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PubMed2004

Molecular identification of the catabolic vinyl chloride reductase from Dehalococcoides sp. strain VS and its environmental distribution.

Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene (vcrA) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.

Amino Acid Sequence↗

PubMed1998

Cytochrome P450 immobilisation as a route to bioremediation/biocatalysis.

The diverse substrate specificity of the cytochrome P450 (P450; CYP) enzyme superfamily offers the opportunity to develop enzymatic systems for environmental detoxification and biotransformations of drugs, pesticides and fine chemicals. Here we report on the immobilisation of a fusion protein between plant cytochrome P450-71B1 (CYP71B1) and its electron donor, plant NADPH cytochrome P450 reductase using an oil-in-water macro-emulsion, termed polyaphron, which contains a proportion of internal organic phase (phi) greater than 0.74. Efficiency of P450 immobilisation was greater than 85%, and in this state enzymatic activity could be measured for more than 24 h at 15 degrees C. Chlortoluron, a recalcitrant herbicide pollutant in the environment, was shown to be metabolised, with the major metabolite (N-monodemethylated chlortoluron) being separated from the substrate due to partitioning into the aqueous phase. The turnovers exhibited superactivity compared with those obtained using free enzyme located in membranes prepared following heterologous expression in Saccharomyces cerevisiae and Escherichia coli. The potential to exploit the unprecedented catalytic diversity of the P450 superfamily in biocatalysis is discussed.

Biodegradation, Environmental↗

PubMed2002

Measurement of triclosan in wastewater treatment systems.

The objective of this study was to investigate the fate and removal of triclosan (TCS; 5-chloro-2-[2,4-dichloro-phenoxy]-phenol), an antimicrobial agent used in a variety of household and personal-care products, in wastewater treatment systems. This objective was accomplished by monitoring the environmental concentrations of TCS, higher chlorinated derivatives of TCS (4,5-dichloro-2-[2,4-dichloro-phenoxy]-phenol [tetra II]; 5,6-dichloro-2-[2,4-dichloro-phenoxy]-phenol [tetra III]; and 4,5,6-trichloro-2-(2,4-dichloro-phenoxy)-phenol [penta]), and a potential biotransformation by-product of TCS (5-chloro-2-[2,4-dicholoro-phenoxy]-anisole [TCS-OMe]) during wastewater treatment. These analytes were isolated from wastewater by using a C18 solid-phase extraction column and from sludge with supercritical fluid CO2. Once the analytes were isolated, they were derivatized to form trimethylsilylethers before quantitation by gas chromatography-mass spectrometry. Recovery of TCS from laboratory-spiked wastewater samples ranged from 79 to 88% for influent, 36 to 87% for final effluent, and 70 to 109% for primary sludge. Field concentrations of TCS in influent wastewater ranged from 3.8 to 16.6 microg/L and concentrations for final effluent ranged from 0.2 to 2.7 microg/L. Removal of TCS by activated-sludge treatment was approximately 96%, whereas removal by trickling-filter treatment ranged from 58 to 86%. The higher chlorinated tetra-II, tetra-III, and penta closans were below quantitation in all of the final effluent samples, except for one sampling event. Digested sludge concentrations of TCS ranged from 0.5 to 15.6 microg/g (dry wt), where the lowest value was from an aerobic digestion process and the highest value was from an anaerobic digestion process. Analysis of these results suggests that TCS is readily biodegradable under aerobic conditions, but not under anaerobic conditions. The higher chlorinated closans were near or below the limit of quantitation in all of the digested sludge samples. Based on results from this study, the chlorinated analogues and biotransformation by-product of TCS are expected to be very low in receiving waters and sludge-amended soils.

Anti-Infective Agents, Local↗

PubMed2001

Initial degradation of dimethylphthalate by esterases from Bacillus species.

Dimethylphthalate (DMP), one of the phthalate esters, is used in the manufacture of plasticizers, insect repellents, and synthetic fibers, and contributes to environmental pollution. In the present study, we report a novel bacterium belonging to the Bacillus sp., which has the ability to utilize DMP as the sole source of carbon. The esterases from the cell-free extract of the Bacillus de-esterified DMP. Native polyacrylamide gel electrophoresis showed the presence of four isoesterases designated Et1--4. The isoesterases Et-4 and Et-1 showed a higher preference towards DMP hydrolysis as compared with Et-2 and 3. A megaplasmid of about 60 kb was detected in this bacterium. The ability of this bacterium to utilize DMP as the sole source of carbon was lost upon plasmid curing. The isoesterases Et-1--4 were absent in the cell-free extracts of the cured bacterium. The results from our studies clearly demonstrate that de-esterification is the initial step in the degradation of DMP and the genes for these esterases seem to be harbored on the plasmid in this bacterium.

Bacillus↗

PubMed1992

Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene.

Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE). The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned. It contained a 2.2-kb EcoRI fragment. With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO. Fragments produced by digestion of DNA with rare cutting restriction endonucleases were separated by pulsed-field gel electrophoresis and transferred to Zeta-Probe membrane (Bio-Rad) for Southern blot analysis. Samples were also analyzed for the presence of soluble MMO by Western blot analysis and the ability to degrade TCE. The physiological groups of methanotrophs in each sample were determined by hybridizing cells with fluorescence-labelled signature probes. Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe. When grown in media with limited copper, all of these bacteria degraded TCE. All of them are type II methanotrophs. The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M. trichosporium OB3b soluble MMO component B gene probe, although M. capsulatus Bath also produces a soluble MMO.

Base Sequence↗

PubMed1987

Relationship between properties of a series of anilines and their transformation by bacteria.

The effect of compound structure on the microbial transformation of a series of substituted anilines was investigated. For the pure-culture and environmental water samples studied, the rate of transformation of the compounds decreased in the following order: aniline greater than 3-bromoaniline greater than 3-chloroaniline greater than 3-methylaniline greater than 3-methoxyaniline greater than 3-nitroaniline greater than 3-cyanoaniline. Second-order rate constants (kb) for each compound was calculated by using bacterial and compound concentrations measured as a function of time. The rate constants correlated with steric parameters. Water samples also were used in kinetic studies with three of the compounds (aniline, 3-chloroaniline, and 3-nitroaniline) to test the relationships with mixed bacterial populations. A simple linear regression of van der Waals radius of the substituent group with log kb gave correlation coefficients (r2) of 0.924 for the river isolate and 0.99 for the mixed populations. Analyses of pure-culture and mixed-population samples by thin-layer chromatography indicate that the primary products are catechols. This finding suggests that the transformation pathway involves oxidative deamination of the anilines.

Aniline Compounds↗

PubMed2004

Expression of laccase IIIb from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica for environmental applications.

Improvement of the catalytic properties of fungal laccases is a current challenge for the efficient bioremediation of natural media polluted by xenobiotics. We developed the heterologous expression of a laccase from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica as a first step for enzyme evolution. The full-length cDNA consisted of a 1,561-bp open reading frame encoding lacIIIb, a 499-amino-acid protein and a 21-amino-acid signal peptide. Native and yeast secretion signals were used to direct the secretion of the enzyme, with the native signal yielding higher enzyme activity in the culture medium. The level of laccase activity secreted by the transformed yeast was similar to that observed for the non-induced wild-type strain of T. versicolor. The identity of the recombinant enzyme was checked by Western blot and matrix-assisted laser desorption/ionization time-of-flight analysis. Electrophoresis separation in native conditions indicated a molecular mass of the recombinant protein slightly higher (5 kDa) than that of the mature T. versicolor laccase IIIb, suggesting a limited excess of glycosylation. The laccase production level reached 2.5 mg/l (0.23 units/ml), which is suitable for engineering purpose.

Base Sequence↗

PubMed2005

Factors controlling pathogen destruction during anaerobic digestion of biowastes.

Anaerobic digestion is the principal method of stabilising biosolids from urban wastewater treatment in the UK, and it also has application for the treatment of other types of biowaste. Increasing awareness of the potential risks to human and animal health from environmental sources of pathogens has focused attention on the efficacy of waste treatment processes at destroying pathogenic microorganisms in biowastes recycled to agricultural land. The degree of disinfection achieved by a particular anaerobic digester is influenced by a variety of interacting operational variables and conditions, which can often deviate from the ideal. Experimental investigations demonstrate that Escherichia coli and Salmonella spp. are not damaged by mesophilic temperatures, whereas rapid inactivation occurs by thermophilic digestion. A hydraulic, biokinetic and thermodynamic model of pathogen inactivation during anaerobic digestion showed that a 2 log10 reduction in E. coli (the minimum removal required for agricultural use of conventionally treated biosolids) is likely to challenge most conventional mesophilic digesters, unless strict maintenance and management practices are adopted to minimise dead zones and by-pass flow. Efficient mixing and organic matter stabilisation are the main factors controlling the rate of inactivation under mesophilic conditions and not a direct effect of temperature per se on pathogenic organisms.

Bacteria, Anaerobic↗

PubMed2000

Environmental risk assessment of human pharmaceuticals in Denmark after normal therapeutic use.

An environmental risk assessment is presented for the 25 most used pharmaceuticals in the primary health sector in Denmark. Predicted environmental concentrations (PECs) for the aquatic environment were calculated using conservative assumptions and all PECs exceeded 1 ng/l. Measured concentrations were in general within a factor of 2-5 of PECs and ranged from approximately 0.5 ng/l to 3 micrograms/l for nine of the pharmaceuticals reported in literature. The calculation of predicted no-effect concentration (PNEC) based on aquatic ecotoxicity data was possible for six of the pharmaceuticals. PEC/PNEC ratio exceeded one for ibuprofen, acetylsalicylic acid, and paracetamol. For estrogens the PEC/PNEC ratio approached one when non-standard test was used. The ratio was below one for estrogens (standard test), diazepam and digoxin. For the terrestrial compartment, toxicity data were not available, and no assessment was carried out. Comparisons of predicted concentrations of furosemide, ibuprofen, oxytetracycline and ciprofloxacin in sludge based on either preliminary experimental sludge-water partition coefficients (Kd), octanol-water coefficients (Kow) or acid-base constants (pKa) revealed large variations.

Acetaminophen↗

PubMedDate not supplied

Bioaugmentation strategies for remediating mixed chemical effluents.

Operationally exhausted metal working fluids are chemically mixed, produced in large quantities (400 000 tonnes year in the U.K.), and potentially environmentally toxic. It is essential to develop more reliable and economical approaches for their disposal. We investigated the effectiveness of a defined bacterial consortium, constructed specifically for treating metal-working fluid (MWF), and contrasted its performance to that of undefined inocula from activated sludge. Construction of the consortium was based on knowledge of the diversity of bacterial communities that naturally colonize MWF and determination of their catabolic abilities and tolerance to the chemical constituents. Chemical analysis of the inoculated MWF bioreactor revealed that, after 100 h at 28 degrees C, the defined inoculum reduced the pollution load by over 80% from an initial chemical oxygen demand of approximately 48 000 mg L(-)(1). The inocula performance was approximately 50% more effective than that of the undefined microbial community from the activated sludge. Furthermore, the performance of the constructed consortium was more reproducible than that of an undefined community, an essential feature for bioaugmentation treatment of industrial wastes.

Bacteria↗

PubMed2005

Phytoremediation: novel approaches to cleaning up polluted soils.

Environmental pollution with metals and xenobiotics is a global problem, and the development of phytoremediation technologies for the plant-based clean-up of contaminated soils is therefore of significant interest. Phytoremediation technologies are currently available for only a small subset of pollution problems, such as arsenic. Arsenic removal employs naturally selected hyperaccumulator ferns, which accumulate very high concentrations of arsenic specifically in above-ground tissues. Elegant two-gene transgenic approaches have been designed for the development of mercury or arsenic phytoremediation technologies. In a plant that naturally hyperaccumulates zinc in leaves, approximately ten key metal homeostasis genes are expressed at very high levels. This outlines the extent of change in gene activities needed in the engineering of transgenic plants for soil clean-up. Further analysis and discovery of genes for phytoremediation will benefit from the recent development of segregating populations for a genetic analysis of naturally selected metal hyperaccumulation in plants, and from comprehensive ionomics data--multi-element concentration profiles from a large number of Arabidopsis mutants.

Arabidopsis↗

PubMed2005

Woody biomass phytoremediation of contaminated brownfield land.

Economic and environmental regeneration of post-industrial landscapes frequently involves some element of re-afforestation or tree planting. We report field trials that evaluate whether woody biomass production is compatible with managing residual trace element contamination in brownfield soils. Large-scale mapping of contamination showed a heterogenous dispersion of metals and arsenic, and highly localised within-site hotspots. Yields of Salix, Populus and Alnus were economically viable, showing that short-rotation coppice has a potentially valuable role in community forestry. Mass balance modelling demonstrated that phytoextraction potentially could reduce contamination hotspots of more mobile elements (Cd and Zn) within a 25-30-year life cycle of the crops. Cd and Zn in stems and foliage of Salix were 4-13 times higher than EDTA-extractable soil concentrations. Lability of other trace elements (As, Pb, Cu, Ni) was not increased 3 years after planting the coppice; woody biomass may provide an effective reduction of exposure (phyto-stabilization) to these less mobile contaminants.

Alnus↗

PubMed2003

Removal of ammonia toxicity in marine sediment TIEs: a comparison of Ulva lactuca, zeolite and aeration methods.

Toxicity Identification Evaluations (TIEs) can be used to determine the specific toxicant(s), including ammonia, causing toxicity observed in marine sediments. Two primary TIE manipulations are available for characterizing and identifying ammonia in marine sediments: Ulva lactuca addition and zeolite addition. In this study, we compared the efficacy of these methods to (1) remove NH(x) and NH(3) from overlying and interstitial waters and (2) reduce toxicity to the amphipod Ampelisca abdita and mysid Americamysis bahia using both spiked and environmentally contaminated sediments. The utility of aeration for removing NH(x) and NH(3) during a marine sediment TIE was also evaluated preliminarily. In general, the U. lactuca and zeolite addition methods performed similarly well at removing spiked NH(x) and NH(3) from overlying and interstitial waters compared to an unmanipulated sediment. Toxicity to the amphipod was reduced approximately the same by both methods. However, toxicity to the mysid was most effectively reduced by the U. lactuca addition indicating this method functions best with epibenthic species exposed to ammonia in the water column. Aeration removed NH(x) and NH(3) from seawater when the pH was adjusted to 10; however, very little ammonia was removed at ambient pHs ( approximately 8.0). This comparison demonstrates both U. lactuca and zeolite addition methods are effective TIE tools for reducing the concentrations and toxicity of ammonia in whole sediment toxicity tests.

Ammonia↗

PubMed2004

The use of plants for remediation of metal-contaminated soils.

The use of green plants to remove, contain, inactivate, or degrade harmful environmental contaminants (generally termed phytoremediation) is an emerging technology. In this paper, an overview is given of existing information concerning the use of plants for the remediation of metal-contaminated soils. Both site decontamination (phytoextraction) and stabilization techniques (phytostabilization) are described. In addition to the plant itself, the use of soil amendments for mobilization (in case of phytoextraction) and immobilization (in case of phytostabilization) is discussed. Also, the economical impacts of changed land-use, eventual valorization of biomass, and cost-benefit aspects of phytoremediation are treated. In spite of the growing public and commercial interest and success, more fundamental research is needed still to better exploit the metabolic diversity of the plants themselves, but also to better understand the complex interactions between metals, soil, plant roots, and micro-organisms (bacteria and mycorrhiza) in the rhizosphere. Further, more demonstration experiments are needed to measure the underlying economics, for public acceptance and last but not least, to convince policy makers.

Belgium↗

PubMed2002

In vitro stability of a novel compliant poly(carbonate-urea)urethane to oxidative and hydrolytic stress.

Poly(ester)urethane and poly(ether)urethane vascular grafts fail in vivo because of hydrolytic and oxidative degradative mechanisms. Studies have shown that poly(carbonate)urethanes have enhanced resistance. There is still a need for a viable, nonrigid, small-diameter, synthetic vascular graft. In this study, we sought to confirm this by exposing a novel formulation of compliant poly(carbonate-urea)urethane (CPU) manufactured by an innovative process, resulting in a stress-free. Small-diameter prosthesis, and a conventional poly(ether)urethane Pulse-Tec graft known to readily undergo oxidation in a variety of degradative solutions, and we assessed them for the development of oxidative and hydrolytic degradation, changes in elastic properties, and chemical stability. To simulate the in vivo environment, we used buffered solutions of phospholipase A(2) and cholesterol esterase; solutions of H(2)O(2)/CoCl(2), t-butyl peroxide/CoCl(2) (t-but/CoCl(2)), and glutathione/t-butyl peroxide/CoCl(2) (Glut/t-but/CoCl(2)); and plasma fractions I-IV, which were derived from fresh human plasma centrifuged in poly(ethylene glycol). To act as a negative control, both graft types were incubated in distilled water. Samples of both graft types (100 mm with a 5.0-mm inner diameter) were incubated in these solutions at 37 degrees C for 70 days before environmental scanning electron microscopy, radial tensile strength and quality control, gel permeation chromatography, and in vitro compliance assessments were performed. Oxidative degradation was ascertained from significant changes in molecular weight with respect to a control on all Pulse-Tec grafts treated with t-but/CoCl(2), Glut/t-but/CoCl(2), and plasma fractions I-III. Pulse-Tec grafts exposed to the H(2)O(2)/CoCl(2) mixture had significantly greater compliance than controls incubated in distilled water (p < 0.001 at 50 mmHg). No changes in molecular weight with respect to the control were observed for the CPU samples; only those immersed in t-but/CoCl(2) and Glut/t-but/CoCl(2) showed an 11% increase in molecular weight to 108,000. Only CPU grafts treated with the Glut/t-but/CoCl(2) mixture exhibited significantly greater compliance (p < 0.05 at 50 mmHg). Overall, results from this study indicate that CPU presents a far greater chemical stability than poly(ether)-urethane grafts do.

Biocompatible Materials↗

PubMed2004

Current issues and uncertainties in the measurement and modelling of air-vegetation exchange and within-plant processing of POPs.

Air-vegetation exchange of POPs is an important process controlling the entry of POPs into terrestrial food chains, and may also have a significant effect on the global movement of these compounds. Many factors affect the air-vegetation transfer including: the physicochemical properties of the compounds of interest; environmental factors such as temperature, wind speed, humidity and light conditions; and plant characteristics such as functional type, leaf surface area, cuticular structure, and leaf longevity. The purpose of this review is to quantify the effects these differences might have on air/plant exchange of POPs, and to point out the major gaps in the knowledge of this subject that require further research. Uptake mechanisms are complicated, with the role of each factor in controlling partitioning, fate and behaviour process still not fully understood. Consequently, current models of air-vegetation exchange do not incorporate variability in these factors, with the exception of temperature. These models instead rely on using average values for a number of environmental factors (e.g. plant lipid content, surface area), ignoring the large variations in these values. The available models suggest that boundary layer conductance is of key importance in the uptake of POPs, although large uncertainties in the cuticular pathway prevents confirmation of this with any degree of certainty, and experimental data seems to show plant-side resistance to be important. Models are usually based on the assumption that POP uptake occurs through the lipophilic cuticle which covers aerial surfaces of plants. However, some authors have recently attached greater importance to the stomatal route of entry into the leaf for gas phase compounds. There is a need for greater mechanistic understanding of air-plant exchange and the 'scaling' of factors affecting it. The review also suggests a number of key variables that researchers should measure in their experiments to allow comparisons to be made between studies in order to improve our understanding of what causes any differences in measured data between sites.

Air Movements↗

PubMed2005

Bioaugmentation for bioremediation: the challenge of strain selection.

Despite its long-term use in bioremediation, bioaugmentation of contaminated sites with microbial cells continues to be a source of controversy within environmental microbiology. This largely results from its notoriously unreliable performance record. In this article, we argue that the unpredictable nature of the approach comes from the initial strain selection step. Up until now, this has been dictated by the search for catabolically competent microorganisms, with little or no consideration given to other essential features that are required to be functionally active and persistent in target habitats. We describe how technical advances in molecular biology and analytical chemistry, now enable assessments of the functional diversity and spatial distribution of microbial communities to be made in situ. These advances now enable microbial populations, targeted for exploitation, to be differentiated to the cell level, an advance that is bound to improve microbial selection and exploitation. We argue that this information-based approach is already proving to be more effective than the traditional 'black-box' approach of strain selection. The future perspectives and opportunities for improving selection of effective microbial strains for bioaugmentation are also discussed.

Bacteria↗

PubMed2002

Bioremediation of oil by marine microbial mats.

Cyanobacterial mats developing in oil-contaminated sabkhas along the African coasts of the Gulf of Suez and in the pristine Solar Lake, Sinai, were collected for laboratory studies. Samples of both mats showed efficient degradation of crude oil in the light, followed by development of an intense bloom of Phormidium spp. and Oscillatoria spp. Isolated cyanobacterial strains, however, did not degrade crude oil in axenic cultures. Strains of sulfate-reducing bacteria and aerobic heterotrophs were capable of degrading model compounds of aliphatic and aromatic hydrocarbons. Results indicate that degradation of oil was done primarily by aerobic heterotrophic bacteria. The oxygenic photosynthesis of oil-insensitive cyanobacteria supplied the molecular oxygen for the efficient aerobic metabolism of organisms, such as Marinobacter sp. The diurnal shifts in environmental conditions at the mat surface, from highly oxic conditions in the light to anaerobic sulfide-rich habitat in the dark, may allow the combined aerobic and anaerobic degradation of crude oil at the mat surface. Hence, coastal cyanobacterial mats may be used for the degradation of coastline oil spills. Oxygen microelectrodes detected a significant inhibition of photosynthetic activity subsequent to oil addition. This prevailed for a few hours and then rapidly recovered. In addition, shifts in bacterial community structure following exposure to oil were determined by denaturing gradient gel electrophoresis of PCR-amplified fractions of 16S rRNA from eubacteria, cyanobacteria and sulfate-reducing bacteria. Since the mats used for the present study were obtained from oil-contaminated environments, they were believed to be preequilibrated for petroleum remediation. The mesocosm system at Eilat provided a unique opportunity to study petroleum degradation by mats formed under different salinities (up to 21%). These mats, dominated by cyanobacteria, can serve as close analogues to the sabkhas contaminated during the Gulf War in Kuwait and Saudi Arabia.

Biodegradation, Environmental↗