Conformational changes allow processing of bulky substrates by a haloalkane dehalogenase with a small and buried active site.

Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure-dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.

Reductive dechlorination of 1,2-dichloroethane in the presence of chloroethenes and 1,2-dichloropropane as co-contaminants.

1,2-Dichloroethane (1,2-DCA) is one of the most abundant manmade chlorinated organic contaminants in the world. Reductive dechlorination of 1,2-DCA by organohalide-respiring bacteria (OHRB) can be impacted by other chlorinated contaminants such as chloroethenes and chloropropanes that can co-exist with 1,2-DCA at contaminated sites. The aim of this study was to evaluate the effect of chloroethenes and 1,2-dichloropropane (1,2-DCP) on 1,2-DCA dechlorination using sediment cultures enriched with 1,2-DCA as the sole chlorinated compound (EA culture) or with 1,2-DCA and tetrachloroethene (PCE) (EB culture), and to model dechlorination kinetics. Both cultures contained Dehalococcoides as most predominated OHRB, and Dehalogenimonas and Geobacter as other known OHRB. In sediment-free enrichments obtained from the EA and EB cultures, dechlorination of 1,2-DCA was inhibited in the presence of the same concentrations of either PCE, vinyl chloride (VC), or 1,2-DCP; however, concurrent dechlorination of dual chlorinated compounds was achieved. In contrast, 1,2-DCA dechlorination completely ceased in the presence of cis-dichloroethene (cDCE) and only occurred after cDCE was fully dechlorinated. In turn, 1,2-DCA did not affect dechlorination of PCE, cDCE, VC, and 1,2-DCP. In sediment-free enrichments obtained from the EA culture, Dehalogenimonas 16S rRNA gene copy numbers decreased 1-3 orders of magnitude likely due to an inhibitory effect of chloroethenes. Dechlorination with and without competitive inhibition fit Michaelis-Menten kinetics and confirmed the inhibitory effect of chloroethenes and 1,2-DCP on 1,2-DCA dechlorination. This study reinforces that the type of chlorinated substrate drives the selection of specific OHRB, and indicates that removal of chloroethenes and in particular cDCE might be necessary before effective removal of 1,2-DCA at sites contaminated with mixed chlorinated solvents.

Rapid Enrichment of Dehalococcoides-Like Bacteria by Partial Hydrophobic Separation.

Organohalide-respiring bacteria can be difficult to enrich and isolate, which can limit research on these important organisms. The goal of this research was to develop a method to rapidly (minutes to days) enrich these organisms from a mixed community. The method presented is based on the hypothesis that organohalide-respiring bacteria would be more hydrophobic than other bacteria as they dehalogenate hydrophobic compounds. The method developed tests this hypothesis by separating a portion of putative organohalide-respiring bacteria, those phylogenetically related to Dehalococcoides mccartyi, at the interface between a hydrophobic organic solvent and an aqueous medium. This novel partial separation technique was tested with a polychlorinated biphenyl-enriched sediment-free culture, a tetrachloroethene-enriched digester sludge culture, and uncontaminated lake sediment. Significantly higher fractions, up to 20.4 times higher, of putative organohalide-respiring bacteria were enriched at the interface between the medium and either hexadecane or trichloroethene. The selective partial separation of these putative organohalide-respiring bacteria occurred after 20 min, strongly suggesting that the separation was a result of physical-chemical interactions between the cell surface and hydrophobic solvent. Dechlorination activity postseparation was verified by the production of cis-dichloroethene when amended with tetrachloroethene. A longer incubation time of 6 days prior to separation with trichloroethene increased the total number of putative organohalide-respiring bacteria. This method provides a way to quickly separate some of the putative organohalide-respiring bacteria from other bacteria, thereby improving our ability to study multiple and different bacteria of potential interest and improving knowledge of these bacteria.IMPORTANCE Organohalide-respiring bacteria, bacteria capable of respiring chlorinated contaminants, can be difficult to enrich, which can limit their predictable use for the bioremediation of contaminated sites. This paper describes a method to quickly separate Dehalococcoides-like bacteria, a group of organisms containing organohalide-respiring bacteria, from other bacteria in a mixed community. From this work, Dehalococcoides-like bacteria appear to have a hydrophobic cell surface, facilitating a rapid (20 min) partial separation from a mixed culture at the surface of a hydrophobic liquid. This method was verified in a polychlorinated biphenyl-enriched sediment-free culture, an anaerobic digester sludge, and uncontaminated sediment. The method described can drastically reduce the amount of time required to partially separate Dehalococcoides-like bacteria from a complex mixed culture, improving researchers' ability to study these important bacteria.

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.

Isolation and characterization of Dehalobacter sp. strain UNSWDHB capable of chloroform and chlorinated ethane respiration.

Dehalobacter sp. strain UNSWDHB can dechlorinate up to 4 mM trichloromethane at a rate of 0.1 mM per day to dichloromethane and 1,1,2-trichloroethane (1 mM, 0.1 mM per day) with the unprecedented product profile of 1,2-dichloroethane and vinyl chloride. 1,1,1-trichloroethane and 1,1-dichloroethane were slowly utilized by strain UNSWDHB and were not completely removed, with minimum threshold concentrations of 0.12 mM and 0.07 mM respectively under growth conditions. Enzyme kinetic experiments confirmed strong substrate affinity for trichloromethane and 1,1,2-trichloroethane (Km = 30 and 62 µM respectively) and poor substrate affinity for 1,1,1-trichloroethane and 1,1-dichloroethane (Km = 238 and 837 µM respectively). Comparison of enzyme kinetic and growth data with other trichloromethane respiring organisms (Dehalobacter sp. strain CF and Desulfitobacterium sp. strain PR) suggests an adaptation of strain UNSWDHB to trichloromethane. The trichloromethane RDase (TmrA) expressed by strain UNSWDHB was identified by BN-PAGE and functionally characterized. Amino acid comparison of homologous RDases from all three organisms revealed only six significant amino acid substitutions/deletions, which are likely to be crucial for substrate specificity. Furthermore, strain UNSWDHB was shown to grow without exogenous supply of cobalamin confirming genomic-based predictions of a fully functional cobalamin synthetic pathway.

Dehalogenimonas sp. Strain WBC-2 Genome and Identification of Its trans-Dichloroethene Reductive Dehalogenase, TdrA.

The Dehalogenimonas population in a dechlorinating enrichment culture referred to as WBC-2 was previously shown to be responsible for trans-dichloroethene (tDCE) hydrogenolysis to vinyl chloride (VC). In this study, blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzymatic assays and protein identification using liquid chromatography coupled with mass spectrometry (LC-MS/MS) led to the functional characterization of a novel dehalogenase, TdrA. This new reductive dehalogenase (RDase) catalyzes the dechlorination of tDCE to VC. A metagenome of the WBC-2 culture was sequenced, and a complete Dehalogenimonas genome, only the second Dehalogenimonas genome to become publicly available, was closed. The tdrA dehalogenase found within the Dehalogenimonas genome appears to be on a genomic island similar to genomic islands found in Dehalococcoides. TdrA itself is most similar to TceA from Dehalococcoides sp. strain FL2 with 76.4% amino acid pairwise identity. It is likely that the horizontal transfer of rdhA genes is not only a feature of Dehalococcoides but also a feature of other Dehalococcoidia, including Dehalogenimonas. A set of primers was developed to track tdrA in WBC-2 subcultures maintained on different electron acceptors. This newest dehalogenase is an addition to the short list of functionally defined RDases sharing the usual characteristic motifs (including an AB operon, a TAT export sequence, two iron-sulfur clusters, and a corrinoid binding domain), substrate flexibility, and evidence for horizontal gene transfer within the Dehalococcoidia.

A New Catabolic Plasmid in Xanthobacter and Starkeya spp. from a 1,2-Dichloroethane-Contaminated Site.

UNLABELLED: 1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria (Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1 and ISPme1) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1 and dhlB2) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1 Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. IMPORTANCE: Earlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more recently isolated DCA-degrading Xanthobacter strains from Australia, in which a relatively small circular plasmid (pDCA) carries both dhlA and dhlB homologs. pDCA represents a true organochlorine-catabolic plasmid, first because its only obvious metabolic phenotype is dehalogenation of organochlorines, and second because acquisition of this plasmid provides both key enzymes required for carbon-chlorine bond cleavage. The discovery of the alternative haloacid dehalogenase dhlB2 in pDCA increases the known genetic diversity of bacterial chloroacetate-hydrolyzing enzymes.

Assessment of biostimulation and bioaugmentation for removing chlorinated volatile organic compounds from groundwater at a former manufacture plant.

Site in a former chemical manufacture plant in China was found contaminated with high level of chlorinated volatile organic compounds (CVOCs). The major contaminants chloroform (CF), 1,2-dichloroethane (1,2-DCA) and vinyl chloride (VC) in groundwater were up to 4.49 × 104, 2.76 × 106 and 4.35 × 104 µg/L, respectively. Ethene and methane were at concentrations up to 2219.80 and 165.85 µg/L, respectively. To test the hypothesis that the CVOCs in groundwater at this site could be removed via biodegradation, biomarker analyses and microcosm studies were conducted. Dehalococcoides 16S rRNA gene and VC-reductase gene vcrA at densities up to 1.5 × 104 and 3.2 × 104 copies/L were detected in some of the groundwater samples, providing strong evidence that dechlorinating bacteria were present in the aquifer. Results from the microcosm studies showed that at moderate concentrations (CF about 4000 µg/L and 1,2-DCA about 100 µg/L), CF was recalcitrant under natural condition but was degraded under biostimulation and bioaugmentation, while 1,2-DCA was degraded under all the three conditions. At high concentration (CF about 1,000,000 µg/L and 1,2-DCA about 20,000 µg/L), CF was recalcitrant under all the three treatments and 1,2-DCA was only degraded under bioaugmentation, indicating that high concentrations of contaminants were inhibitory to the bacteria. Electron donors had influence on the degradation of contaminants. Of the four fatty acids (pyruvate, acetate, propionate and lactate) examined, all could stimulate the degradation of 1,2-DCA at both moderate and high concentrations, whereas only pyruvate and acetate could stimulate the degradation of CF at moderate concentration. In the microcosms, the observed first-order degradation rates of CF and 1,2-DCA were up to 0.12 and 0.11/day, respectively. Results from the present study provided scientific basis for remediating CVOCs contaminated groundwater at the site.

Kinetics of heavy metal inhibition of 1,2-dichloroethane biodegradation in co-contaminated water.

Sites co-contaminated with heavy metals and 1,2-DCA may pose a greater challenge for bioremediation, as the heavy metals could inhibit the activities of microbes involved in biodegradation. Therefore, this study was undertaken to quantitatively assess the effects of heavy metals (arsenic, cadmium, mercury, and lead) on 1,2-DCA biodegradation in co-contaminated water. The minimum inhibitory concentrations (MICs) and concentrations of the heavy metals that caused half-life doubling (HLDs) of 1,2-DCA as well as the degradation rate coefficient (k(1)) and half-life (t(½)) of 1,2-DCA were measured and used to predict the toxicity of the heavy metals in the water microcosms. An increase in heavy metal concentration resulted in a progressive increase in the t(½) and relative t(½) and a decrease in k(1). The MICs and HLDs of the heavy metals were found to vary, depending on the heavy metals type. In addition, the presence of heavy metals was shown to inhibit 1,2-DCA biodegradation in a dose-dependent manner, with the following order of decreasing inhibitory effect: Hg(2+) > As(3+) > Cd(2+) > Pb(2+). Findings from this study have significant implications for the development of bioremediation strategies for effective degradation of 1,2-DCA and other related compounds in wastewater co-contaminated with heavy metals.

Enzyme activity and gene expression profiles of Xanthobacter autotrophicus GJ10 during aerobic biodegradation of 1,2-dichloroethane.

Xanthobacter autotrophicus GJ10 has been widely studied because of its ability to degrade halogenated compounds, especially 1,2-dichloroethane (1,2-DCA), which is achieved through chromosomal as well as plasmid pAUX1 encoded 1,2-DCA degrading genes. This work described the gene expression and enzyme activity profiles as well as the intermediates formed during the 1,2-DCA degradation by this organism. A correlation between gene expression, enzyme activity and metabolic intermediates, after the induction of GJ10 grown culture with 1,2-DCA, was established at different time intervals. Haloalkane dehalogenase (dhlA) and haloacid dehalogenase (dhlB) were constitutively expressed while the expression of alcohol dehydrogenase (max) and aldehyde dehydrogenase (ald) was found to be inducible. The DhlA and DhlB activities were relatively higher compared to that of the inducible enzymes, Max and Ald. To the best of our knowledge, this is the first study to correlate gene expression profiles with enzyme activity and metabolite formation during 1,2-DCA degradation process in GJ10. Findings from this study may assist in fully understanding the mechanism of 1,2-DCA degradation by GJ10. It could also assist in the design and implementation of appropriate bioaugmentation strategies for complete removal of 1,2-DCA from contaminated environment.

Absorption of ethanol, acetone, benzene and 1,2-dichloroethane through human skin in vitro: a test of diffusion model predictions.

The overall goal of this research was to further develop and improve an existing skin diffusion model by experimentally confirming the predicted absorption rates of topically-applied volatile organic compounds (VOCs) based on their physicochemical properties, the skin surface temperature, and the wind velocity. In vitro human skin permeation of two hydrophilic solvents (acetone and ethanol) and two lipophilic solvents (benzene and 1,2-dichloroethane) was studied in Franz cells placed in a fume hood. Four doses of each (14)C-radiolabed compound were tested - 5, 10, 20, and 40µLcm(-2), corresponding to specific doses ranging in mass from 5.0 to 63mgcm(-2). The maximum percentage of radiolabel absorbed into the receptor solutions for all test conditions was 0.3%. Although the absolute absorption of each solvent increased with dose, percentage absorption decreased. This decrease was consistent with the concept of a stratum corneum deposition region, which traps small amounts of solvent in the upper skin layers, decreasing the evaporation rate. The diffusion model satisfactorily described the cumulative absorption of ethanol; however, values for the other VOCs were underpredicted in a manner related to their ability to disrupt or solubilize skin lipids. In order to more closely describe the permeation data, significant increases in the stratum corneum/water partition coefficients, Ksc, and modest changes to the diffusion coefficients, Dsc, were required. The analysis provided strong evidence for both skin swelling and barrier disruption by VOCs, even by the minute amounts absorbed under these in vitro test conditions.

Cloning, expression, purification and three-dimensional structure prediction of haloalkane dehalogenase from a recently isolated Ancylobacter aquaticus strain UV5.

Haloalkane dehalogenase (DhlA) converts 1,2-dichloroethane (1,2-DCA) to 2-chloroethane in the genus Ancylobacter and Xanthobacter autotrophicus GJ10 (XaDhlA) and allows these organisms to utilise 1,2-DCA and some other halogenated alkanes for growth. The DhlA encoding gene (dhlA) was PCR-amplified from the genomic DNA of a recently isolated Ancylobacter aquaticus UV5 strain, cloned and overexpressed in Escherichiacoli BL21 (DE3). The recombinant enzyme was purified by using Amicon ultra-15 centrifugal filter units, an anion-exchange QFF column followed by a gel-filtration column (Sephacryl HR100). Enzyme activity was determined by using 1,2-DCA as a substrate. Three-dimensional structure of the enzyme was predicted using SWISS-MODEL workspace and the biophysical properties were predicted by submitting the amino acid sequence of DhlA on ExPASy server. DhlA (Mr 35kDa) exhibited optimum activity at temperature 37°C and pH 9.0. The enzyme retained approximately 50% of its activity after 1h of incubation at 50°C, and showed moderate stability against denaturing agent urea. The DhlA displayed a Km value of 842µM and kcat/Km ratio of 168mM(-)(1)min(-)(1) for its substrate 1,2-DCA. This DhlA was found to belong to the α/ß hydrolase family with a catalytic triad composed of Asp-His-Asp in its active site. This is the first study reporting on the characterisation and reaction kinetics of purified DhlA from A.aquaticus UV5 indigenous to contaminated site in Africa.

Substrate interactions in dehalogenation of 1,2-dichloroethane, 1,2-dichloropropane, and 1,1,2-trichloroethane mixtures by Dehalogenimonas spp.

When chlorinated alkanes are present as soil or groundwater pollutants, they often occur in mixtures. This study evaluated substrate interactions during the anaerobic reductive dehalogenation of chlorinated alkanes by the type strains of two Dehalogenimonas species, D. lykanthroporepellens and D. alkenigignens. Four contaminant mixtures comprised of combinations of the chlorinated solvents 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were assessed for each species. Chlorinated solvent depletion and daughter product formation determined as a function of time following inoculation into anaerobic media revealed preferential dechlorination of 1,1,2-TCA over both 1,2-DCA and 1,2-DCP for both species. 1,2-DCA in particular was not dechlorinated until 1,1,2-TCA reached low concentrations. In contrast, both species concurrently dechlorinated 1,2-DCA and 1,2-DCP over a comparably large concentration range. This is the first report of substrate interactions during chlorinated alkane dehalogenation by pure cultures, and the results provide insights into the chlorinated alkane transformation processes that may be expected for contaminant mixtures in environments where Dehalogenimonas spp. are present.

[To the question of the optimization of methods for detection of vinyl chloride and 1,2-dichloroethane, and their metabolites in biological fluids in workers involved in production of polyvinyl chloride].

There is considered the improvement of methodological approaches to the gas chromatographic methods- of the detection of vinyl chloride and 1,2-dichloroethane and their metabolites--chloroethanol and monochloroacetic acid in biological fluids. There were evaluated such metrological characteristics of methods, as repeatability, interlaboratoty precision, relevance and accuracy. The value of relative expanded uncertainty does not exceed 30%. There are reported optimal regimes of gas chromatographic analysis, conditions for sample preparation. The results of the contents ofthese chemical compounds and their metabolites in biological fluids from persons working in contact with chlorinated hydrocarbons are presented These techniques can be used for the detection ofthe fact of exposure to toxic substances, assessment of the level of exposure and biomonitoring.

The complete degradation of 1,2-dichloroethane in Escherichia coli by metabolic engineering.

1,2-Dichloroethane (1,2-DCA), a widely utilized chemical intermediate and organic solvent in industry, frequently enters the environment due to accidental leaks and mishandling during application processes. Thus, the in-situ remediation of contaminated sites has become increasingly urgent. However, traditional remediation methods are inefficient and costly, while bioremediation presents a green, efficient, and non-secondary polluting alternative. In this study, an engineered strain capable of completely degrading 1,2-DCA was constructed. We introduced six exogenous genes of the 1,2-DCA degradation pathway into E. coli and confirmed their normal transcription and efficient expression in this engineered strain through qRT-PCR and proteomics. The degradation experiments showed that the strain completely degraded 2 mM 1,2-DCA within 12 h. Furthermore, the results of isotope tracing verified that the final degradation product, malic acid, entered the tricarboxylic acid cycle (TCA) of E. coli and was ultimately fully metabolized. Also, morphological changes in the engineered strain and control strain exposed to 1,2-DCA were observed under SEM, and the results revealed that the engineered strain is more tolerant to 1,2-DCA than the control strain. In conclusion, this study paved a new way for humanity to deal with the increasingly complex environmental challenges.

Synergistic algal/bacterial interaction in membrane bioreactor for detoxification of 1,2-dichloroethane-rich petroleum wastewater.

The study addressed the challenge of treating petroleum industry wastewater with high concentrations of 1,2-dichloroethane (1,2-DCA) ranging from 384 to 1654 mg/L, which poses a challenge for bacterial biodegradation and algal photodegradation. To overcome this, a collaborative approach using membrane bioreactors (MBRs) that combine algae and bacteria was employed. This synergistic method effectively mitigated the toxicity of 1,2-DCA and curbed MBR fouling. Two types of MBRs were tested: one (B-MBR) used bacterial cultures and the other (AB-MBR) incorporated a mix of algal and bacterial cultures. The AB-MBR significantly contributed to 1,2-DCA removal, with algae accounting for over 20% and bacteria for approximately 49.5% of the dechlorination process. 1,2-DCA metabolites, including 2-chloroethanol, 2-chloro-acetaldehyde, 2-chloroacetic acid, and acetic acid, were partially consumed as carbon sources by algae. Operational efficiency peaked at a 12-hour hydraulic retention time (HRT) in AB-MBR, enhancing enzyme activities crucial for 1,2-DCA degradation such as dehydrogenase (DH), alcohol dehydrogenase (ADH), and acetaldehyde dehydrogenase (ALDH). The microbial diversity in AB-MBR surpassed that in B-MBR, with a notable increase in Proteobacteria, Bacteroidota, Planctomycetota, and Verrucomicrobiota. Furthermore, AB-MBR showed a significant rise in the dominance of 1,2-DCA-degrading genus such as Pseudomonas and Acinetobacter. Additionally, algal-degrading phyla (e.g., Nematoda, Rotifera, and Streptophyta) were more prevalent in AB-MBR, substantially reducing the issue of membrane fouling.

Kinetics of 1,2-dichloroethane and 1,2-dibromoethane biodegradation in anaerobic enrichment cultures.

1,2-Dichloroethane (1,2-DCA) and 1,2-dibromoethane (ethylene dibromide [EDB]) contaminate groundwater at many hazardous waste sites. The objectives of this study were to measure yields, maximum specific growth rates (µ), and half-saturation coefficients (K(S)) in enrichment cultures that use 1,2-DCA and EDB as terminal electron acceptors and lactate as the electron donor and to evaluate if the presence of EDB has an effect on the kinetics of 1,2-DCA dehalogenation and vice versa. Biodegradation was evaluated at the high concentrations found at some industrial sites (>10 mg/liter) and at lower concentrations found at former leaded-gasoline sites (1.9 to 3.7 mg/liter). At higher concentrations, the Dehalococcoides yield was 1 order of magnitude higher when bacteria were grown with 1,2-DCA than when they were grown with EDB, while µ's were similar for the two compounds, ranging from 0.19 to 0.52 day(-1) with 1,2-DCA to 0.28 to 0.36 day(-1) for EDB. K(S) was larger for 1,2-DCA (15 to 25 mg/liter) than for EDB (1.8 to 3.7 mg/liter). In treatments that received both compounds, EDB was always consumed first and adversely impacted the kinetics of 1,2-DCA utilization. Furthermore, 1,2-DCA dechlorination was interrupted by the addition of EDB at a concentration 100 times lower than that of the remaining 1,2-DCA; use of 1,2-DCA did not resume until the EDB level decreased close to its maximum contaminant level (MCL). In lower-concentration experiments, the preferential consumption of EDB over 1,2-DCA was confirmed; both compounds were eventually dehalogenated to their respective MCLs (5 µg/liter for 1,2-DCA, 0.05 µg/liter for EDB). The enrichment culture grown with 1,2-DCA has the advantage of a more rapid transition to 1,2-DCA after EDB is consumed.

Adaptation of a membrane bioreactor to 1,2-dichloroethane revealed by 16S rDNA pyrosequencing and dhlA qPCR.

A pilot-scale membrane bioreactor (MBR) was tested for bioremediation of 1,2-dichloroethane (DCA) in groundwater. Pyrosequencing of 16S rDNA was used to study changes in the microbiology of the MBR over 137 days, including a 67 day initial adaptation phase of increasing DCA concentration. The bacterial community in the MBR was distinct from those in soil and groundwater at the same site, and was dominated by alpha- and beta- proteobacteria, including Rhodobacter, Methylibium, Rhodopseudomonas, Methyloversatilis, Caldilinea, Thiobacillus, Azoarcus, Hyphomicrobium, and Leptothrix. Biodegradation of DCA in the MBR began after 26 days, and was sustained for the remainder of the experiment. A quantitative PCR (qPCR) assay for the dehalogenase gene dhlA was developed to monitor DCA-degrading bacteria in the MBR, and a positive correlation was seen between dhlA gene abundance and the cumulative amount of DCA that had entered the MBR. Genera previously associated with aerobic DCA biodegradation (Xanthobacter, Ancylobacter, Azoarcus) were present in the MBR, and the abundance of Azoarcus correlated well with dhlA gene abundance. This study shows that MBRs can be an effective method for removal of DCA from groundwater, and that the dhlA qPCR is a rapid and sensitive method for detection of DCA-degrading bacteria.

Modeling trichloroethene reduction in a hydrogen-based biofilm.

We constructed a multispecies biofilm model for simultaneous reduction of trichloroethene (TCE) and nitrate (NO3(-)) in the biofilm of a H2-based membrane biofilm reactor (MBfR). The one-dimensional model includes dual-substrate Monod kinetics for a steady-state biofilm with multiple solid and dissolved components. The model has five solid components: autotrophic denitrifying bacteria (ADB), heterotrophic denitrifying bacteria (HDB), Dehalococcoides (DHC), inert biomass (IB), and extracellular polymeric substances (EPS). The model has eight dissolved components: NO3(-), TCE, dichloroethene (DCE), vinyl chloride (VC), ethene, hydrogen (H2), substrate-utilization-associated products (UAP), and biomass-associated products (BAP). We used this model to simulate a bench-scale experiment in a H2-based MBfR. The model simulated the trends well: almost complete removal of nitrate, incomplete reduction of TCE, and almost no accumulation of DCE and VC. To gain insight into reductive dehalogenation in a H2-based MBfR, we also simulated the concentrations of nitrate, TCE, DCE, VC, and ethene in the reactor effluent while varying the influent nitrate concentration. Simultaneous low concentrations of nitrate and the three chlorinated ethenes can occur as long as the influent ratio of NO3(-) to TCE is not too large, so that DHC are a significant fraction of the biofilm.

Distribution of blood and tissue concentrations in rats by inhalation exposure to 1,2-dichloroethane.

The compound 1,2-dichloroethane (DCE) is a ubiquitous environmental contaminant. The primary route of exposure of humans to DCE is inhalation of its vapor. The present investigation was undertaken to determine the distribution and accumulation of DCE in the blood, lung, liver, brain, kidney and abdominal fat of rats during and after inhalation exposure. Male rats were exposed to 160 ppm (v/v) of DCE vapor for 360 min and the concentrations of DCE in the blood and tissues during the inhalation exposure period and after the end of the exposure period were measured. DCE accumulation in the abdominal fat was much greater than that in the blood and other tissues. The information we obtained in this study is useful basic data pertaining to the pharmacokinetics of DCE and DCE-mediated carcinogenicity: Our results suggest that one of the factors involved in the induction of peritoneal tumors in rats exposed to DCE vapor by inhalation is DCE accumulation in the abdominal fat.