Benzene dynamics and biodegradation in alluvial aquifers affected by river fluctuations.

The spatial distribution and temporal dynamics of a benzene plume in an alluvial aquifer strongly affected by river fluctuations was studied. Benzene concentrations, aquifer geochemistry datasets, past river morphology, and benzene degradation rates estimated in situ using stable carbon isotope enrichment were analyzed in concert with aquifer heterogeneity and river fluctuations. Geochemistry data demonstrated that benzene biodegradation was on-going under sulfate reducing conditions. Long-term monitoring of hydraulic heads and characterization of the alluvial aquifer formed the basis of a detailed modeled image of aquifer heterogeneity. Hydraulic conductivity was found to strongly correlate with benzene degradation, indicating that low hydraulic conductivity areas are capable of sustaining benzene anaerobic biodegradation provided the electron acceptor (SO4 (2-) ) does not become rate limiting. Modeling results demonstrated that the groundwater flux direction is reversed on annual basis when the river level rises up to 2 m, thereby forcing the infiltration of oxygenated surface water into the aquifer. The mobilization state of metal trace elements such as Zn, Cd, and As present in the aquifer predominantly depended on the strong potential gradient within the plume. However, infiltration of oxygenated water was found to trigger a change from strongly reducing to oxic conditions near the river, causing mobilization of previously immobile metal species and vice versa. MNA appears to be an appropriate remediation strategy in this type of dynamic environment provided that aquifer characterization and targeted monitoring of redox conditions are adequate and electron acceptors remain available until concentrations of toxic compounds reduce to acceptable levels.

Evidence for in situ degradation of mono-and polyaromatic hydrocarbons in alluvial sediments based on microcosm experiments with 13C-labeled contaminants.

A microcosm study was conducted to investigate the degradation of mono- and polyaromatic hydrocarbons under in situ-like conditions using alluvial sediments from the site of a former cokery. Benzene, naphthalene, or acenaphthene were added to the sediments as (13)C-labeled substrates. Based on the evolution of (13)C-CO(2) determined by gas chromatography isotope-ratio mass spectrometry (GC-IRMS) it was possible to prove mineralization of the compound of interest in the presence of other unknown organic substances of the sediment material. This new approach was suitable to give evidence for the intrinsic biodegradation of benzene, naphthalene, and acenaphthene under oxic and also under anoxic conditions, due to the high sensitivity and reproducibility of (13)C/(12)C stable isotope analysis. This semi-quantitative method can be used to screen for biodegradation of any slowly degrading, strongly sorbing compound in long-term experiments.

Deposition, persistence and turnover of pollutants: First results from the EU project AquaTerra for selected river basins and aquifers.

Deposition, turnover and movement of persistent organic pollutants (POP) were investigated in the EU integrated project "AquaTerra", which is among the first funded environmental projects within the 6th Framework Program by the European Commission. Project work integrates across various disciplines that range from biogeochemistry, environmental engineering, computer modelling and chemistry to socio-economic sciences. Field study areas are the river basins of the Ebro, the Meuse, the Elbe and the Danube as well as the 3-km(2) French catchment of the Brévilles Spring. Within the first 2 years of the project more than 1700 samples of atmospherically deposited particles, sediments, and water have been collected in the above-mentioned systems. Results show clear spatial patterns of deposition of polyaromatic hydrocarbons (PAHs) with the highest rates in the Meuse Basin. For local inputs, in the Brévilles sandy aquifer, the contamination of the groundwater by the pesticides atrazine (AT) and deethylatrazine did not decrease even 5 years after their agricultural inputs were stopped. On the other hand, herbicides such as mecroprop (MCPP), and PAHs, were at least partially degraded microbiologically in laboratory studies with soils and aquifer material from selected sites. For sediment transport of contaminants, new flood sampling techniques revealed highest deposition rates of beta-hexachlorocyclohexane (beta-HCH) in river sediments at hotspot areas on the Mulde River in the Bitterfeld region (Elbe Basin, Germany). These selected preliminary results of AquaTerra help to improve fundamental understanding of persistent organic pollutants (POP) in the environment.

Stable hydrogen and carbon isotope fractionation during microbial toluene degradation: mechanistic and environmental aspects.

Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d(8) and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d(8) and nondeuterated toluene. The D/H isotope fractionation varied slightly for sulfate-reducing strain TRM1 (slope of curve [b] = -1.219), Desulfobacterium cetonicum (b = -1.196), Thauera aromatica (b = -0.816), and Geobacter metallireducens (b = -1.004) and was greater for the aerobic bacterium Pseudomonas putida mt-2 (b = -2.667). The D/H isotope fractionation was 3 orders of magnitude greater than the (13)C/(12)C carbon isotope fractionation reported previously. Hydrogen isotope fractionation with nonlabeled toluene was 1.7 and 6 times less than isotope fractionation with per-deuterated toluene-d(8) and nonlabeled toluene for sulfate-reducing strain TRM1 (b = -0.728) and D. cetonicum (b = -0.198), respectively. Carbon and hydrogen isotope fractionation during toluene degradation by D. cetonicum remained constant over a growth temperature range of 15 to 37 degrees C but varied slightly during degradation by P. putida mt-2, which showed maximum hydrogen isotope fractionation at 20 degrees C (b = -4.086) and minimum fractionation at 35 degrees C (b = -2.138). D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecule which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study. Use of ring-labeled toluene-d(5) in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in cell extracts of D. cetonicum with an initial activity of 3.63 mU (mg of protein)(-1). The D/H isotope fractionation (b = -1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.

The use of a solid adsorber resin for enrichment of bacteria with toxic substrates and to identify metabolites: degradation of naphthalene, O-, and m-xylene by sulfate-reducing bacteria.

Anaerobic sulfate-reducing bacteria were enriched from contaminated aquifer samples with naphthalene, o-, and m-xylene as sole carbon and energy source in the presence of Amberlite-XAD7, a solid adsorber resin. XAD7 served as a substrate reservoir maintaining a constantly low substrate concentration in the culture medium. In equilibration experiments with XAD7, the aromatic hydrocarbons needed up to 5 days to achieve equilibrium between the water and the XAD7 phase. The equilibrium concentration was directly correlated with the amount of added substrate and XAD7. In the enrichments presented here, XAD7 and aromatic hydrocarbons were adjusted to maintain substrate concentrations of 100 microM m-, or o-xylene, or 50 microM naphthalene. After five subsequent transfers, the three cultures were able to grow with higher substrate concentrations in the absence of XAD7 although they grew best with lower hydrocarbon concentrations. Two new xylene-degrading cultures were obtained that could not utilise toluene as carbon source. O-xylene was degraded anaerobically by a culture, which could also oxidise m-xylene but not p-xylene. Eighty-three percent of the electrons from o-xylene oxidation were recovered in the produced sulfide, indicating a complete oxidation to CO2. Another sulfate-reducing enrichment culture oxidised m-xylene completely to CO2 but not o-, or p-xylene. A naphthalene-degrading sulfate-reducing enrichment culture oxidised naphthalene completely to CO2. Metabolites of naphthalene degradation were recovered from the XAD7 phase and subjected to GC/MS analysis. Besides the metabolites 2-naphthoic acid and decahydro-2-naphthoic acid which were identified by the mass spectrum and coelution with chemically synthesised reference compounds, the reduced 2-naphthoic acid derivatives 5,6,7,8-tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. Cultivation of bacterial cultures in the presence of XAD7 and subsequent derivatisation and extraction of metabolites directly from the solid XAD7 resin provides a new method for the isolation of sensitive bacteria and identification of metabolites.

13C/12C isotope fractionation of aromatic hydrocarbons during microbial degradation.

The influence of microbial degradation on the 13C/12C isotope composition of aromatic hydrocarbons is presented using toluene as a model compound. Four different toluene-degrading bacterial strains grown in batch culture with oxygen, nitrate, ferric iron or sulphate as electron acceptors were studied as representatives of different environmental redox conditions potentially prevailing in contaminated aquifers. The biological degradation induced isotope shifts in the residual, non-degraded toluene fraction and the kinetic isotope fractionation factors alphaC for toluene degradation by Pseudomonas putida (1.0026 +/- 0.00017), Thauera aromatica (1.0017 +/- 0.00015), Geobacter metallireducens (1.0018 +/- 0.00029) and the sulphate-reducing strain TRM1 (1.0017 +/- 0.00016) were in the same range for all four species, although they use at least two different degradation pathways. A similar 13C/12C isotope fractionation factor (alphaC = 1.0015 +/- 0.00015) was observed in situ in a non-sterile soil column in which toluene was degraded under sulphate-reducing conditions. No carbon isotope shifts resulting from soil-hydrocarbon interactions were observed in a non-degrading soil column control with aquifer material under the same conditions. The results imply that microbial degradation of toluene can produce a 13C/12C isotope fractionation in the residual hydrocarbon fraction under different environmental conditions.