Pilot tests for the optimization of the bioremediation strategy of a multi-layered aquifer at a multi-focus site impacted with chlorinated ethenes.

A multi-layered aquifer in an industrial area in the north of the Iberian Peninsula is severely contaminated with the chlorinated ethenes (CEs) tetrachloroethylene, trichloroethylene, cis-1,2-dichloroethylene, and vinyl chloride. Both shallow and deep aquifers are polluted, with two differentiated north and south CEs plumes. Hydrogeochemical and isotopic data (δ13C of CEs) evidenced natural attenuation of CEs. To select the optimal remediation strategy to clean-up the contamination plumes, laboratory treatability studies were performed, which confirmed the intrinsic biodegradation potential of the north and south shallow aquifers to fully dechlorinate CEs to ethene after injection of lactate, but also the combination of lactate and sulfidized mZVI as an alternative treatment for the north deep aquifer. In the lactate-amended microcosms, full dechlorination of CEs was accompanied by an increase in 16S rRNA gene copies of Dehalococcoides and Dehalogenimonas, and the tceA, vcrA and bvcA reductive dehalogenases. Three in situ pilot tests were implemented, which consisted in injections of lactate in the north and south shallow aquifers, and injections of lactate and sulfidized mZVI in the north deep aquifer. The hydrogeochemical, isotopic and molecular analyses used to monitor the pilot tests evidenced that results obtained mimicked the laboratory observations, albeit at different dechlorination rates. It is likely that the efficiency of the injections was affected by the amendment distribution. In addition, monitoring of the pilot tests in the shallow aquifers showed the release of CEs due to back diffusion from secondary sources, which limited the use of isotopic data for assessing treatment efficiency. In the pilot test that combined the injection of lactate and sulfidized mZVI, both biotic and abiotic pathways contributed to the production of ethene. This study demonstrates the usefulness of integrating different chemical, isotopic and biomolecular approaches for a more robust selection and implementation of optimal remediation strategies in CEs polluted sites.

Dual C-Cl isotope fractionation offers potential to assess biodegradation of 1,2-dichloropropane and 1,2,3-trichloropropane by Dehalogenimonas cultures.

1,2-dichloropropane (1,2-DCP) and 1,2,3-trichloropropane (1,2,3-TCP) are hazardous chemicals frequently detected in groundwater near agricultural zones due to their historical use in chlorinated fumigant formulations. In this study, we show that the organohalide-respiring bacterium Dehalogenimonas alkenigignens strain BRE15 M can grow during the dihaloelimination of 1,2-DCP and 1,2,3-TCP to propene and allyl chloride, respectively. Our work also provides the first application of dual isotope approach to investigate the anaerobic reductive dechlorination of 1,2-DCP and 1,2,3-TCP. Stable carbon and chlorine isotope fractionation values for 1,2-DCP (ƐC = -13.6 ± 1.4 ‰ and ƐCl = -27.4 ± 5.2 ‰) and 1,2,3-TCP (ƐC = -3.8 ± 0.6 ‰ and ƐCl = -0.8 ± 0.5 ‰) were obtained resulting in distinct dual isotope slopes (Λ12DCP = 0.5 ± 0.1, Λ123TCP = 4 ± 2). However direct comparison of ΛC-Cl among different substrates is not possible and investigation of the C and Cl apparent kinetic isotope effects lead to the hypothesis that concerted dichloroelimination mechanism is more likely for both compounds. In fact, whole cell activity assays using cells suspensions of the Dehalogenimonas-containing culture grown with 1,2-DCP and methyl viologen as electron donor suggest that the same set of reductive dehalogenases was involved in the transformation of 1,2-DCP and 1,2,3-TCP. This study opens the door to the application of isotope techniques for evaluating biodegradation of 1,2-DCP and 1,2,3-TCP, which often co-occur in groundwaters near agricultural fields.

Toluene-driven anaerobic biodegradation of chloroform in a continuous-flow bioelectrochemical reactor.

Subsurface co-contamination by multiple pollutants can be challenging for the design of bioremediation strategies since it may require promoting different and often antagonistic degradation pathways. Here, we investigated the simultaneous degradation of toluene and chloroform (CF) in a continuous-flow anaerobic bioelectrochemical reactor. As a result, 47 µmol L-1 d-1 of toluene and 60 µmol L-1 d-1 of CF were concurrently removed, when the anode was polarized at +0.4 V vs. Standard Hydrogen Electrode (SHE). Analysis of the microbial community structure and key functional genes allowed to identify the involved degradation pathways. Interestingly, when acetate was supplied along with toluene, to simulate the impact of a readily biodegradable substrate on process performance, toluene degradation was adversely affected, likely due to competitive inhibition effects. Overall, this study proved the efficacy of the developed bioelectrochemical system in simultaneously treating multiple groundwater contaminants, paving the way for the application in real-world scenarios.

Respiratory protein interactions in Dehalobacter sp. strain 8M revealed through genomic and native proteomic analyses.

Dehalobacter (Firmicutes) encompass obligate organohalide-respiring bacteria used for bioremediation of groundwater contaminated with halogenated organics. Various aspects of their biochemistry remain unknown, including the identities and interactions of respiratory proteins. Here, we sequenced the genome of Dehalobacter sp. strain 8M and analysed its protein expression. Strain 8M encodes 22 reductive dehalogenase homologous (RdhA) proteins. RdhA D8M_v2_40029 (TmrA) was among the two most abundant proteins during growth with trichloromethane and 1,1,2-trichloroethane. To examine interactions of respiratory proteins, we used blue native gel electrophoresis together with dehalogenation activity tests and mass spectrometry. The highest activities were found in gel slices with the highest abundance of TmrA. Protein distributions across gel lanes provided biochemical evidence that the large and small subunits of the membrane-bound [NiFe] uptake hydrogenase (HupL and HupS) interacted strongly and that HupL/S interacted weakly with RdhA. Moreover, the interaction of RdhB and membrane-bound b-type cytochrome HupC was detected. RdhC proteins, often encoded in rdh operons but without described function, migrated in a protein complex not associated with HupL/S or RdhA. This study provides the first biochemical evidence of respiratory protein interactions in Dehalobacter, discusses implications for the respiratory architecture and advances the molecular comprehension of this unique respiratory chain.

Proteogenomics of the novel Dehalobacterium formicoaceticum strain EZ94 highlights a key role of methyltransferases during anaerobic dichloromethane degradation.

Dichloromethane (DCM, methylene chloride) is a toxic, high-volume industrial pollutant of long-standing. Anaerobic biodegradation is crucial for its removal from contaminated environments, yet prevailing mechanisms remain unresolved, especially concerning dehalogenation. In this study, we obtained an assembled genome of a novel DCM-degrading strain, Dehalobacterium formicoaceticum strain EZ94, from a stable DCM-degrading consortium, and we analyzed its proteome during degradation of DCM. A gene cluster recently predicted to play a major role in anaerobic DCM catabolism (the mec cassette) was found. Methyltransferases and other proteins encoded by the mec cassette were among the most abundant proteins produced, suggesting their involvement in DCM catabolism. Reductive dehalogenases were not detected. Genes and corresponding proteins for a complete Wood-Ljungdahl pathway, which could enable further metabolism of DCM carbon, were also found. Unlike for the anaerobic DCM degrader "Ca. F. warabiya," no genes for metabolism of the quaternary amines choline and glycine betaine were identified. This work provides independent and supporting evidence that mec-associated methyltransferases are key to anaerobic DCM metabolism.

Dual C-Br Isotope Fractionation Indicates Distinct Reductive Dehalogenation Mechanisms of 1,2-Dibromoethane in Dehalococcoides- and Dehalogenimonas-Containing Cultures.

Brominated organic compounds such as 1,2-dibromoethane (1,2-DBA) are highly toxic groundwater contaminants. Multi-element compound-specific isotope analysis bears the potential to elucidate the biodegradation pathways of 1,2-DBA in the environment, which is crucial information to assess its fate in contaminated sites. This study investigates for the first time dual C-Br isotope fractionation during in vivo biodegradation of 1,2-DBA by two anaerobic enrichment cultures containing organohalide-respiring bacteria (i.e., either Dehalococcoides or Dehalogenimonas). Different εbulkC values (-1.8 ± 0.2 and -19.2 ± 3.5‰, respectively) were obtained, whereas their respective εbulkBr values were lower and similar to each other (-1.22 ± 0.08 and -1.2 ± 0.5‰), leading to distinctly different trends (ΛC-Br = Δδ13C/Δδ81Br ≈ εbulkC/εbulkBr) in a dual C-Br isotope plot (1.4 ± 0.2 and 12 ± 4, respectively). These results suggest the occurrence of different underlying reaction mechanisms during enzymatic 1,2-DBA transformation, that is, concerted dihaloelimination and nucleophilic substitution (SN2-reaction). The strongly pathway-dependent ΛC-Br values illustrate the potential of this approach to elucidate the reaction mechanism of 1,2-DBA in the field and to select appropriate εbulkC values for quantification of biodegradation. The results of this study provide valuable information for future biodegradation studies of 1,2-DBA in contaminated sites.

Combining nanoscale zero-valent iron and anaerobic dechlorinating bacteria to degrade chlorinated methanes and 1,2-dichloroethane.

Nanoscale zero-valent iron (nZVI) has the potential to degrade a diversity of chlorinated compounds, and it is widely used for remediation of contaminated groundwaters. However, some frequently detected contaminants such as dichloromethane (DCM) and 1,2-dichloroethane (1,2-DCA) have shown nearly no reactivity with nZVI. Here, we tested the feasibility of combining anaerobic dechlorinating bacteria, Dehalobacterium and Dehalogenimonas, and nZVI as a treatment train to detoxify chlorinated methanes (i.e., chloroform-CF- and DCM), and 1,2-DCA. First, we showed that CF (500 µM) was fully degraded by 1 g/L nZVI to DCM as a major by-product, which was susceptible to fermentation by Dehalobacterium to innocuous products. Our results indicate that soluble compounds released by nZVI might cause an inhibitory impact on Dehalobacterium activity, avoiding DCM depletion. The DCM dechlorination activity was recovered when transferred to a fresh medium without nZVI. The increase in H2 production and pH was discarded as potential inhibitors. Similarly, a Dehalogenimonas-containing culture was unable to dichloroeliminate 1,2-DCA when exposed to 1 g/L nZVI, but dechlorinating activity was also recovered when transferred to nZVI-free media. The recovery of the dechlorinating activity of Dehalobacterium and Dehalogenimonas suggests that combination of nZVI and bioremediation techniques can be feasible under field conditions where dilution processes can alleviate the impact of the potential inhibitory soluble compounds.

Bioelectrochemically-assisted degradation of chloroform by a co-culture of Dehalobacter and Dehalobacterium.

Using bioelectrochemical systems (BESs) to provide electrochemically generated hydrogen is a promising technology to provide electron donors for reductive dechlorination by organohalide-respiring bacteria. In this study, we inoculated two syntrophic dechlorinating cultures containing Dehalobacter and Dehalobacterium to sequentially transform chloroform (CF) to acetate in a BES using a graphite fiber brush as the electrode. In this co-culture, Dehalobacter transformed CF to stoichiometric amounts of dichloromethane (DCM) via organohalide respiration, whereas the Dehalobacterium-containing culture converted DCM to acetate via fermentation. BES were initially inoculated with Dehalobacter, and sequential cathodic potentials of -0.6, -0.7, and -0.8 V were poised after consuming three CF doses (500 µM) per each potential during a time-span of 83 days. At the end of this period, the accumulated DCM was degraded in the following seven days after the inoculation of Dehalobacterium. At this point, four consecutive amendments of CF at increasing concentrations of 200, 400, 600, and 800 µM were sequentially transformed by the combined degradation activity of Dehalobacter and Dehalobacterium. The Dehalobacter 16S rRNA gene copies increased four orders of magnitude during the whole period. The coulombic efficiencies associated with the degradation of CF reached values > 60% at a cathodic potential of -0.8 V when the degradation rate of CF achieved the highest values. This study shows the advantages of combining syntrophic bacteria to fully detoxify chlorinated compounds in BESs and further expands the use of this technology for treating water bodies impacted with pollutants.

Assessment of aerobic biodegradation of lower-chlorinated benzenes in contaminated groundwater using field-derived microcosms and compound-specific carbon isotope fractionation.

Biodegradation of lower chlorinated benzenes (tri-, di- and monochlorobenzene) was assessed at a coastal aquifer contaminated with multiple chlorinated aromatic hydrocarbons. Field-derived microcosms, established with groundwater from the source zone and amended with a mixture of lower chlorinated benzenes, evidenced biodegradation of monochlorobenzene (MCB) and 1,4-dichlorobenzene (1,4-DCB) in aerobic microcosms, whereas the addition of lactate in anaerobic microcosms did not enhance anaerobic reductive dechlorination. Aerobic microcosms established with groundwater from the plume consumed several doses of MCB and concomitantly degraded the three isomers of dichlorobenzene with no observable inhibitory effect. In the light of these results, we assessed the applicability of compound stable isotope analysis to monitor a potential aerobic remediation treatment of MCB and 1,4-DCB in this site. The carbon isotopic fractionation factors (ε) obtained from field-derived microcosms were -0.7‰ ± 0.1 ‰ and -1.0‰ ± 0.2 ‰ for MCB and 1,4-DCB, respectively. For 1,4-DCB, the carbon isotope fractionation during aerobic biodegradation was reported for the first time. The weak carbon isotope fractionation values for the aerobic pathway would only allow tracing of in situ degradation in aquifer parts with high extent of biodegradation. However, based on the carbon isotope effects measured in this and previous studies, relatively high carbon isotope shifts (i.e., ∆δ13C > 4.0 ‰) of MCB or 1,4-DCB in contaminated groundwater would suggest that their biodegradation is controlled by anaerobic reductive dechlorination.

Trichloromethane dechlorination by a novel Dehalobacter sp. strain 8M reveals a third contrasting C and Cl isotope fractionation pattern within this genus.

Trichloromethane (TCM) is a pollutant frequently detected in contaminated aquifers, and only four bacterial strains are known to respire it. Here, we obtained a novel Dehalobacter strain capable of transforming TCM to dichloromethane, which was denominated Dehalobacter sp. strain 8M. Besides TCM, strain 8M also completely transformed 1,1,2-trichloroethane to vinyl chloride and 1,2-dichloroethane. Quantitative PCR analysis for the 16S rRNA genes confirmed growth of Dehalobacter with TCM and 1,1,2-trichloroethane as electron acceptors. Carbon and chlorine isotope fractionation during TCM transformation was studied in cultured cells and in enzymatic assays with cell suspensions and crude protein extracts. TCM transformation in the three studied systems resulted in small but significant carbon (εC = -2.7 ± 0.1‰ for respiring cells, -3.1 ± 0.1‰ for cell suspensions, and - 4.1 ± 0.5‰ for crude protein extracts) and chlorine (εCl = -0.9 ± 0.1‰, -1.1 ± 0.1‰, and - 1.2 ± 0.2‰, respectively) isotope fractionation. A characteristic and consistent dual CCl isotope fractionation pattern was observed for the three systems (combined ΛC/Cl = 2.8 ± 0.3). This ΛC/Cl differed significantly from previously reported values for anaerobic dechlorination of TCM by the corrinoid cofactor vitamin B12 and other Dehalobacter strains. These findings widen our knowledge on the existence of different enzyme binding mechanisms underlying TCM-dechlorination within the genus Dehalobacter and demonstrates that dual isotope analysis could be a feasible tool to differentiate TCM degraders at field studies.

Enhanced dechlorination of 1,2-dichloropropane to propene in a bioelectrochemical system mediated by Dehalogenimonas.

Bioelectrochemical systems (BES) are promising technologies to enhance the growth of organohalide-respiring bacteria and to treat chlorinated aliphatic hydrocarbons. In this study, two carbon-based cathodic electrode materials, a graphite brush and a carbon cloth, were used as hydrogen suppliers to couple growth of Dehalogenimonas and dechlorination of 1,2-DCP to nontoxic propene in the cathode vessel. The BES with graphite brush electrode consumed ~4000 µM 1,2-DCP during 110 days and exhibited a degradation rate 5.6-fold higher than the maximum value obtained with the carbon cloth electrode, with a cathode potential set at -0.7 V. Quantitative PCR confirmed that Dehalogenimonas gene copies increased by two orders of magnitude in the graphite brush BES, with an average yield of 1.2·108±5·107 cells per µmol of 1,2-DCP degraded. The use of a pulsed voltage operation (cathode potential set at -0.6 V for 16 h and -1.1 V for 8 h) increased the coulombic efficiency and degradation of 1,2-DCP when compared with a continuous voltage operation of -1.1 V. Bacterial cell aggregates were observed in the surface of the graphite brush electrodes by electron scanning microscopy, suggesting biofilm formation. This study expands the range of chlorinated compounds degradable and organohalide-respiring bacteria capable of growing in BES.

Genome Sequence, Proteome Profile, and Identification of a Multiprotein Reductive Dehalogenase Complex in Dehalogenimonas alkenigignens Strain BRE15M.

Bacteria of the genus Dehalogenimonas respire with vicinally halogenated alkanes via dihaloelimination. We aimed to describe involved proteins and their supermolecular organization. Metagenomic sequencing of a Dehalogenimonas-containing culture resulted in a 1.65 Mbp draft genome of Dehalogenimonas alkenigignens strain BRE15M. It contained 31 full-length reductive dehalogenase homologous genes (rdhA), but only eight had cognate rdhB gene coding for membrane-anchoring proteins. Shotgun proteomics of cells grown with 1,2-dichloropropane as an electron acceptor identified 1152 proteins representing more than 60% of the total proteome. Ten RdhA proteins were detected, including a DcpA ortholog, which was the strongest expressed RdhA. Blue native gel electrophoresis (BNE) demonstrating maximum activity was localized in a protein complex of 146-242 kDa. Protein mass spectrometry revealed the presence of DcpA, its membrane-anchoring protein DcpB, two hydrogen uptake hydrogenase subunits (HupL and HupS), an iron-sulfur protein (HupX), and subunits of a redox protein with a molybdopterin-binding motif (OmeA and OmeB) in the complex. BNE after protein solubilization with different detergent concentrations revealed no evidence for an interaction between the putative respiratory electron input module (HupLS) and the OmeA/OmeB/HupX module. All detected RdhAs comigrated with the organohalide respiration complex. Based on genomic and proteomic analysis, we propose quinone-independent respiration in Dehalogenimonas.

Interspecies interaction and effect of co-contaminants in an anaerobic dichloromethane-degrading culture.

An anaerobic stable mixed culture dominated by bacteria belonging to the genera Dehalobacterium, Acetobacterium, Desulfovibrio, and Wolinella was used as a model to study the microbial interactions during DCM degradation. Physiological studies indicated that DCM was degraded in this mixed culture at least in a three-step process: i) fermentation of DCM to acetate and formate, ii) formate oxidation to CO2 and H2, and iii) H2/CO2 reductive acetogenesis. The 16S rRNA gene sequencing of cultures enriched with formate or H2 showed that Desulfovibrio was the dominant population followed by Acetobacterium, but sequences representing Dehalobacterium were only present in cultures amended with DCM. Nuclear magnetic resonance analyses confirmed that acetate produced from 13C-labelled DCM was marked at the methyl ([2-13C]acetate), carboxyl ([1-13C]acetate), and both ([1,2-13C]acetate) positions, which is in accordance to acetate formed by both direct DCM fermentation and H2/CO2 acetogenesis. The inhibitory effect of ten different co-contaminants frequently detected in groundwaters on DCM degradation was also investigated. Complete inhibition of DCM degradation was observed when chloroform, perfluorooctanesulfonic acid, and diuron were added at 838, 400, and 107 µM, respectively. However, the inhibited cultures recovered the DCM degradation capability when transferred to fresh medium without co-contaminants. Findings derived from this work are of significant relevance to provide a better understanding of the synergistic interactions among bacteria to accomplish DCM degradation as well as to predict the effect of co-contaminants during anaerobic DCM bioremediation in groundwater.

Integrative isotopic and molecular approach for the diagnosis and implementation of an efficient in-situ enhanced biological reductive dechlorination of chlorinated ethenes.

Based on the previously observed intrinsic bioremediation potential of a site originally contaminated with perchloroethene (PCE), field-derived lactate-amended microcosms were performed to test different lactate isomers and concentrations, and find clearer isotopic and molecular parameters proving the feasibility of an in-situ enhanced reductive dechlorination (ERD) from PCE-to-ethene (ETH). According to these laboratory results, which confirmed the presence of Dehalococcoides sp. and the vcrA gene, an in-situ ERD pilot test consisting of a single injection of lactate in a monitoring well was performed and monitored for 190 days. The parameters used to follow the performance of the ERD comprised the analysis of i) hydrochemistry, including redox potential (Eh), and the concentrations of redox sensitive species, chlorinated ethenes (CEs), lactate, and acetate; ii) stable isotope composition of carbon of CEs, and sulphur and oxygen of sulphate; and iii) 16S rRNA gene sequencing from groundwater samples. Thus, it was proved that the injection of lactate promoted sulphate-reducing conditions, with the subsequent decrease in Eh, which allowed for the full reductive dechlorination of PCE to ETH in the injection well. The biodegradation of CEs was also confirmed by the enrichment in 13C and carbon isotopic mass balances. The metagenomic results evidenced the shift in the composition of the microbial population towards the predominance of fermentative bacteria. Given the success of the in-situ pilot test, a full-scale ERD with lactate was then implemented at the site. After one year of treatment, PCE and trichloroethene were mostly depleted, whereas vinyl chloride (VC) and ETH were the predominant metabolites. Most importantly, the shift of the carbon isotopic mass balances towards more positive values confirmed the complete reductive dechlorination, including the VC-to-ETH reaction step. The combination of techniques used here provides complementary lines of evidence for the diagnosis of the intrinsic biodegradation potential of a polluted site, but also to monitor the progress, identify potential difficulties, and evaluate the success of ERD at the field scale.

Dual carbon - chlorine isotope fractionation during dichloroelimination of 1,1,2-trichloroethane by an enrichment culture containing Dehalogenimonas sp.

Chlorinated ethanes are frequent groundwater contaminants but compound specific isotope analysis (CSIA) has been scarcely applied to investigate their degradation pathways. In this study, dual carbon and chlorine isotope fractionation was used to investigate for the first time the anoxic biodegradation of 1,1,2-trichloroethane (1,1,2-TCA) using a Dehalogenimonas-containing culture. The isotopic fractionation values obtained for the biodegradation of 1,1,2-TCA were ɛC = -6.9 ±â€¯0.4‰ and ɛCl = -2.7 ±â€¯0.3‰. The detection of vinyl chloride (VC) as unique byproduct and a closed carbon isotopic mass balance corroborated that dichloroelimination was the degradation pathway used by this strain. Combining the values of δ13C and δ37Cl resulted in a dual element C-Cl isotope slope of Λ = 2.5 ±â€¯0.2‰. Investigation of the apparent kinetic isotope effects (AKIEs) expected for cleavage of a CCl bond showed an important masking of the intrinsic isotope fractionation. Theoretical calculation of Λ suggested that dichloroelimination of 1,1,2-TCA was taking place via simultaneous cleavage of two CCl bonds (concerted reaction mechanism). The isotope data obtained in this study can be useful to monitor natural attenuation of 1,1,2-TCA via dichloroelimination and provide insights into the source and fate of VC in contaminated groundwaters.

Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain).

The bioremediation potential of an aquifer contaminated with tetrachloroethene (PCE) was assessed by combining hydrogeochemical data of the site, microcosm studies, metabolites concentrations, compound specific-stable carbon isotope analysis and the identification of selected reductive dechlorination biomarker genes. The characterization of the site through 10 monitoring wells evidenced that leaked PCE was transformed to TCE and cis-DCE via hydrogenolysis. Carbon isotopic mass balance of chlorinated ethenes pointed to two distinct sources of contamination and discarded relevant alternate degradation pathways in the aquifer. Application of specific-genus primers targeting Dehalococcoides mccartyi species and the vinyl chloride-to-ethene reductive dehalogenase vcrA indicated the presence of autochthonous bacteria capable of the complete dechlorination of PCE. The observed cis-DCE stall was consistent with the aquifer geochemistry (positive redox potentials; presence of dissolved oxygen, nitrate, and sulphate; absence of ferrous iron), which was thermodynamically favourable to dechlorinate highly chlorinated ethenes but required lower redox potentials to evolve beyond cis-DCE to the innocuous end product ethene. Accordingly, the addition of lactate or a mixture of ethanol plus methanol as electron donor sources in parallel field-derived anoxic microcosms accelerated dechlorination of PCE and passed cis-DCE up to ethene, unlike the controls (without amendments, representative of field natural attenuation). Lactate fermentation produced acetate at near-stoichiometric amounts. The array of techniques used in this study provided complementary lines of evidence to suggest that enhanced anaerobic bioremediation using lactate as electron donor source is a feasible strategy to successfully decontaminate this site.

Hydrogen Isotope Fractionation during the Biodegradation of 1,2-Dichloroethane: Potential for Pathway Identification Using a Multi-element (C, Cl, and H) Isotope Approach.

Even though multi-element isotope fractionation patterns provide crucial information with which to identify contaminant degradation pathways in the field, those involving hydrogen are still lacking for many halogenated groundwater contaminants and degradation pathways. This study investigates for the first time hydrogen isotope fractionation during both aerobic and anaerobic biodegradation of 1,2-dichloroethane (1,2-DCA) using five microbial cultures. Transformation-associated isotope fractionation values (εbulkH) were -115 ± 18‰ (aerobic C-H bond oxidation), -34 ± 4‰ and -38 ± 4‰ (aerobic C-Cl bond cleavage via hydrolytic dehalogenation), and -57 ± 3‰ and -77 ± 9‰ (anaerobic C-Cl bond cleavage via reductive dihaloelimination). The dual-element C-H isotope approach (ΛC-H = Δδ2H/Δδ13C ≈ εbulkH/εbulkC, where Δδ2H and Δδ13C are changes in isotope ratios during degradation) resulted in clearly different ΛC-H values: 28 ± 4 (oxidation), 0.7 ± 0.1 and 0.9 ± 0.1 (hydrolytic dehalogenation), and 1.76 ± 0.05 and 3.5 ± 0.1 (dihaloelimination). This result highlights the potential of this approach to identify 1,2-DCA degradation pathways in the field. In addition, distinct trends were also observed in a multi- (i.e., Δδ2H versus Δδ37Cl versus Δδ13C) isotope plot, which opens further possibilities for pathway identification in future field studies. This is crucial information to understand the mechanisms controlling natural attenuation of 1,2-DCA and to design appropriate strategies to enhance biodegradation.

Detoxification of 1,1,2-trichloroethane to ethene in a bioreactor co-culture of Dehalogenimonas and Dehalococcoides mccartyi strains.

1,1,2-Trichloroethane (1,1,2-TCA) is a non-flammable organic solvent and common environmental contaminant in groundwater. Organohalide-respiring bacteria are key microorganisms to remediate 1,1,2-TCA because they can gain metabolic energy during its dechlorination under anaerobic conditions. However, all current isolates produce hazardous end products such as vinyl chloride, monochloroethane or 1,2-dichloroethane that accumulate in the medium. Here, we constructed a syntrophic co-culture of Dehalogenimonas and Dehalococcoides mccartyi strains to achieve complete detoxification of 1,1,2-TCA to ethene. In this co-culture, Dehalogenimonas transformed 1,1,2-TCA via dihaloelimination to vinyl chloride, whereas Dehalococcoides reduced vinyl chloride via hydrogenolysis to ethene. Molasses, pyruvate, and lactate supported full dechlorination of 1,1,2-TCA in serum bottle co-cultures. Scale up of the cultivation to a 5-L bioreactor operating for 76d in fed-batch mode was successful with pyruvate as substrate. This synthetic combination of bacteria with known complementary metabolic capabilities demonstrates the potential environmental relevance of microbial cooperation to detoxify 1,1,2-TCA.

Molecular and carbon isotopic characterization of an anaerobic stable enrichment culture containing Dehalobacterium sp. during dichloromethane fermentation.

Biodegradation of dichloromethane (DCM) under reducing conditions is of major concern due to its widespread detection in contaminated groundwaters. Here, we report an anaerobic enrichment culture derived from a membrane bioreactor operating in an industrial wastewater treatment plant, capable of fermenting DCM and the brominated analogue dibromomethane (DBM). Comparative analysis of bacterial 16S rDNA-DGGE profiles from fresh liquid medium inoculated with single colonies picked from serial dilution-to-extinction agar vials showed that cultures degrading DCM contained a predominant band belonging to Dehalobacterium, however this band was absent in cultures unable to degrade DCM. Analysis of the microbial composition of the enrichment by bacterial 16S rRNA gene amplicon paired-end sequencing confirmed the presence of Dehalobacterium together with three additional phylotypes belonging to Acetobacterium, Desulfovibrio, and Wolinella, representing all four operational taxonomic units >99.9% of the retrieved sequences. The carbon isotopic fractionation (ε) determined for DCM degradation in this culture was -27±2‰. This value differs from the ε previously reported for the DCM-fermentative bacteria Dehalobacter (-15.5±1.5‰) but they are both significantly different from those reported for facultative methylotrophic organisms (ranging from -45 to -61‰). This significant difference in the ε allows differentiating between hydrolytic transformation of DCM via glutathione-dependent dehalogenases and fermentation pathway. CAPSULE: The carbon isotopic fractionation of dichloromethane by an enriched Dehalobacterium-containing culture has significant potential to monitor biodegradation of DCM in groundwaters.

Isotopes in geobiochemistry: tracing metabolic pathways in microorganisms of environmental relevance with stable isotopes.

Stable isotopes are flexibly used as tracers to investigate environmental processes, microorganisms responsible for environmental transformations, syntrophic relationships in consortia, and metabolic pathways. With the advent of widely accessible high-resolution, highly accurate and sensitive mass spectrometers connected to liquid chromatography (LC-MS/MS) and the explosion of microbial genome sequence information the options to apply stable isotope tracers to geobiochemical topics have multiplied. With methods at hand to analyze biochemical pathways and enzymatic functions of yet-uncultivated microorganisms even in mixed cultures, a wide field of new discoveries can be expected. Applications rely both on the high sensitivity to detect trace amounts of biological material in slow or non-growing cultures and on the high multi-dimensional resolution of LC-MS/MS to allow the separation of complex samples and to retrieve phylogenetic information. Challenges and examples of stable isotope applications to describe geobiochemical processes are reviewed. Overall, the potential is not yet sufficiently deployed.