Key factors controlling microbial distribution on a DNAPL source area.

Chlorinated solvents are among the common groundwater contaminants that show high complexity in their distribution in the subsoil. Microorganisms play a vital role in the natural attenuation of chlorinated solvents. Thus far, how the in situ soil microbial community responds to chlorinated solvent contamination has remained unclear. In this study, the microbial community distribution within two boreholes located in the source area of perchloroethene (PCE) was investigated via terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis. Microbial data were related to the lithological and geochemical data and the concentration and isotopic composition of chloroethenes to determine the key factors controlling the distribution of the microbial communities. The results indicated that Proteobacteria, Actinobacteria, and Firmicutes were the most abundant phylums in the sediment. The statistical correlation with the environmental data proved that fine granulometry, oxygen tolerance, terminal electron-acceptor processes, and toxicity control microbial structure. This study improves our understanding of how the microbial community in the subsoil responds to high concentrations of chlorinated solvents.

The role of ecotones in the dehalogenation of chloroethenes in alluvial fan aquifers.

The presence of ecotones in transition zones between geological strata (e.g. layers of gravel and sand interbedded with layers of silt in distal alluvial fan deposits) in aquifers plays a significant role in regulating the flux of matter and energy between compartments. Ecotones are characterised by steep physicochemical and biological gradients and considerable biological diversity. However, the link between organic pollutants and degradation potential in ecotones has scarcely been studied. The aim of this study is to relate the presence of ecotones with the dehalogenation of chloroethenes. A field site was selected where chloroethene contamination occurs in a granular aquifer with geological heterogeneities. The site is monitored by multilevel and conventional wells. Groundwater samples were analysed by chemical, isotopic, and molecular techniques. The main results were as follows: (1) two ecotones were characterised in the source area, one in the upper part of the aquifer and the second in the transition zone to the bottom aquitard, where the aged pool is located; (2) the ecotone located in the transition zone to the bottom aquitard has greater microbial diversity, due to higher geological heterogeneities; (3) both ecotones show the reductive dehalogenation of perchloroethylene and trichloroethylene; and (4) these ecotones are the main zones of the reductive dehalogenation of the pollutants, given the more reductive conditions at the centre of the plume. These findings suggest that ecotones are responsible for natural attenuation, where oxic conditions prevailed at the aquifer and bioremediation strategies could be applied more effectively in these zones to promote complete reductive dehalogenation.

Anaerobic biotransformation of hexachlorocyclohexane isomers by Dehalococcoides species and an enrichment culture.

The biotransformation of hexachlorocyclohexane isomers (HCH) by two Dehalococcoides mccartyi strains (195 and BTF08) and an enrichment culture was investigated and compared to conversion by the obligate anaerobic strain Clostridium pasteurianum strain DSMZ 525. The D. mccartyi strains preferentially transformed γ-HCH over α-HCH and δ-HCH isomers while ß-HCH biotransformation was not significant. In case of the enrichment culture, γ-HCH was preferentially transformed over the δ-HCH, ß-HCH and α-HCH isomers. Major observed metabolites in both cases were tetrachlorocyclohexene and as end products monochlorobenzene (MCB) and benzene. Dechlorination of the γ-HCH isomer was linked to an increase in cell numbers for strain 195. γ-HCH transformation was linked to considerable carbon stable isotope fractionation with the enrichment factor εc = - 5.5 ± 0.8‰ for D. mccartyi strain 195, εc = - 3.1 ± 0.4‰ for the enrichment culture and εc = - 4.1 ± 0.6‰ for co-metabolic transformation by C. pasteurianum.

Evaluation of carbon isotope fractionation during anaerobic reductive dehalogenation of chlorinated and brominated benzenes.

Compound specific stable isotope analysis (CSIA) has been established as a useful tool to evaluate in situ biodegradation. Here, CSIA was used to determine microbial dehalogenation of chloro- and bromobenzenes in microcosms derived from Hackensack River sediments. Gas chromatography-isotope ratio mass spectrometry (GC-IRMS) was used to measure carbon isotope fractionation during reductive dehalogenation of hexachlorobenzene (HCB), pentachlorobenzene (PeCB), 1,2,3,5-tetrachlorobenzene (TeCB), 1,2,3,5-tetrabromobenzene (TeBB), and 1,3,5-tribromobenzene (TriBB). Strong evidence of isotope fractionation coupled to dehalogenation was not observed in the substrate, possibly due to the low solubilities of the highly halogenated benzene substrates and a dilution of the isotope signal. Nonetheless, we could measure a depletion of the δ13C value in the dichlorobenzene product during dechlorination of HCB, the sequential depletion and enrichment of δ13C value for trichlorobenzene in TeCB dechlorinating cultures, and the enrichment of δ13C during debromination of TriBB. This indicates that a measurable isotope fractionation occurred during reductive dehalogenation of highly halogenated chloro- and bromobenzenes in aquatic sediments. Thus, although more quantitative measurements will be needed, the data suggests that CSIA may have application for monitoring in situ microbial reductive dehalogenation of highly halogenated benzenes.

Variable dual carbon-bromine stable isotope fractionation during enzyme-catalyzed reductive dehalogenation of brominated ethenes.

The potential of compound-specific stable isotope analysis (CSIA) to characterize biotransformation of brominated organic compounds (BOCs) was assessed and compared to chlorinated analogues. Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S catalyzed the dehalogenation of tribromoethene (TBE) to either vinyl bromide (VB) or ethene, respectively. Significantly lower isotope fractionation was observed for TBE dehalogenation by S. multivorans (εC = -1.3 ± 0.2‰) compared to D. hafniense (εC = -7.7 ± 1.5‰). However, higher fractionation was observed for dibromoethene (DBE) dehalogenation by S. multivorans (εC = -16.8 ± 1.8‰ and -21.2 ± 1.6‰ for trans- and cis-1,2- (DBE) respectively), compared to D. hafniense PCE-S (εC = -9.5 ± 1.2‰ and -14.5 ± 0.7‰ for trans-1,2-DBE and cis-1,2-DBE, respectively). Significant, but similar, bromine fractionation was observed for for S. multivorans (εBr = -0.53 ± 0.15‰, -1.03 ± 0.26‰, and -1.18 ± 0.13‰ for trans-1,2-DBE, cis-1,2-DBE and TBE, respectively) and D. hafniense PCE-S (εBr = -0.97 ± 0.28‰, -1.16 ± 0.36‰, and -1.34 ± 0.32‰ for cis-1,2-DBE, TBE and trans-1,2-DBE, respectively). Variable CBr dual-element slopes were estimated at Λ (εC/εBr) = 1.03 ± 0.2, 17.9 ± 5.8, and 29.9 ± 11.0 for S. multivorans debrominating TBE, cis-1,2-DBE and trans-1,2-DBE, respectively, and at 7.14 ± 1.6, 8.27 ± 3.7, and 8.92 ± 2.4 for D. hafniense PCE-S debrominating trans-1,2-DBE, TBE and cis-1,2-DBE, respectively. A high variability in isotope fractionation, which was substrate property related, was observed for S. multivorans but not D. hafniense, similar as observed for chlorinated ethenes, and may be due to rate-limiting steps preceding the bond-cleavage or differences in the reaction mechanism. Overall, significant isotope fractionation was observed and, therefore, CSIA can be applied to monitor the fate of brominated ethenes in the environment. Isotope effects differences, however, are not systematically comparable to chlorinated ethenes.

Dual Carbon-Bromine Stable Isotope Analysis Allows Distinguishing Transformation Pathways of Ethylene Dibromide.

The present study investigated dual carbon-bromine isotope fractionation of the common groundwater contaminant ethylene dibromide (EDB) during chemical and biological transformations, including aerobic and anaerobic biodegradation, alkaline hydrolysis, Fenton-like degradation, debromination by Zn(0) and reduced corrinoids. Significantly different correlation of carbon and bromine isotope fractionation (ΛC/Br) was observed not only for the processes following different transformation pathways, but also for abiotic and biotic processes with, the presumed, same formal chemical degradation mechanism. The studied processes resulted in a wide range of ΛC/Br values: ΛC/Br = 30.1 was observed for hydrolysis of EDB in alkaline solution; ΛC/Br between 4.2 and 5.3 were determined for dibromoelimination pathway with reduced corrinoids and Zn(0) particles; EDB biodegradation by Ancylobacter aquaticus and Sulfurospirillum multivorans resulted in ΛC/Br = 10.7 and 2.4, respectively; Fenton-like degradation resulted in carbon isotope fractionation only, leading to ΛC/Br ∞. Calculated carbon apparent kinetic isotope effects ((13)C-AKIE) fell with 1.005 to 1.035 within expected ranges according to the theoretical KIE, however, biotic transformations resulted in weaker carbon isotope effects than respective abiotic transformations. Relatively large bromine isotope effects with (81)Br-AKIE of 1.0012-1.002 and 1.0021-1.004 were observed for nucleophilic substitution and dibromoelimination, respectively, and reveal so far underestimated strong bromine isotope effects.

Reductive dechlorination in recalcitrant sources of chloroethenes in the transition zone between aquifers and aquitards.

In the transition zone between aquifers and basal aquitards, the perchloroethene pools at an early time in their evolution are more recalcitrant than those elsewhere in the aquifer. The aim of this study is to demonstrate that the biodegradation of chloroethenes from aged pools (i.e., pools after decades of continuous groundwater flushing and dissolution) of perchloroethene is favored in the transition zone. A field site was selected where an aged pool exists at the bottom of a transition zone. Two boreholes were drilled to obtain sediment and groundwater samples to perform chemical, isotopic, molecular, and clone library analyses and microcosm experiments. The main results were as follows: (i) the transition zone is characterized by a high microbial richness; (ii) reductively dechlorinating microorganisms are present and partial reductive dechlorination coexists with denitrification, Fe and Mn reduction, and sulfate reduction; (iii) reductively dechlorinating microorganisms were also present in the zone of the aged pool; (v) the high concentrations of perchloroethene in this zone resulted in a decrease in microbial richness; (vi) however, the presence of fermenting microorganisms supplying electrons for the reductively dechlorinating microorganisms prevented the reductive dechlorination to be inhibited. These findings suggest that biostimulation and/or bioaugmentation could be applied to promote complete reductive dechlorination and to enhance the dissolution of more nonaqueous phase liquids (DNAPL).

Functional Gene Markers for Fumarate-Adding and Dearomatizing Key Enzymes in Anaerobic Aromatic Hydrocarbon Degradation in Terrestrial Environments.

Anaerobic degradation is a key process in many environments either naturally or anthropogenically exposed to petroleum hydrocarbons. Considerable advances into the biochemistry and physiology of selected anaerobic degraders have been achieved over the last decades, especially for the degradation of aromatic hydrocarbons. However, researchers have only recently begun to explore the ecology of complex anaerobic hydrocarbon degrader communities directly in their natural habitats, as well as in complex laboratory systems using tools of molecular biology. These approaches have mainly been facilitated by the establishment of a suite of targeted marker gene assays, allowing for rapid and directed insights into the diversity as well as the identity of intrinsic degrader populations and degradation potentials established at hydrocarbon-impacted sites. These are based on genes encoding either peripheral or central key enzymes in aromatic compound breakdown, such as fumarate-adding benzylsuccinate synthases or dearomatizing aryl-coenzyme A reductases, or on aromatic ring-cleaving hydrolases. Here, we review recent advances in this field, explain the different detection methodologies applied, and discuss how the detection of site-specific catabolic gene markers has improved the understanding of processes at contaminated sites. Functional marker gene-based strategies may be vital for the development of a more elaborate population-based assessment and prediction of aromatic degradation potentials in hydrocarbon-impacted environments.

Anaerobic microbial dehalogenation of organohalides-state of the art and remediation strategies.

Contamination and remediation of groundwater with halogenated organics and understanding of involved microbial reactions still poses a challenge. Over the last years, research in anaerobic microbial dehalogenation has advanced in many aspects providing information about the reaction, physiology of microorganisms as well as approaches to investigate the activity of microorganisms in situ. Recently published crystal structures of reductive dehalogenases (Rdh), heterologous expression systems and advanced analytical, proteomic and stable isotope approaches allow addressing the overall reaction and specific enzymes as well as co-factors involved during anaerobic microbial dehalogenation. In addition to Dehalococcoides spp., Dehalobacter and Dehalogenimonas strains have been recognized as important and versatile organohalide respirers. Together, these provide perspectives for integrated concepts allowing to improve and monitor in situ biodegradation.

Evidence for benzylsuccinate synthase subtypes obtained by using stable isotope tools.

We studied the benzylsuccinate synthase (Bss) reaction mechanism with respect to the hydrogen-carbon bond cleavage at the methyl group of toluene by using different stable isotope tools. Λ values (slopes of linear regression curves for carbon and hydrogen discrimination) for two-dimensional compound-specific stable isotope analysis (2D-CSIA) of toluene activation by Bss-containing cell extracts (in vitro studies) were found to be similar to previously reported data from analogous experiments with whole cells (in vivo studies), proving that Λ values generated by whole cells are caused by Bss catalysis. The Bss enzymes of facultative anaerobic bacteria produced smaller Λ values than those of obligate anaerobes. In addition, a partial exchange of a single deuterium atom in benzylsuccinate with hydrogen was observed in experiments with deuterium-labeled toluene. In this study, the Bss enzymes of the tested facultative anaerobes showed 3- to 8-fold higher exchange probabilities than those for the enzymes of the tested obligate anaerobic bacteria. The phylogeny of the Bss variants, determined by sequence analyses of BssA, the gene product corresponding to the α subunit of Bss, correlated with the observed differences in Λ values and hydrogen exchange probabilities. In conclusion, our results suggest subtle differences in the reaction mechanisms of Bss isoenzymes of facultative and obligate anaerobes and show that the putative isoenzymes can be differentiated by 2D-CSIA.

Enzymes involved in the anaerobic degradation of meta-substituted halobenzoates.

Organohalides are environmentally relevant compounds that can be degraded by aerobic and anaerobic microorganisms. The denitrifying Thauera chlorobenzoica is capable of degrading halobenzoates as sole carbon and energy source under anaerobic conditions. LC-MS/MS-based coenzyme A (CoA) thioester analysis revealed that 3-chloro- or 3-bromobenzoate were preferentially metabolized via non-halogenated CoA-ester intermediates of the benzoyl-CoA degradation pathway. In contrast, 3-fluorobenzoate, which does not support growth, was converted to dearomatized fluorinated CoA ester dead-end products. Extracts from cells grown on 3-chloro-/3-bromobenzoate catalysed the Ti(III)-citrate- and ATP-dependent reductive dehalogenation of 3-chloro/3-bromobenzoyl-CoA to benzoyl-CoA, whereas 3-fluorobenzoyl-CoA was converted to a fluorinated cyclic dienoyl-CoA compound. The reductive dehalogenation reactions were identified as previously unknown activities of ATP-dependent class I benzoyl-CoA reductases (BCR) present in all facultatively anaerobic, aromatic compound degrading bacteria. A two-step dearomatization/H-halide elimination mechanism is proposed. A halobenzoate-specific carboxylic acid CoA ligase was characterized in T. chlorobenzoica; however, no such enzyme is present in Thauera aromatica, which cannot grow on halobenzoates. In conclusion, it appears that the presence of a halobenzoate-specific carboxylic acid CoA ligase rather than a specific reductive dehalogenase governs whether an aromatic compound degrading anaerobe is capable of metabolizing halobenzoates.

Combined application of PCR-based functional assays for the detection of aromatic-compound-degrading anaerobes.

To explore the reliability of assays that detect aromatic-compound-degrading anaerobes, a combination of three functional-gene-targeting assays was applied to microcosms from benzene-contaminated aquifers. Results of the assays were consistent and suggest that species related to the genera Azoarcus and Geobacter dominated benzene degradation at the individual sites.

Occurrence, genes and expression of the W/Se-containing class II benzoyl-coenzyme A reductases in anaerobic bacteria.

Benzoyl-coenzyme A (CoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds and catalyse the reductive dearomatization of benzoyl-CoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA. Class I BCRs are ATP-dependent FeS enzymes, whereas class II BCRs are supposed to be ATP-independent and contain W, FeS clusters, and most probably selenocysteine. The active site components of a putative eight subunit class II BCR, BamBCDEFGHI, were recently characterized in Geobacter metallireducens. In this organism bamB was identified as structural gene for the W-containing active site subunit; bamF was predicted to code for a selenocysteine containing electron transfer subunit. In this work the occurrence and expression of BCRs in a number of anaerobic, aromatic compound degrading model microorganisms was investigated with a focus on the BamB and BamF components. Benzoate-induced class II BCR in vitro activities were determined in the soluble protein fraction in all obligately anaerobic bacteria tested. Where applicable, the results were in agreement with Western blot analysis using BamB targeting antibodies. By establishing a specific bamB targeting PCR assay, bamB homologues were identified in all tested obligately anaerobic bacteria with the capacity to degrade aromatic compounds; a number of bamB sequences from Gram-negative/positive sulfate-reducing bacteria were newly sequenced. In several organisms at least two bamB paralogues per genome were identified; however, in nearly all cases only one of them was transcribed during growth on an aromatic substrate. These benzoate-induced bamB genes are proposed to code for the active site subunit of class II BCRs; the major part of them group into a phylogenetic subcluster within the bamB homologues. Results from in silico analysis suggested that all class II BCRs contain selenocysteine in the BamF, and in many cases also in the BamE subunit. The results obtained indicate that the distribution of the two classes of BCRs in anaerobic bacteria appears to be strictly ruled by the available free energy from the oxidation of the aromatic carbon source rather than by phylogenetic relationships.

Identification of the Geobacter metallireducens bamVW two-component system, involved in transcriptional regulation of aromatic degradation.

Regulation of aromatic degradation in obligate anaerobes was studied in the Fe(III)-respiring model organism Geobacter metallireducens GS-15. A two-component system and a sigma54-dependent promoter were identified that are both involved in the regulation of the gene coding for benzoate-coenzyme A ligase, catalyzing the initial step of benzoate degradation.

6-Oxocyclohex-1-ene-1-carbonyl-coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes.

In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m-xylene-degrading enrichment culture demonstrating its potential use in less defined bacterial communities. The gene probe established in this work provides for the first time a general tool for the detection of a central functionality in aromatic compound-degrading anaerobes.