Laboratory evidence of MTBE biodegradation in Borden aquifer material

Mainly due to intrinsic biodegradation, monitored natural attenuation can be an effective and inexpensive remediation strategy at petroleum release sites. However, gasoline additives such as methyl tert-butyl ether (MTBE) can jeopardize this strategy because these compounds often degrade, if at all, at a slower rate than the collectively benzene, toluene, ethylbenzene and the xylene (BTEX) compounds. Investigation of whether a compound degrades under certain conditions, and at what rate, is therefore important to the assessment of the intrinsic remediation potential of aquifers. A natural gradient experiment with dissolved MTBE-containing gasoline in the shallow, aerobic sand aquifer at Canadian Forces Base (CFB) Borden (Ontario, Canada) from 1988 to 1996 suggested that biodegradation was the main cause of attenuation for MTBE within the aquifer. This laboratory study demonstrates biologically catalyzed MTBE degradation in Borden aquifer-like environments, and so supports the idea that attenuation due to biodegradation may have occurred in the natural gradient experiment. In an experiment with batch microcosms of aquifer material, three of the microcosms ultimately degraded MTBE to below detection, although this required more than 189 days (or >300 days in one case). Failure to detect the daughter product tert-butyl alcohol (TBA) in the field and the batch experiments could be because TBA was more readily degradable than MTBE under Borden conditions.     

Retardation of organo-bromides in a fractured chalk aquitard

This study investigates the mechanisms controlling the distribution of 3-bromo-2,2-bis(bromomethyl)propanol (TBNPA) and 2,2-bis(bromomethyl)propan-1,3-diol (DBNPG) in a fractured chalk aquitard. An extensive monitoring program showed a systematic decrease in the TBNPA/DBNPG ratio with distance from the contamination source. Sorption of TBNPA on the white and/or gray chalks comprising the aquitard is approximately one order of magnitude greater than that of DBNPG. This results in more efficient removal of TBNPA from the fracture into the porous matrix and thus decreases the TBNPA/DBNPG ratio in the fracture water. Mathematical modeling of solute transport in the fracture domain illustrates the probable importance of sorption in controlling the spatial variation in TBNPA and DBNPG ratio.     

Microbiological analysis of multi-level borehole samples from a contaminated groundwater system

A range of bacteriological, geochemical process-related and molecular techniques have been used to assess the microbial biodegradative potential in groundwater contaminated with phenol and other tar acids. The contaminant plume has travelled 500 m from the pollutant source over several decades. Samples were obtained from the plume using a multi-level sampler (MLS) positioned in two boreholes (boreholes 59 and 60) which vertically transected two areas of the plume. Activity of the microbial community, as represented by phenol degradation potential and ability to utilise a range of substrates, was found to be influenced by the plume. Phenol degradation potential appeared to be influenced more by the concentration of the contaminants than the total bacterial cell numbers. However, in the areas of highest phenol concentration, the depression of cell numbers clearly had an effect. The types of bacteria present were assessed by culture and DNA amplification by polymerase chain reaction (PCR). Bacterial groups or processes associated with major geochemical processes, such as methanogenesis, sulphate reduction and denitrification, that have the potential to drive contaminant degradation, were detected at various borehole levels. A comparative molecular analysis of the microbial community between samples obtained from the MLS revealed the microbial community was diverse. The examination of microbial activity complemented those results obtained through chemical analysis, and when combined with hydrological data, showed that MLS samples provided a realistic profile of plume effects and could be related to the potential for natural attenuation of the site.     

Reactive transport modeling of processes controlling the distribution and natural attenuation of phenolic compounds in a deep sandstone aquifer

Reactive solute transport modeling was utilized to evaluate the potential for natural attenuation of a contaminant plume containing phenolic compounds at a chemical producer in the West Midlands, UK. The reactive transport simulations consider microbially mediated biodegradation of the phenolic compounds (phenols, cresols, and xylenols) by multiple electron acceptors. Inorganic reactions including hydrolysis, aqueous complexation, dissolution of primary minerals, formation of secondary mineral phases, and ion exchange are considered. One-dimensional (1D) and three-dimensional (3D) simulations were conducted. Mass balance calculations indicate that biodegradation in the saturated zone has degraded approximately 1-5% of the organic contaminant plume over a time period of 47 years. Simulations indicate that denitrification is the most significant degradation process, accounting for approximately 50% of the organic contaminant removal, followed by sulfate reduction and fermentation reactions, each contributing 15-20%. Aerobic respiration accounts for less than 10% of the observed contaminant removal in the saturated zone. Although concentrations of Fe(III) and Mn(IV) mineral phases are high in the aquifer sediment, reductive dissolution is limited, producing only 5% of the observed mass loss. Mass balance calculations suggest that no more than 20-25% of the observed total inorganic carbon (TIC) was generated from biodegradation reactions in the saturated zone. Simulations indicate that aerobic biodegradation in the unsaturated zone, before the contaminant entered the aquifer, may have produced the majority of the TIC observed in the plume. Because long-term degradation is limited to processes within the saturated zone, use of observed TIC concentrations to predict the future natural attenuation may overestimate contaminant degradation by a factor of 4-5.     

Biofilm formation and microbial activity in a biofilter system in the presence of MTBE, ETBE and TAME

Emerging water contaminants derived from unleaded gasoline such as methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), are in need of effective bioremediation technologies for restoring water resources. In order to design the conditions of a future groundwater bioremediating biofilter, this work assesses the potential use of Acinetobacter calcoaceticus M10, Rhodococcus ruber E10 and Gordonia amicalis T3 for the removal of MTBE, ETBE and TAME in consortia or as individual strains. Biofilm formation on an inert polyethylene support material was assessed with scanning electron microscopy, and consortia were also analysed with fluorescent in situ hybridisation to examine the relation between the strains. A. calcoaceticus M10 was the best coloniser, followed by G. amicalis T3, however, biofilm formation of pair consortia favoured consortium M10-E10 both in formation and activity. However, degradation batch studies determined that neither consortium exhibited higher degradation than individual strain degradation. The physiological state of the three strains was also determined through flow cytometry using propidium iodide and 3′-dihexylocarbocyanine iodide thus gathering information on their viability and activity with the three oxygenates since previous microbial counts revealed slow growth. Strain E10 was observed to have the highest physiological activity in the presence of MTBE, and strain M10 activity with TAME was only maintained for 24 h, thus we believe that biotransformation of MTBE occurs within the active periods established by the cytometry analyses. Viable cell counts and oxygenate removal were determined in the presence of the metabolites tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA), resulting in TBA biotransformation by M10 and E10, and TAA by M10. Our results show that A. calcoaceticus M10 and the consortium M10-E10 could be adequate inocula in MTBE and TAME bioremediating technologies.     

Trimethylbenzoic acids as metabolite signatures in the biogeochemical evolution of an aquifer contaminated with jet fuel hydrocarbons

Evolution of trimethylbenzoic acids in the KC-135 aquifer at the former Wurtsmith Air Force Base (WAFB), Oscoda, MI was examined to determine the functionality of trimethylbenzoic acids as key metabolite signatures in the biogeochemical evolution of an aquifer contaminated with JP-4 fuel hydrocarbons. Changes in the composition of trimethylbenzoic acids and the distribution and concentration profiles exhibited by 2,4,6- and 2,3,5-trimethylbenzoic acids temporally and between multilevel wells reflect processes indicative of an actively evolving contaminant plume. The concentration levels of trimethylbenzoic acids were 3-10 orders higher than their tetramethylbenzene precursors, a condition attributed to slow metabolite turnover under sulfidogenic conditions. The observed degradation of tetramethylbenzenes into trimethylbenzoic acids obviates the use of these alkylbenzenes as non-labile tracers for other degradable aromatic hydrocarbons, but provides rare field evidence on the range of high molecular weight alkylbenzenes and isomeric assemblages amenable to anaerobic degradation in situ. The coupling of actual tetramethylbenzene loss with trimethylbenzoic acid production and the general decline in the concentrations of these compounds demonstrate the role of microbially mediated processes in the natural attenuation of hydrocarbons and may be a key indicator in the overall rate of hydrocarbon degradation and the biogeochemical evolution of the KC-135 aquifer.     

甲基叔丁基醚(MTBE)对环境的污染及其对我国汽油生产的影响

本文简要介绍了汽油添加剂MTBE对环境的污染及减少MTBE污染机理研究的进展 ,同时介绍了对我国汽油生产的影响     

Assessing the natural attenuation of organic contaminants in aquifers using plume-scale electron and carbon balances: model development with analysis of uncertainty and parameter sensitivity

A quantitative methodology is described for the field-scale performance assessment of natural attenuation using plume-scale electron and carbon balances. This provides a practical framework for the calculation of global mass balances for contaminant plumes, using mass inputs from the plume source, background groundwater and plume residuals in a simplified box model. Biodegradation processes and reactions included in the analysis are identified from electron acceptors, electron donors and degradation products present in these inputs. Parameter values used in the model are obtained from data acquired during typical site investigation and groundwater monitoring studies for natural attenuation schemes. The approach is evaluated for a UK Permo-Triassic Sandstone aquifer contaminated with a plume of phenolic compounds. Uncertainty in the model predictions and sensitivity to parameter values was assessed by probabilistic modelling using Monte Carlo methods. Sensitivity analyses were compared for different input parameter probability distributions and a base case using fixed parameter values, using an identical conceptual model and data set. Results show that consumption of oxidants by biodegradation is approximately balanced by the production of CH4 and total dissolved inorganic carbon (TDIC) which is conserved in the plume. Under this condition, either the plume electron or carbon balance can be used to determine contaminant mass loss, which is equivalent to only 4% of the estimated source term. This corresponds to a first order, plume-averaged, half-life of > 800 years. The electron balance is particularly sensitive to uncertainty in the source term and dispersive inputs. Reliable historical information on contaminant spillages and detailed site investigation are necessary to accurately characterise the source term. The dispersive influx is sensitive to variability in the plume mixing zone width. Consumption of aqueous oxidants greatly exceeds that of mineral oxidants in the plume, but electron acceptor supply is insufficient to meet the electron donor demand and the plume will grow. The aquifer potential for degradation of these contaminants is limited by high contaminant concentrations and the supply of bioavailable electron acceptors. Natural attenuation will increase only after increased transport and dilution.     

Isotopic modelling of the significance of bacterial sulphate reduction for phenol attenuation in a contaminated aquifer

A Triassic sandstone aquifer polluted with a mixture of phenolic hydrocarbons has been investigated by means of high-resolution groundwater sampling. Samples taken at depth intervals of 1 m have revealed the presence of a diving pollutant plume with a sharply defined upper margin. Concentrations of pollutant phenols exceed 4 g/l in the plume core, rendering it sterile but towards the diluted upper margin evidence for bacterial sulphate reduction (BSR) has been obtained. Groundwaters have been analysed for both delta34S-SO4 and delta18O-SO4. Two reservoirs have been identified with distinct sulphate oxygen isotope ratios. Groundwater sulphate (delta18O-SO4 = 3-5/1000) outside the plume shows a simple linear mixing trend with an isotopically uniform pollutant sulphate reservoir (delta18O-SO4 = 10-12/1000) across the plume margin. The sulphur isotope ratios do not always obey a simple mixing relation, however, at one multilevel borehole, enrichment in 34SO4 at the plume margin is inversely correlated with sulphate concentration. This and the presence of 34S-depleted dissolved sulphide indicate that enrichment in 34SO4 is the result of bacterial sulphate reduction. Delta34S analysis of trace hydrogen sulphide within the plume yielded an isotope enrichment factor (epsilon) of -9.4/1000 for present-day bacterial sulphate reduction. This value agrees with a long-term estimate (-9.9/1000) obtained from a Rayleigh model of the sulphate reduction process. The model was also used to obtain an estimate of the pre-reduction sulphate concentration profile with depth. The difference between this and the present-day profiles then gave a mass balance for sulphate consumption. The organic carbon mineralisation that would account for this sulphate loss is shown to represent only 0.1/1000 of the phenol concentration in this region of the plume. Hence, the contribution of bacterial sulphate reduction to biodegradation has thus far been small. The highest total phenolic concentration (TPC) at which there is sulphur isotope evidence of bacterial sulphate reduction is 2000 mg/l. We suggest that above this concentration, the bactericidal properties of phenol render sulphate-reducing bacteria inactive. Dissolved sulphate trapped in the concentrated plume core will only be utilised by sulphate reducers when toxic phenols in the plume are diluted by dispersion during migration.     

Attenuation of landfill leachate by UK Triassic sandstone aquifer materials: 2. Sorption and degradation of organic pollutants in laboratory columns

The sorption and degradation of dissolved organic matter (DOM) and 13 organic micropollutants (BTEX, aromatic hydrocarbons, chloro-aromatic and -aliphatic compounds, and pesticides) in acetogenic and methanogenic landfill leachate was studied in laboratory columns containing Triassic sandstone aquifer materials from the English Midlands. Solute sorption and degradation relationships were evaluated using a simple transport model. Relative to predictions, micropollutant sorption was decreased up to eightfold in acetogenic leachate, but increased up to sixfold in methanogenic leachate. This behaviour reflects a combination of interactions between the micropollutants, leachate DOM and aquifer mineral fraction. Sorption of DOM was not significant. Degradation of organic fractions occurred under Mn-reducing and SO₄-reducing conditions. Degradation of some micropollutants occurred exclusively under Mn-reducing conditions. DOM and benzene were not significantly degraded under the conditions and time span (up to 280 days) of the experiments. Most micropollutants were degraded immediately or after a lag phase (32–115 days). Micropollutant degradation rates varied considerably (half-lives of 8 to >2000 days) for the same compounds (e.g., TeCE) in different experiments, and for compounds (e.g., naphthalene, DCB and TeCA) within the same experiment. Degradation of many micropollutants was both simultaneous and sequential, and inhibited by the utilisation of different substrates. This mechanism, in combination with lag phases, controls micropollutant degradation potential in these systems more than the degradation rate. These aquifer materials have a potentially large capacity for in situ bioremediation of organic pollutants in landfill leachate and significant degradation may occur in the Mn-reducing zones of leachate plumes. However, degradation of organic pollutants in acetogenic leachate may be limited in aquifers with low pH buffering capacity and reducible Mn oxides. Contaminants in this leachate present a greater risk to groundwater resources in these aquifers than methanogenic leachate.     

Aerobic biodegradation of vinyl chloride and cis-1,2-dichloroethylene in aquifer sediments

Laboratory batch experiments have been performed with sediment and groundwater obtained from two sites in Denmark to study the aerobic biodegradation of vinyl chloride (VC) and cis-1,2-dichloroethylene (c-1,2-DCE) to assess the natural aerobic biodegradation potential at two sites. The experiments revealed that VC was degraded to below the detection limit within 204 and 57 days at the two sites. c-1,2-DCE was also degraded in the experiments but not completely. At the two sites 50% and 35% was removed by the end of the experimental period of 204 and 274 days. The removal of c-1,2-DCE seems to occur concomitantly with VC indicating that the biodegradation of c-1,2-DCE may depend on the biodegradation of VC. However, in both cases natural groundwater was mixed with sediment and consequently there may be other compounds (e.g. ammonium, natural organic compound etc.) that serves as primary substrates for the co-metabolic biodegradation of c-1,2-DCE. At one of the sites methane was supplied to try to enhance the biodegradation of VC and c-1,2-DCE. That was successful since the time for complete biodegradation of VC decreased from 204 days in the absence of methane to 84 days in the presence of methane. For c-1,2-DCE the amount that was biodegraded after 204 days increased from 50% to 90% as a result of the addition of methane. It seems like a potential for natural biodegradation exists at least for VC at these two sites and also to some degree for c-1,2-DCE.     

Engineered and subsequent intrinsic in situ bioremediation of a diesel fuel contaminated aquifer

A diesel fuel contaminated aquifer in Menziken, Switzerland was treated for 4.5 years by injecting aerated groundwater, supplemented with KNO3 and NH4H2PO4 to stimulate indigenous populations of petroleum hydrocarbon (PHC) degrading microorganisms. After dissolved PHC concentrations had stabilized at a low level, engineered in situ bioremediation was terminated. The main objective of this study was to evaluate the efficacy of intrinsic in situ bioremediation as a follow-up measure to remove PHC remaining in the aquifer after terminating engineered in situ bioremediation. In the first 7 months of intrinsic in situ bioremediation, redox conditions in the source area became more reducing as indicated by lower concentrations of SO4(2-) and higher concentrations of Fe(II) and CH4. In the core of the source area, strongly reducing conditions prevailed during the remaining study period (3 years) and dissolved PHC concentrations were higher than during engineered in situ bioremediation. This suggests that biodegradation in the core zone was limited by the availability of oxidants. In lateral zones of the source area, however, gradually more oxidized conditions were reestablished again, suggesting that PHC availability increasingly limited biodegradation. The total DIC production rate in the aquifer decreased within 2 years to about 25% of that during engineered in situ bioremediation and remained at that level. Stable carbon isotope analysis confirmed that the produced DIC mainly originated from PHC mineralization. The total rate of DIC and CH4 production in the source area was more than 300 times larger than the rate of PHC elution. This indicates that biodegradation coupled to consumption of naturally occurring oxidants was an important process for removal of PHC which remained in the aquifer after terminating engineered measures.     

Biodegradation kinetics and toxicity of vegetable oil triacylglycerols under aerobic conditions

The aerobic biodegradation of five triacylglycerols (TAGs), three liquids [triolein (OOO), trilinolein (LLL), and trilinolenin (LnLnLn)] and two solids [tripalmitin (PPP) and tristearin (SSS)] was studied in water. Respirometry tests were designed and conducted to determine the biochemical oxygen demand (BOD) parameters of the compounds. In the case of the solid lipids, the degradation process was limited by their extremely non-polar nature. When added to water, PPP and SSS formed irregular clumps or gumballs, not a fine and uniform suspension required for the lipase activity. After 30 days, appreciable mineralization was not achieved; therefore, first-order biodegradation coefficients could not be determined. The bioavailability of the liquid TAGs was restricted due to the presence of double bonds in the fatty acids (FAs). An autoxidation process occurred in the allylic chains, resulting in the production of hydroperoxides. These compounds polymerized and became non-biodegradable. Nevertheless, the non-oxidized fractions were readily mineralized, and BOD rate constants were estimated by non-linear regression: LLL (k = 0.0061 h⁻¹) and LnLnLn (k = 0.0071 h⁻¹) were degraded more rapidly than OOO (k = 0.0025 h⁻¹). Lipids strongly partitioned to the biomass and, therefore, Microtox^® toxicity was not observed in the water column. However, EC₅₀ values (<15% sample volume) were measured in the solid phase.     

Stable isotope fractionation analysis as a tool to monitor biodegradation in contaminated acquifers

The assessment of biodegradation in contaminated aquifers has become an issue of increasing importance in the recent years. To some extent, this can be related to the acceptance of intrinsic bioremediation or monitored natural attenuation as a means to manage contaminated sites. Among the few existing methods to detect biodegradation in the subsurface, stable isotope fractionation analysis (SIFA) is one of the most promising approaches which is pronounced by the drastically increasing number of applications. This review covers the recent laboratory and field studies assessing biodegradation of contaminants via stable isotope analysis. Stable isotope enrichment factors have been found that vary from no fractionation for dioxygenase reactions converting aromatic hydrocarbons over moderate fractionation by monooxygenase reactions (epsilon=-3 per thousand) and some anaerobic studies on microbial degradation of aromatic hydrocarbons (epsilon=-1.7 per thousand) to larger fractionations by anaerobic dehalogenation reactions of chlorinated solvents (epsilon=between -5 per thousand and -30 per thousand). The different isotope enrichment factors can be related to the respective biochemical reactions. Based on that knowledge, we discuss under what circumstances SIFA can be used for a qualitative or even a quantitative assessment of biodegradation in the environment. In a steadily increasing number of cases, it was possible to explain biodegradation processes in the field based on isotope enrichment factors obtained from laboratory experiments with pure cultures and measured isotope values from the field. The review will focus on the aerobic and anaerobic degradation of aromatic hydrocarbons and chlorinated solvents as the major contaminants of groundwater. Advances in the instrumental development for stable isotope analysis are only mentioned if it is important for the understanding of the application.     

Biodegradation of triclosan and formation of methyl-triclosan in activated sludge under aerobic conditions

Triclosan is an antimicrobial agent which is widely used in household and personal care products. Widespread use of this compound has led to the elevated concentrations of triclosan in wastewater, wastewater treatment plants (WWTPs) and receiving waters. Removal of triclosan and formation of triclosan-methyl was investigated in activated sludge from a standard activated sludge WWTP equipped with enhanced biological phosphorus removal. The removal was found to occur mainly under aerobic conditions while under anoxic (nitrate reducing) and anaerobic conditions rather low removal rates were determined. In a laboratory-scale activated sludge reactor 75% of the triclosan was removed under aerobic conditions within 150 h, while no removal was observed under anaerobic or anoxic conditions. One percent of the triclosan was converted to triclosan-methyl under aerobic conditions, less under anoxic (nitrate reducing) and none under anaerobic conditions.     

Biological degradation of triclocarban and triclosan in a soil under aerobic and anaerobic conditions and comparison with environmental fate modelling

Triclocarban and triclosan are two antimicrobial agents widely used in many personal care products. Their biodegradation behaviour in soil was investigated by laboratory degradation experiments and environmental fate modelling. Quantitative structure-activity relationship (QSAR) analyses showed that triclocarban and triclosan had a tendency to partition into soil or sediment in the environment. Fate modelling suggests that either triclocarban or triclosan "does not degrade fast" with its primary biodegradation half-life of "weeks" and ultimate biodegradation half-life of "months". Laboratory experiments showed that triclocarban and triclosan were degraded in the aerobic soil with half-life of 108 days and 18 days, respectively. No negative effect of these two antimicrobial agents on soil microbial activity was observed in the aerobic soil samples during the experiments. But these two compounds persisted in the anaerobic soil within 70 days of the experimental period.     

Geology and sediment geochemistry of a landfill leachate contaminated aquifer (Grindsted, Denmark)

A landfill leachate affected aquifer was investigated with respect to the geology and sediment geochemistry (solid organic carbon, cation exchange capacity, oxidation capacity, reduced iron and sulfur species) involving 185 sediment samples taken along a 305-m-long and 10–12-m-deep transect downgradient from the landfill. The geology showed two distinct sand layers (upper Quaternary, Weichselian and a lower Tertiary, Miocene) sandwiching thin layers of silt/clay deposits, peat and brown coal. The organic carbon content (TOC) and the cation exchange capacity (CEC) of the sandy sediments were low (TOC, 100–300 μg C (g DW)⁻¹ ; CEC, 0.1–0.5 meq per 100 g DW) and correlated fairly well with the geology. Processes in the contaminant plume caused depletion of oxidation capacity and precipitation of reduced iron and sulfur species. However, some of these parameters were also affected by the geology, e.g. the oxidation capacity (OXC) was significantly higher in the Quaternary layer (OXC, 14–35 μeq g DW⁻¹) than in the Tertiary sand layer (OXC, <5 μeq g DW⁻¹). The intermediate layers (silt/clay and brown coal) have significantly higher values of most of the parameters investigated. This work demonstrates the need for a small scale geological model and a detailed mapping of the geochemistry of the sediments in order to distinguish impacts caused by the contaminant plumes from natural variations in the aquifer geochemistry.     

Detection of methylquinoline transformation products in microcosm experiments and in tar oil contaminated groundwater using LC-NMR

N-heterocyclic compounds are known pollutants at tar oil contaminated sites. Here we report the degradation of methyl-, and hydroxy-methyl-substituted quinolines under nitrate-, sulfate- and iron-reducing conditions in microcosms with aquifer material of a former coke manufacturing site. Comparison of degradation potential and rate under different redox conditions revealed highest degradation activities under sulfate-reducing conditions, the prevailing conditions in the field. Metabolites of methylquinolines, with the exception of 2-methylquinolines, were formed in high amounts in the microcosms and could be identified by ¹H NMR spectroscopy as 2(1H)-quinolinone analogues. 4-Methyl-, 6-methyl-, and 7-methyl-3,4-dihydro-2(1H)-quinolinone, the hydrogenated metabolites in the degradation of quinoline compounds, were identified by high resolution LC-MS. Metabolites of methylquinolines showed persistence, although for the first time a transformation of 4-methylquinoline and its metabolite 4-methyl-2(1H)-quinolinone is described. The relevance of the identified metabolites is supported by the detection of a broad spectrum of them in groundwater of the field site using LC-NMR technique. LC-NMR allowed the differentiation of isomers and identification without reference compounds. A variety of methylated 2(1H)-quinolinones, as well as methyl-3,4-dihydro-2(1H)-quinolinone isomers were not identified before in groundwater.     

Activity-dependent labeling of oxygenase enzymes in a trichloroethene-contaminated groundwater site

A variety of naturally occurring bacteria produce enzymes that cometabolically degrade trichloroethene (TCE), including organisms with aerobic oxygenases. Groundwater contaminated with TCE was collected from the aerobic region of the Test Area North site of the Idaho National Laboratory. Samples were evaluated with enzyme activity probes, and resulted in measurable detection of toluene oxygenase activity (6-79% of the total microbial cells). Wells from both inside and outside contaminated plume showed activity. Toluene oxygenase-specific PCR primers determined that toluene-degrading genes were present in all groundwater samples evaluated. In addition, bacterial isolates were obtained and possessed toluene oxygenase enzymes, demonstrated activity, and were dominated by the phylotype Pseudomonas. This study demonstrated, through the use of enzymatic probes and oxygenase gene identification, that indigenous microorganisms at a contaminated site were cometabolically active. Documentation such as this can be used to substantiate observations of natural attenuation of TCE-contaminated groundwater plumes.     

Changes in enantiomeric fraction as evidence of natural attenuation of mecoprop in a limestone aquifer

Natural attenuation of the chiral pesticide mecoprop [2-(2-methyl-4-chlorophenoxy)propionic acid] has been studied by determining changes in its enantiomeric fraction in different redox environments down gradient of a landfill in the Lincolnshire Limestone. Previous studies have shown that mecoprop degrades predominantly aerobically and that differences in the biological behaviour of the two enantiomers will change their relative proportions during biodegradation. Originally deposited as a racemic mixture, there has been no change in the enantiomeric fraction in the most polluted part of the landfill plume where conditions are sulphate reducing/methanogenic. In the nitrate-reducing zone, the proportion of (S)-mecoprop increases, suggesting preferential degradation of (R)-mecoprop; while in the aerobic zone, the proportion of (R)-mecoprop increases, suggesting faster degradation of (S)-mecoprop. Mecoprop persistence in the confined Lincolnshire Limestone further downdip is explained by inhibition of degradation in sulphate-reducing conditions, which develop naturally. Laboratory microcosm experiments using up to 10 mg l(-1) of mecoprop confirm these inferences and show that under aerobic conditions, (S)-mecoprop and (R)-mecoprop degrade with zero-order kinetics at rates of 1.90 and 1.32 mg l(-1) day(-1), respectively. Under nitrate-reducing conditions (S)-mecoprop does not degrade, but (R)-mecoprop degrades with zero-order kinetics at 0.65 mg l(-1) day(-1) to produce a stoichiometric equivalent amount of 4-chloro-2-methylphenol. This metabolite only degrades when the (R)-mecoprop has disappeared. The addition of nitrate to a dormant iron-reducing microcosm devoid of nitrate stimulated anaerobic degradation of (R)-mecoprop after a lag period of 21 days. There was no evidence for enantiomeric inversion. The study demonstrates the sensitivity of changes in enantiomeric fraction for detecting natural attenuation, and reveals subtle differences in mecoprop degradation in different redox environments within the Lincolnshire Limestone aquifer.