At a field site in the industrial area of Vilvoorde, Belgium, we investigated the capacity of the indigenous microbial community of a eutrophic river sediment to biodegrade chlorinated aliphatic hydrocarbons (CAHs) originating from discharging, polluted groundwater using a compound-specific isotope approach. We specifically targeted the site's major pollutants cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC). Analysis of Rayleigh correlation plots enabled us to assess the extent to which microbial and abiotic natural attenuation processes contributed to the mitigation of a pollution of the surface water due to discharging CAH-contaminated groundwater. Our results provide evidence for (i) the occurrence of biodegradation of cis-DCE and VC by reductive dechlorination in parts of the aquifer and at several positions in the river sediment, (ii) the presence of river sediment zones exhibiting attenuation of chloroethenes by a combination of biodegradation and dilution through unpolluted water, (iii) the existence of zones in the river sediment lacking significant biodegradation, and thus (iv) a pronounced spatial heterogeneity in the occurrence and extent of biodegradation in the aquifer and river sediment. We conclude that at many investigated positions in the river sediment, the indigenous microbial community failed to facilitate complete biodegradation of the groundwater-sourced chloroethenes. The overall intrinsic bioremediation capacity of the river sediment was thus not high enough to completely prevent the release of these pollutants into the surface water. These findings and conclusions are thus in agreement with those of our companion paper (1), which investigated the river sediments at the Vilvoorde study site by a combination of stable hydrogen and oxygen isotope analysis of water and the detection of chlorinated aliphatic hydrocarbons (CAHs) and their dechlorination products.

Polluted groundwater in urban and industrial areas often represents a continuous source of diffuse contamination of surface waters. However, the fate of the infiltrating groundwater pollutants might be influenced by the sediment ill eutropbic water bodies since they might possess characteristic biological and physicochemical properties influencing pollutant degradation. In a part of the eutrophic river Zenne (Vilvoorde, Belgium) we studied the intrinsic capacity of river sediment microbial communities to degrade chlorinated aliphatic hydrocarbons (CAHs). The CAH concentrations deten-nined in groundwater sampled at screenpoints and using piston sampler equipment indicate that the Zenne locally drains groundwater polluted with vinyl chloride (VC) and cis-1,2-dichloroethene (cis-DCE). To further monitor the CAH influx zone into the Zenne, Teflon pore water samplers were installed at certain positions and at a depth of 20, 60, and 120 cm in the sediments of the Zenne. The CAH concentrations in the interstitial water in the sediments will in the future be compared with CAH concentrations in groundwater sampled from monitoring wells positioned 5 and 20 rn away from the Zenne. Anaerobic microcosm tests with sediment material from the Zenne showed a rapid microbial dehalogenation of both VC and cisDCE to nontoxic ethene (1 mg/L in 19 days). In congruence, chloroethene degrading Dehalococcoides sp. were present in the sediment as shown by detection of their 16S rRNA genes. Furthermore, the VC reductive dehalogenases, vcrA and bvcA, from respectively Dehalococcoides sp. strain VS and BAV1 were detected in the river sediment by polymerase chain reaction (PCR) analysis. Both Dehalococcoides sp. can grow on VC and cis-DCE as a sole energy source and are probably responsible for the observed degradation of these groundwater pollutants in the microcosms and in situ in the riverbed. Our results indicate that the interface between groundwater and surface water harbors a unique microbial community structure that changes with depth and that is capable of degrading CAHs. However, at specific locations in the studied test area with high groundwater influx rates, VC and cis-DCE seem to reach the surface water and discharge into the river. At these locations, the observed microbial degradation potential of the river sediment apparently is inadequate to prevent the CAHs from reaching the surface water. Future research will therefore focus on how to stimulate the degradation of the CAHs, both in the river sediment and in the aquifer material upstream of the river Zenne.

Various approaches to clean contaminated aquatic environments have been proposed. In recent years, natural attenuation has received increasing attention and it is generally accepted that microorganisms are the principal mediators of the natural attenuation of many pollutants. However, the complexity of environmental systems such as sediments requires a multifaceted approach to understand microbial processes and their potential. This is even more so under in situ conditions, where the activity of pollutant degrading microorganisms is generally slow, partial and constrained spatially and/or temporally. Recent developments in molecular biology and genomics are offering tools to explore microbial processes at a level that encompasses the genetic characteristics of the local microbial players, culturable or not, as well as their organization into complex communities and their interactions both with each other and with the target chemicals. It is now possible to study microbes directly in their environments at the population level as well as at the single cell level and to link biology to geochemistry. Integrative knowledge from culture independent studies based on functional characters and assessment of the diversity and quantity of catabolic genes in response to pollution, will allow a deeper understanding of and a rational intervention in environmental processes. Moreover, the use of genomic libraries to retrieve genes from natural bacterial communities without cultivation will allow a breakthrough in accessing new microbial capabilities. In this chapter, the main features, advantages and limitations of these innovative approaches to the biomonitoring and analysis of intrinsic bioremediation potential of polluted environments and sediments are critically reviewed. Then, the potential of the same strategies in the integrated chemical, physical and biological monitoring and characterization of polluted sediments subjected to natural decontamination is shown through the description of the main results of case studies performed on a) polychlorinated biphenyl (PCB)-contaminated marine sediments of the Porto Marghera area of Venice Lagoon (Italy) in which the occurrence of PCB-reductive dechlorination processes has been demonstrated for the first time in the literature, b) sediments contaminated by chlorinated aliphatic hydrocarbons (CAHs) collected from different positions of the eutrophic river Zenne (Vilvoorde, Belgium), where they have been found to act as a natural biobarrier for the CAHs occurring in the groundwater that is passing through the sediment zone, hereby reducing the risk of surface water contamination, and c) other environmental contaminated systems subjected to ex-situ and in situ active bioremediation, where these processes are described on the basis of the experience accumulated in pilot and real-life systems.

Various approaches to clean contaminated aquatic environments have been proposed. In recent years, natural attenuation has received increasing attention and it is generally accepted that microorganisms are the principal mediators of the natural attenuation of many pollutants. However, the complexity of environmental systems such as sediments requires a multifaceted approach to understand microbial processes and their potential. This is even more so under in situ conditions, where the activity of pollutant degrading microorganisms is generally slow, partial and constrained spatially and/or temporally. Recent developments in molecular biology and genomics are offering tools to explore microbial processes at a level that encompasses the genetic characteristics of the local microbial players, culturable or not, as well as their organization into complex communities and their interactions both with each other and with the target chemicals. It is now possible to study microbes directly in their environments at the population level as well as at the single cell level and to link biology to geochemistry. Integrative knowledge from culture independent studies based on functional characters and assessment of the diversity and quantity of catabolic genes in response to pollution, will allow a deeper understanding of and a rational intervention in environmental processes. Moreover, the use of genomic libraries to retrieve genes from natural bacterial communities without cultivation will allow a breakthrough in accessing new microbial capabilities. In this chapter, the main features, advantages and limitations of these innovative approaches to the biomonitoring and analysis of intrinsic bioremediation potential of polluted environments and sediments are critically reviewed. Then, the potential of the same strategies in the integrated chemical, physical and biological monitoring and characterization of polluted sediments subjected to natural decontamination is shown through the description of the main results of case studies performed on a) polychlorinated biphenyl (PCB)-contaminated marine sediments of the Porto Marghera area of Venice Lagoon (Italy) in which the occurrence of PCB-reductive dechlorination processes has been demonstrated for the first time in the literature, b) sediments contaminated by chlorinated aliphatic hydrocarbons (CAHs) collected from different positions of the eutrophic river Zenne (Vilvoorde, Belgium), where they have been found to act as a natural biobarrier for the CAHs occurring in the groundwater that is passing through the sediment zone, hereby reducing the risk of surface water contamination, and c) other environmental contaminated systems subjected to ex-situ and in situ active bioremediation, where these processes are described on the basis of the experience accumulated in pilot and real-life systems.

The pathway of 2-chloroethanol degradation in the denitrifying strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the mu(max) was 0.31 h(-1). NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.

Soil column experiments were performed to investigate the transformation of chlorinated hydrocarbons in the presence of different electron acceptors or electron donors including NaNO3, Fe(OH)(3), MnO2, or a mixture of volatile fatty acids (VFAs), respectively. The results indicated that 1,2-dichloroethane (1,2-DCA) and vinyl chloride (VC) could be oxidized anaerobically under manganese reducing and denitrifying conditions. Profile measurements in the MnO2-amended column revealed half-lives of 1.3 h for 1,2-DCA, and 4.7 h for VC degradation. Under iron reduction, only 1,2-DCA was degraded. In a control column, without the addition of donors and acceptors, not only 1,2-DCA and VC, but also chlorobenzene (MCB) was removed. In the column supplied with VFAs, sequential reductive dechlorination of tetrachloroethene (PCE) to ethene was observed, and profile measurements revealed that half-lives of PCE, trichloroethene, cis-1,2-dichloroethene and VC were 0.8 h, 1.3 h, 1.6 h and 3.4 h, respectively.

A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis-dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a 'biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium.

Batch and soil column experiments were performed to investigate the potential of anaerobic oxidation processes for natural and stimulated degradation of chlorinated and non-chlorinated hydrocarbons. Model pollutants which included 1,2-dichloroethane (DCA), 1,1,1-trichloroethane (TCA), vinylchloride (VC), cis-1,2-dichloroethene (CIS), trans-1,2-dichloroethene (TRANS), trichloroethene (TCE), monochlororbenzene (MCB), 2-chloroethanol (CE) and octane were supplied as electron donor under iron (III), manganese (IV) or nitrate reducing conditions. Evidence was obtained that DCA, VC, MCB, CE and octane can be oxidised under anoxic conditions. Particularly nitrate-reducing conditions may have great potential for natural or stimulated remediation of sites contaminated with these particular hydrocarbons.