Distribution of ETBE-degrading microorganisms and functional capability in groundwater, and implications for characterising aquifer ETBE biodegradation potential.

Microbes in aquifers are present suspended in groundwater or attached to the aquifer sediment. Groundwater is often sampled at gasoline ether oxygenate (GEO)-impacted sites to assess the potential biodegradation of organic constituents. However, the distribution of GEO-degrading microorganisms between the groundwater and aquifer sediment must be understood to interpret this potential. In this study, the distribution of ethyl tert-butyl ether (ETBE)-degrading organisms and ETBE biodegradation potential was investigated in laboratory microcosm studies and mixed groundwater-aquifer sediment samples obtained from pumped monitoring wells at ETBE-impacted sites. ETBE biodegradation potential (as determined by quantification of the ethB gene) was detected predominantly in the attached microbial communities and was below detection limit in the groundwater communities. The copy number of ethB genes varied with borehole purge volume at the field sites. Members of the Comamonadaceae and Gammaproteobacteria families were identified as responders for ETBE biodegradation. However, the detection of the ethB gene is a more appropriate function-based indicator of ETBE biodegradation potential than taxonomic analysis of the microbial community. The study shows that a mixed groundwater-aquifer sediment (slurry) sample collected from monitoring wells after minimal purging can be used to assess the aquifer ETBE biodegradation potential at ETBE-release sites using this function-based concept.

Biodegradation potential of MTBE in a fractured chalk aquifer under aerobic conditions in long-term uncontaminated and contaminated aquifer microcosms.

The potential for aerobic biodegradation of MTBE in a fractured chalk aquifer is assessed in microcosm experiments over 450 days, under in situ conditions for a groundwater temperature of 10 degrees C, MTBE concentration between 0.1 and 1.0 mg/L and dissolved O2 concentration between 2 and 10 mg/L. Following a lag period of up to 120 days, MTBE was biodegraded in uncontaminated aquifer microcosms at concentrations up to 1.2 mg/L, demonstrating that the aquifer has an intrinsic potential to biodegrade MTBE aerobically. The MTBE biodegradation rate increased three-fold from a mean of 6.6+/-1.6 microg/L/day in uncontaminated aquifer microcosms for subsequent additions of MTBE, suggesting an increasing biodegradation capability, due to microbial cell growth and increased biomass after repeated exposure to MTBE. In contaminated aquifer microcosms which also contained TAME, MTBE biodegradation occurred after a shorter lag of 15 or 33 days and MTBE biodegradation rates were higher (max. 27.5 microg/L/day), probably resulting from an acclimated microbial population due to previous exposure to MTBE in situ. The initial MTBE concentration did not affect the lag period but the biodegradation rate increased with the initial MTBE concentration, indicating that there was no inhibition of MTBE biodegradation related to MTBE concentration up to 1.2 mg/L. No minimum substrate concentration for MTBE biodegradation was observed, indicating that in the presence of dissolved O2 (and absence of inhibitory factors) MTBE biodegradation would occur in the aquifer at MTBE concentrations (ca. 0.1 mg/L) found at the front of the ether oxygenate plume. MTBE biodegradation occurred with concomitant O2 consumption but no other electron acceptor utilisation, indicating biodegradation by aerobic processes only. However, O2 consumption was less than the stoichiometric requirement for complete MTBE mineralization, suggesting that only partial biodegradation of MTBE to intermediate organic metabolites occurred. The availability of dissolved O2 did not affect MTBE biodegradation significantly, with similar MTBE biodegradation behaviour and rates down to ca. 0.7 mg/L dissolved O2 concentration. The results indicate that aerobic MTBE biodegradation could be significant in the plume fringe, during mixing of the contaminant plume and uncontaminated groundwater and that, relative to the plume migration, aerobic biodegradation is important for MTBE attenuation. Moreover, should the groundwater dissolved O2 concentration fall to zero such that MTBE biodegradation was inhibited, an engineered approach to enhance in situ bioremediation could supply O2 at relatively low levels (e.g. 2-3 mg/L) to effectively stimulate MTBE biodegradation, which has significant practical advantages. The study shows that aerobic MTBE biodegradation can occur at environmentally significant rates in this aquifer, and that long-term microcosm experiments (100s days) may be necessary to correctly interpret contaminant biodegradation potential in aquifers to support site management decisions.

Hydrochemical and isotopic effects associated with petroleum fuel biodegradation pathways in a chalk aquifer.

Hydrochemical data, compound specific carbon isotope analysis and isotopic enrichment trends in dissolved hydrocarbons and residual electron acceptors have been used to deduce BTEX and MTBE degradation pathways in a fractured chalk aquifer. BTEX compounds are mineralised sequentially within specific redox environments, with changes in electron acceptor utilisation being defined by the exhaustion of specific BTEX components. A zone of oxygen and nitrate exhaustion extends approximately 100 m downstream from the plume source, with residual sulphate, toluene, ethylbenzene and xylene. Within this zone complete removal of the TEX components occurs by bacterial sulphate reduction, with sulphur and oxygen isotopic enrichment of residual sulphate (epsilon(s) = -14.4 per thousand to -16.0 per thousand). Towards the plume margins and at greater distance along the plume flow path nitrate concentrations increase with delta15N values of up to +40 per thousand indicating extensive denitrification. Benzene and MTBE persist into the denitrification zone, with carbon isotope enrichment of benzene indicating biodegradation along the flow path. A Rayleigh kinetic isotope enrichment model for 13C-enrichment of residual benzene gives an apparent epsilon value of -0.66 per thousand. MTBE shows no significant isotopic enrichment (delta13C = -29.3 per thousand to -30.7 per thousand) and is isotopically similar to a refinery sample (delta13C = -30.1 per thousand). No significant isotopic variation in dissolved MTBE implies that either the magnitude of any biodegradation-induced isotopic fractionation is small, or that relatively little degradation has taken place in the presence of BTEX hydrocarbons. It is possible, however, that MTBE degradation occurs under aerobic conditions in the absence of BTEX since no groundwater samples were taken with co-existing MTBE and oxygen. Low benzene delta13C values are correlated with high sulphate delta34S, indicating that little benzene degradation has occurred in the sulphate reduction zone. Benzene degradation may be associated with denitrification since increased benzene delta13C is associated with increased delta15N in residual nitrate. Re-supply of electron acceptors by diffusion from the matrix into fractures and dispersive mixing is an important constraint on degradation rates and natural attenuation capacity in this dual-porosity aquifer.

Determination of interstitial water chemistry and porosity in consolidated aquifer materials by diffusion equilibrium-exchange.

Diffusion equilibrium exchange (DEE) is presented as a novel, practical alternative to centrifugation for the recovery and chemical analysis of interstitial water in contaminated core samples from consolidated rocks and aquifers. The methodology is suitable for sampling organic and inorganic compounds, including redox sensitive species such as SO4(2-), NO3-, NO2-, Mn(II), Fe(II), and sulfide (HS-). DEE also permits analyte extraction from kilogram quantities of core, which avoids extended centrifugation or sample amalgamation and provides analyte masses appropriate for stable isotope analysis. The procedure involves simple and rapid on-site sectioning of representative core samples, which are preserved in the field by storage in airtight bottles filled with deoxygenated deionized water containing a conservative tracer (Br-). Equilibration times for individual solutes can be estimated in advance to reduce the need for time-series analysis; for an effective diffusion coefficient of 2.5 x 10(-10) m2 s(-1) (Br- in chalk rock) equilibration was >90% completed after 30 h, consistent with the predicted equilibration time. The DEE method presented minimizes sampling errors from temperature changes, oxidation of reduced chemical species, and loss of volatile compounds, which can occur with other interstitial water sampling techniques. It also gives superior resolution of in situ solute distributions and geochemical processes in consolidated sediments than centrifugation and can provide estimates of aquifer porosity in core samples. Laboratory experiments using chalk rock core and simulated extraction procedures confirm the superior performance of the DEE method over centrifugation for a range of solutes. The method has been used to generate detailed interstitial water profiles of electron acceptor and contaminant concentrations along the flow path of a petroleum hydrocarbon plume in the U.K. Upper Chalk aquifer as part of a natural attenuation assessment.

Oncostatin M induces tissue-type plasminogen activator and plasminogen activator inhibitor-1 in Calu-1 lung carcinoma cells.

Oncostatin M (OSM) is a glycoprotein cytokine that is produced by activated T-lymphocytes, monocytes, and macrophages. In a DNA synthesis assay, OSM reduced tritiated thymidine incorporation by 53% in Calu-1 lung carcinoma cells. Radiolabeled cDNAs from untreated Calu-1 cells and 30-h OSM-treated cells were used to probe duplicate nylon membrane cDNA expression arrays. This study revealed OSM-mediated expression of mRNAs encoding tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Northern blot analysis showed that the steady-state level of tPA mRNA is nearly undetectable in Calu-1 cells. Exposure of these cells to OSM for 30 h increased tPA mRNA expression by 20-fold and PAI-1 mRNA expression by 5-fold. Exposure of these cells to other gp130 receptor family cytokines, including leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and IL-11, do not significantly affect DNA synthesis or induction of tPA/PAI-1. Western blot studies demonstrated that OSM mediates a marked increase in secretion of the tPA protein. Secreted tPA was present in the conditioned medium almost exclusively as tPA/PAI-1 complexes. Inhibitor studies demonstrated that OSM-mediated induction of tPA and PAI-1 mRNAs is largely dependent upon activation of the MEK1/2 pathway. The JAK3/STAT3 pathway potentially serves a secondary role in these regulatory events.

Arginine to lysine 108 substitution in recombinant CYP1A2 abolishes methoxyresorufin metabolism in lymphoblastoid cells.

1. Cytochrome P4501A2 (CYP1A2) activates a large number of procarcinogens to carcinogens. Phytochemicals such as flavones can inhibit CYP1A2 activity competitively, and hydroxylated derivatives of flavone (galangin) may be potent, selective inhibitors of CYP1A2 activity relative to CYP1A1 activity. Molecular modelling of the CYP1A2 interaction with hydroxylated derivatives of flavone suggests that a number of hydrophobic residues of the substrate-binding domain engage in hydrogen bonding with such inhibitors. 2. We have tested this model using site-directed mutagenesis of these residues in expression plasmids transfected into the human B-lymphoblastoid cell line, AHH-1 TK+/-. 3. Consistent with the molecular model's predicted placement in the active site, amino acid substitutions at the predicted residues abolished CYP1A2 enzymatic activity. 4. Transfected cell lines contained equal amounts of immunoreactive CYP1A2. 5. Our results support the molecular model's prediction of the critical amino acid residues present in the hydrophobic active site, residues that can hydrogen bond with CYP1A2 inhibitors and modify substrate binding and/or turnover.