiling of ammonia-oxidizer communities in acidic soils subjected to nitrogen and sulphur deposition.

Communities of ammonia-oxidizing bacteria (AOB) were characterized in two acidic soil sites experimentally subjected to varying levels of nitrogen and sulphur deposition. The sites were an acidic spruce forest soil in Deepsyke, Southern Scotland, with low background deposition, and a nitrogen-saturated upland grass heath in Pwllpeiran, North Wales. Betaproteobacterial ammonia-oxidizer 16S rRNA and ammonia monooxygenase (amoA) genes were analysed by cloning, sequencing and denaturing gradient gel electrophoresis (DGGE). DGGE profiles of amoA and 16S rRNA gene fragments from Deepsyke soil in 2002 indicated no effect of nitrogen deposition on AOB communities, which contained both Nitrosomonas europaea and Nitrosospira. In 2003, only Nitrosospira could be detected, and no amoA sequences could be retrieved. These results indicate a decrease in the relative abundance of AOB from the year 2002 to 2003 in Deepsyke soil, which may be the result of the exceptionally low rainfall in spring 2003. Nitrosospira-related sequences from Deepsyke soil grouped in all clusters, including cluster 1, which typically contains only sequences from marine environments. In Pwllpeiran soil, 16S rRNA gene libraries were dominated by nonammonia oxidizers and no amoA sequences were detectable. This indicates that autotrophic AOB play only a minor role in these soils even at high nitrogen deposition.

Persistence, dissipation, and activity of Escherichia coli O157:H7 within sand and seawater environments.

Runoff from agricultural land into watercourses may transport and deposit animal-derived waste contaminated with Escherichia coli O157:H7 onto beaches, which may in turn lead to human infection. To simulate contamination, freshwater mixed with cattle slurry containing E. coli O157:H7 was added to sand from three recreational beaches. The sand was then maintained in a dry state (nontidal) or subjected to a repeated seawater tidal simulation. The pathogen could still be recovered from all sands by day 5. Although survival of the pathogen did not statistically vary between sands of different origin under nontidal conditions, significant differences in numbers occurred between sands when subject to tidal simulation. In the tidal simulations, a considerable proportion of the E. coli O157:H7 rapidly dissipated from sand into the seawater. In a separate experiment, the activity of bioluminescent (lux-marked) E. coli O157:H7 cells was monitored in various mixtures of contaminated runoff water and seawater over 5 days. Pathogen activity declined with increasing seawater concentration; however, cells remained viable in all treatments over the 5-day period. The addition of nutrients to water rapidly increased pathogen activity in all treatments. Our findings highlight the resilience of E. coli O157:H7 in aquatic and marine environments.

Studying plant-microbe interactions using stable isotope technologies.

Interactions between plants and microorganisms in the rhizosphere are complex and varied. They include the general transfer of nutrients and specific interactions mediated by the release of signalling molecules from plant roots. Until recently, understanding the nature of these interactions was limited by a reliance on traditional, cultivation-based techniques. Stable isotope probing provides the potential for cultivation-independent characterisation of organisms actively assimilating carbon derived from plant root exudate or added to the soil. Current applications have focused on interactions with relatively low-level specificity, but there is significant potential for mechanistic studies of more specific interactions, particularly if the sensitivity of the technique can be increased.

Rapid quantification of polycyclic aromatic hydrocarbons in hydroxypropyl-beta-cyclodextrin (HPCD) soil extracts by synchronous fluorescence spectroscopy (SFS).

Synchronous fluorescence spectroscopy (SFS) was directly applied to rapidly quantify selected polycyclic aromatic hydrocarbons (PAHs: benzo[a]pyrene and pyrene) in aqueous hydroxypropyl-beta-cyclodextrin (HPCD) soil extract solutions from a variety of aged contaminated soils containing four different PAHs. The method was optimized and validated. The results show that SFS can be used to analyse benzo[a]pyrene and pyrene in HPCD based soil extracts with high sensitivity and selectivity. The linear calibration ranges were 4.0x10(-6)-1.0x10(-3)mM for benzo[a]pyrene and 6.0x10(-6)-1.2x10(-3)mM for pyrene in 10mM HPCD aqueous solution alone. The detection limits according to the error propagation theory for benzo[a]pyrene and pyrene were 3.9x10(-6) and 5.4x10(-6)mM, respectively. A good agreement between SFS and HPLC was reached for both determinations of PAHs in HPCD alone and in soil HPCD extracts. Hence, SFS is a potential means to simplify the present non-exhaustive hydroxypropyl-beta-cyclodextrin (HPCD)-based extraction technique for the evaluation of PAH bioavailability in soil.

Endobacteria in some ectomycorrhiza of Scots pine (Pinus sylvestris).

The diversity of cultivable endobacteria associated with four different ectomycorrhizal morphotypes (Suillus flavidus, Suillus variegatus, Russula paludosa and Russula sp.) of Scots pine (Pinus sylvestris) was analysed by restriction fragment length polymorphism profiling of PCR-amplified rDNA intergenic spacer regions and by sequence analyses of 16S rRNA genes. Ectomycorrhizal root tip surface-sterilization methods were developed and assessed for their efficiencies. Bacterial communities from surface-sterilized ectomycorrhizal root tips were different from those of ectomycorrhizal root tips without surface-sterilization for all the morphotypes studied. Endobacteria belonging to the genera Pseudomonas, Burkholderia and Bacillus were isolated from more than one ectomycorrhizal morphotype, whereas species of Rahnella, Janthinobacterium and Rhodococcus were only isolated from the single morphotypes of S. variegatus, R. paludosa and Russula sp., respectively. Some of the isolated endobacteria utilized fungal sugars more readily than typical plant sugars in carbon utilization assays.

Earthworms as vectors of Escherichia coli O157:H7 in soil and vermicomposts.

Survival and movement of Escherichia coli O157:H7 in both soil and vermicompost is of concern with regards to human health. Whilst it is accepted that E. coli O157:H7 can persist for considerable periods in soils, it is not expected to survive thermophilic composting processes. However, the natural behavior of earthworms is increasingly utilized for composting (vermicomposting), and the extent to which earthworms promote the survival and dispersal of the bacterium within such systems is unknown. The faecal material produced by earthworms provides a ready supply of labile organic substrates to surrounding microbes within soil and compost, thus promoting microbial activity. Earthworms can also cause significant movement of organisms through the channels they form. Survival and dispersal of E. coli O157:H7 were monitored in contaminated soil and farmyard manure subjected to earthworm digestion over 21 days. Our findings lead to the conclusion that anecic earthworms such as Lumbricus terrestris may significantly aid vertical movement of E. coli O157 in soil, whereas epigeic earthworms such as Dendrobaena veneta significantly aid lateral movement within compost. Although the presence of earthworms in soil and compost may aid proliferation of E. coli O157 in early stages of contamination, long-term persistence of the pathogen appears to be unaffected.

Potential application of synchronous fluorescence spectroscopy to determine benzo[a]pyrene in soil extracts.

Benzo[a]pyrene (BaP) is a significant environmental pollutant and rapid, accurate methods to quantify this compound in soil for both research and environmental investigation purposes are required. In this work, solvent extracts from five contrasting soils spiked with four different polycyclic aromatic hydrocarbons (PAHs) were rapidly analysed by using a synchronous fluorescence spectroscopy (SFS) method. The SFS method was validated using HPLC with ultraviolet detection. A good correlation for the quantification of BaP in soil extracts by the two methods was observed. The detection limit of the SFS method was 1.6 x 10(-9) g/ml in CTAB micellar medium (7.8 mmol/l). The work demonstrates that SFS has potential as a sensitive, accurate, rapid, simple and economic methodology and an efficient alternative to HPLC for fast confirmation and quantification of BaP in complex soil extracts.

Degradation of the polycyclic aromatic hydrocarbon (PAH) fluorene is retarded in a Scots pine ectomycorrhizosphere.

• Polycyclic aromatic hydrocarbons (PAHs) are an important class of persistent organic pollutants (POPs) in the environment and accumulate in forest soils. These soils are often dominated by ectomycorrhizal (EcM) roots, but little is known about how EcM fungi degrade PAHs, or the overall effect of field colonized EcM roots on the fate of PAHs. • The ability of eight EcM fungi to degrade PAHs in liquid culture spiked with 14 C labelled PAHs was investigated. Microcosms were used to determine the impact of naturally colonized mycorrhizal pine seedlings on PAH mineralization and volatilization. • Only two EcM fungi (Thelephora terrestris and Laccaria laccata) degraded at least one PAH and none were able to mineralize the PAHs in pure culture. Where degradation occurred, the compounds were only mono-oxygenated. EcM pine seedlings did not alter naphthalene mineralization or volatilization but retarded fluorene mineralization by 35% compared with unplanted, ectomycorrhizosphere soil inoculated, microcosms. • The EcM fungi possessed limited PAH degrading abilities, which may explain why EcM dominated microcosms retarded fluorene mineralization. This observation is considered in relation to the 'Gadgil-effect', where retarded litter decomposition has been observed in the presence of EcM roots.

Survival of Escherichia coli O157:H7 in private drinking water wells: influences of protozoan grazing and elevated copper concentrations.

The survival characteristics of Escherichia coli O157:H7 in private drinking water wells were investigated to assess the potential for human exposure. A non-toxigenic, chromosomally lux-marked strain of E. coli O157:H7 was inoculated into well water from four different sites in the North East of Scotland. These waters differed significantly in their heavy metal contents as well as nutrient and bacterial grazer concentrations. Grazing and other biological factors were studied using filtered (3 and 0.2 microm) and autoclaved water. The survival of E. coli O157:H7 was primarily decreased by elevated copper concentrations. This hypothesis was supported by acute toxicity assay data. In addition, significant protozoan predation effects were observed in untreated water when compared with survival rates in filtered water. The combination of these two factors in particular determines the survival time of the pathogen in a private water well. It therefore appears that wells with higher water quality as assessed using the European Union Drinking Water Directive standards will also allow survival of E. coli O157:H7 for much longer periods.

A tripartite microbial reporter gene system for real-time assays of soil nutrient status.

Plant-derived carbon is the substrate which drives the rate of microbial assimilation and turnover of nutrients, in particular N and P, within the rhizosphere. To develop a better understanding of rhizosphere dynamics, a tripartite reporter gene system has been developed. We used three lux-marked Pseudomonas fluorescens strains to report on soil (1) assimilable carbon, (2) N-status, and (3) P-status. In vivo studies using soil water, spiked with C, N and P to simulate rhizosphere conditions, showed that the tripartite reporter system can provide real-time assessment of carbon and nutrient status. Good quantitative agreement for bioluminescence output between reference material and soil water samples was found for the C and P reporters. With regard to soil nitrate, the minimum bioavailable concentration was found to be greater than that analytically detectable in soil water. This is the first time that bioavailable soil C, N and P have been quantified using a tripartite reporter gene system.

Factors affecting benthic impacts at Scottish fish farms.

The factors affecting patterns of benthic [seabed] biology and chemistry around 50 Scottish fish farms were investigated using linear mixed-effects models that account for inherent correlations between observations from the same farm. The abundance of benthic macrofauna and sediment concentrations of organic carbon were both influenced by a significant, albeit weak, interaction between farm size, defined as the maximum weight of fish permitted on site at any one time, and current speed. Above a farm size threshold of between 800 and 1000 t, the magnitude of effects at farms located in areas of elevated current speeds were greater than at equivalent farms located in more quiescent waters. Sediment concentrations of total organic matter were influenced by an interaction between distance and depth, indicating that wind-driven resuspension events may help reduce the accumulation of organic waste at farms located in shallow waters. The analyses presented here demonstrate that the production and subsequent fate of organic waste at fish farms is more complex than is often assumed; in isolation, current speed, water depth, and farr size are not necessarily good predictors of benthic impact.

Potential use of DNA adducts to detect mutagenic compounds in soil.

In this study, three different soils with contrasting features, spiked with 300 mg benzo[a]pyrene (BaP)/kg dry soil, were incubated at 20 degrees C and 60% water holding capacity for 540 days. At different time points, BaP and DNA were extracted and quantified, and DNA adducts were quantified by (32)P-postlabelling. After 540 days incubation, 69.3, 81.6 and 83.2% of initial BaP added remained in Cruden Bay, Boyndie and Insch soils, respectively. Meanwhile, a significantly different amount of DNA-BaP adducts were found in the three soils exposed to BaP over time. The work demonstrates the concept that DNA adducts can be detected on DNA extracted from soil. Results suggest the technique is not able to directly reflect bioavailability of BaP transformation products. However, this new method provides a potential way to detect mutagenic compounds in contaminated soil and to assess the outcomes of soil remediation.

Heat and lime-treatment as effective control methods for E. coli O157:H7 in organic wastes.

Land-application of abattoir wastes is economically appealing and may provide an effective means of closing the nutrient cycling loop. This practise is constrained, however, by legislation which necessitates pre-treatment to remove pathogenic micro-organisms prior to land-spreading. Here we investigated whether heat-treatment or lime addition could eliminate Escherichia coli O157:H7 from three contrasting abattoir wastes. We found that treatment at 60 degrees C for 10 min effectively eradicated the organism while treatment for the same length of time at 50 degrees C led to 2-4 log reductions, but not a complete kill. Temperatures of 72 degrees C induced waste solidification rendering its use impractical. The potential for re-growth in heat-treated and untreated wastes was also investigated. Survival was significantly greater in heat-treated wastes, although the difference was less than half a log unit in magnitude. This effect of heat-treatment on pathogen survival appeared to be ameliorated when wastes were mixed with soil. No viable E. coli O157:H7 cells were recovered from any waste after application of lime (CaO) at a rate of 10 gl(-1), even after enrichment. Our results indicate that pasteurisation-style or liming treatments may provide a suitable alternative method for reducing pathogen loads in abattoir wastes, so that they can be applied to land with minimal biological risk.

Mycorrhizas and biomass crops: opportunities for future sustainable development.

Central to soil health and plant productivity in natural ecosystems are in situ soil microbial communities, of which mycorrhizal fungi are an integral component, regulating nutrient transfer between plants and the surrounding soil via extensive mycelial networks. Such networks are supported by plant-derived carbon and are likely to be enhanced under coppiced biomass plantations, a forestry practice that has been highlighted recently as a viable means of providing an alternative source of energy to fossil fuels, with potentially favourable consequences for carbon mitigation. Here, we explore ways in which biomass forestry, in conjunction with mycorrhizal fungi, can offer a more holistic approach to addressing several topical environmental issues, including 'carbon-neutral' energy, ecologically sustainable land management and CO(2) sequestration.

Diversity of predominant endophytic bacteria in European deciduous and coniferous trees.

The diversity of endophytic bacteria residing in root, stem, and leaf tissues was examined in coniferous and deciduous tree species, Scots pine (Pinus sylvestris L.), silver birch (Betula pendula Roth), and rowan (Sorbus aucuparia L.). Using cultivation-dependent and -independent analyses, the bacterial communities were observed to be significantly different in the belowground (roots and rhizosphere) and aboveground (leaves and stems) samples of the respective host trees. No significant differences, with respect to the different tree species, were observed in the associated communities. Predominant cultivable endophytes isolated included bacteria closely related to Bacillus subtilis, Bacillus licheniformis, Paenibacillus spp., and Acinetobacter calcoaceticus. Comparisons of the most abundant cultivable bacteria in the rhizosphere and root samples suggested that root endophytic bacteria may be in residence through processes of selection or active colonization rather than by passive diffusion from the rhizosphere.

The role of ecological theory in microbial ecology.

Microbial ecology is currently undergoing a revolution, with repercussions spreading throughout microbiology, ecology and ecosystem science. The rapid accumulation of molecular data is uncovering vast diversity, abundant uncultivated microbial groups and novel microbial functions. This accumulation of data requires the application of theory to provide organization, structure, mechanistic insight and, ultimately, predictive power that is of practical value, but the application of theory in microbial ecology is currently very limited. Here we argue that the full potential of the ongoing revolution will not be realized if research is not directed and driven by theory, and that the generality of established ecological theory must be tested using microbial systems.

An ecotoxicological approach to assessing the impact of tanning industry effluent on river health.

A study was conducted to investigate the sediment health and water quality of the River Sagana, Kenya, as impacted by the local tanning industry. Chemical analysis identified the main chemical pollutants (pentachlorophenols and chromium) while a bioassay addressed pollutant bioavailability. The bioassay, exploiting the luminescence response of a lux marked bacterial biosensor, was coupled to a dehydrogenase and Dapnia magna test to determine toxicity effects on sediments. Results highlighted the toxicity of the tannery effluent to the sediments at the point of discharge (64% of control bioluminescence) with gradual improvement downstream. There was a significant increase in dehydrogenase downstream, with the enzyme activity attaining a peak at 600 m, also indicating a gradual reduction of toxicity. Biological oxygen demand (19.56 mg L(-1)) dissolved oxygen (3.97 mg L(-1)) and high lethal dose value (85%) of D. magna also confirmed an initial stress at the point of discharge and recovery downstream. Optical density of surface water demonstrated an increase in suspended particulates and colour after the discharge point, eventually decreasing beyond 400 m. In conclusion, the study highlighted the importance of understanding the biogeochemistry of river systems impacted by industries discharging effluent into them and the invaluable role of a biosensor-based ecotoxicological approach to address effluent hazards, particularly in relation to river sediments.