Discovery of an Inducible Toluene Monooxygenase That Cooxidizes 1,4-Dioxane and 1,1-Dichloroethylene in Propanotrophic Azoarcus sp. Strain DD4.

Cometabolic degradation plays a prominent role in bioremediation of commingled groundwater contamination (e.g., chlorinated solvents and the solvent stabilizer 1,4-dioxane [dioxane]). In this study, we untangled the diversity and catalytic functions of multicomponent monooxygenases in Azoarcus sp. strain DD4, a Gram-negative propanotroph that is effective in degrading dioxane and 1,1-dichloroethylene (1,1-DCE). Using a combination of knockout mutagenesis and heterologous expression, a toluene monooxygenase (MO) encoded by the tmoABCDEF gene cluster was unequivocally proved to be the key enzyme responsible for the cometabolism of both dioxane and 1,1-DCE. Interestingly, in addition to utilizing toluene as a primary substrate, this toluene MO can also oxidize propane into 1-propanol. Expression of this toluene MO in DD4 appears inducible by both substrates (toluene and propane) and their primary hydroxylation products (m-cresol, p-cresol, and 1-propanol). These findings coherently explain why DD4 can grow on propane and express toluene MO for active cooxidation of dioxane and 1,1-DCE. Furthermore, upregulation of tmo transcription by 1-propanol underlines the implication potential of using 1-propanol as an alternative auxiliary substrate for DD4 bioaugmentation. The discovery of this toluene MO in DD4 and its degradation and induction versatility can lead to broad applications, spanning from environmental remediation and water treatment to biocatalysis in green chemistry.IMPORTANCE Toluene MOs have been well recognized given their robust abilities to degrade a variety of environmental pollutants. Built upon previous research efforts, this study ascertained the untapped capability of a toluene MO in DD4 for effective cooxidation of dioxane and 1,1-DCE, two of the most prevailing yet challenging groundwater contaminants. This report also aligns the induction of a toluene MO with nontoxic and commercially accessible chemicals (e.g., propane and 1-propanol), extending its implications in the field of environmental microbiology and beyond.

Aerobic degradation of cis-dichloroethene by the marine bacterium Marinobacter salsuginis strain 5N-3.

An aerobic bacterium, designated strain 5N-3 (NBRC 113055), that degrades cis-dichloroethene (cDCE) was isolated from a sea sediment in Japan. Strain 5N-3 was able to degrade a certain amount of cDCE in the presence of pyruvate without the action of inducers. In the presence of inducers, such as phenol and benzene, the strain completely removed cDCE. By the application of 16S ribosomal RNA (16S rRNA) gene sequencing and average nucleotide identity analyses, the strain 5N-3 was identified as Marinobacter salsuginis. On the other hand, identified species of Marinobacter are not known to degrade cDCE at all. A draft genome sequence analysis of the strain 5N-3 suggested that the dmp-homologous operon (operon for phenol degradation) may be contributing to the aerobic degradation of cDCE. This is the first report on an aerobic marine bacterium that has been found to degrade cDCE.

Effect of nickel, cobalt, and iron on methanogenesis from methanol and cometabolic conversion of 1,2-dichloroethene by Methanosarcina barkeri.

Methanogens are responsible for the last step in anaerobic digestion (AD), in which methane (a biofuel) is produced. Some methanogens can cometabolize chlorinated pollutants, contributing for their removal during AD. Methanogenic cofactors involved in cometabolic reductive dechlorination, such as F430 and cobalamin, contain metal ions (nickel, cobalt, iron) in their structure. We hypothesized that the supplementation of trace metals could improve methane production and the cometabolic dechlorination of 1,2-dichloroethene (DCE) by pure cultures of Methanosarcina barkeri. Nickel, cobalt, and iron were added to cultures of M. barkeri growing on methanol and methanol plus DCE. Metal amendment improved DCE dechlorination to vinyl chloride (VC): assays with 20 µM of Fe3+ showed the highest final concentration of VC (5× higher than in controls without Fe3+ ), but also in assays with 5.5 µM of Co2+ and 5 µM of Ni2+ VC formation was improved (3.5-4× higher than in controls without the respective metals). Dosing of metals could be useful to improve anaerobic removal of chlorinated compounds, and more importantly decrease the detrimental effect of DCE on methane production in anaerobic digesters.

In-situ reductive degradation of chlorinated DNAPLs in contaminated groundwater using polyethyleneimine-modified zero-valent iron nanoparticles.

Zero-valent iron nanoparticles (ZVIN) have found applications in many strategies for on-site soil and groundwater decontamination. A number of studies have reported the prospective utilization of ZVIN in the reduction of chlorinated organic compounds such as dense non-aqueous phase liquids (DNAPLs) in groundwater. Due to their bioaccumulation and carcinogenesis, DNAPLs in groundwater are a human health hazard and pose environmental risks. Therefore, decontamination of these contaminants is necessary. This study presents the in-situ remediation of trichloroethylene (TCE), perchloroethene (PCE), and 1,2-dichloroethene (1,2-DCE) DNAPLs through the direct injection of polyethylenimine (PEI)-coated ZVIN (PEI-ZVIN composite materials) to facilitate the reduction of contaminants in low-permeability media. A field test was conducted at the premises of a petrochemical company, situated in the Miaoli County of Northern Taiwan that discharged significant amounts of DNAPLs. After in-situ injection and one-day of reaction with groundwater contaminants, ZVIN was further characterized to examine its efficacy in the reduction of pollutants. After the direct injection of PEI-ZVIN, a notable reduction in the concentration of DNAPLs was recorded with conversion from toxic to non-toxic substances. Use of resistivity image profiling (RIP) technique suggested similar conductivity data for the PEI-coated ZVIN suspension and groundwater samples. X-ray absorption near edge structure (XANES) and X-ray absorption fine structure (EXAFS) studies depicted that the oxidation of ZVIN and PEI-ZVIN was occurring after the reductive reaction with contaminated groundwater. The reacted samples had bond distance values of 1.98, 2.00, 1.96, and 1.94 Å. Combining floating surface-coated ZVIN and RIP technique seems promising and environmentally attractive.

Enhanced reductive de-chlorination of a solvent contaminated aquifer through addition and apparent fermentation of cyclodextrin.

In a field study, aqueous cyclodextrin (CD) was investigated for its ability to extract chlorinated volatile organic compounds (cVOC), such as trichloroethylene (TCE), 1,1,1-trichloroethane (TCA), and dichloroethene (DCE) through in-situ flushing of a sandy aquifer. After cessation of aquifer flushing, a plume of CD was left. Changes in CD, cVOC, and inorganic terminal electron acceptors (TEAs) (DO, nitrate, sulfate, iron) were monitored in four rounds of wellwater sampling (20, 210, 342, and 425days after cessation of active pumping). Post-CD flushing VOC levels rebounded (850% for TCE, 190% for TCA, and 53% for DCE) between the first two sampling rounds, apparently due to rate-limited desorption from aquifer media and dissolution from remaining NAPL. However, substantial reduction in the mass of TCE (6.3 to 0.11mol: 98%) and TCA (2.8 to 0.73mol: 74%) in groundwater was observed between 210 and 425days. DCE should primarily be produced from the degradation of TCE and is expected to subsequently degrade to chloroethene. Since DCE levels decreased only slightly (0.23 to 0.17mol: 26%), its degradation rate should be similar to that produced from the decaying TCE. Cyclodextrin was monitored starting from day 210. The mass of residual CD (as measured by Total Organic Carbon) decreased from 150mol (day 210) to 66 (day 425) (56% decrease). The naturally anaerobic zone within the aquifer where residual CD mass decreased coincided with a loss of other major potential TEAs: nitrate (97% loss), sulfate (31%) and iron (31%). In other studies, TCE and 1,1,1-TCA have been found to be more energetically favorable TEAs than sulfate and iron and their degradation via reductive dechlorination has been found to be enhanced by the fermentation of carbohydrates. Such processes can explain these observations, but more investigation is needed to evaluate whether residual levels of CD can facilitate the anaerobic degradation of chlorinated VOCs.

Natural attenuation of chlorinated ethenes in hyporheic zones: A review of key biogeochemical processes and in-situ transformation potential.

Chlorinated ethenes (CEs) are legacy contaminants whose chemical footprint is expected to persist in aquifers around the world for many decades to come. These organohalides have been reported in river systems with concerning prevalence and are thought to be significant chemical stressors in urban water ecosystems. The aquifer-river interface (known as the hyporheic zone) is a critical pathway for CE discharge to surface water bodies in groundwater baseflow. This pore water system may represent a natural bioreactor where anoxic and oxic biotransformation process act in synergy to reduce or even eliminate contaminant fluxes to surface water. Here, we critically review current process understanding of anaerobic CE respiration in the competitive framework of hyporheic zone biogeochemical cycling fuelled by in-situ fermentation of natural organic matter. We conceptualise anoxic-oxic interface development for metabolic and co-metabolic mineralisation by a range of aerobic bacteria with a focus on vinyl chloride degradation pathways. The superimposition of microbial metabolic processes occurring in sediment biofilms and bulk solute transport delivering reactants produces a scale dependence in contaminant transformation rates. Process interpretation is often confounded by the natural geological heterogeneity typical of most riverbed environments. We discuss insights from recent field experience of CE plumes discharging to surface water and present a range of practical monitoring technologies which address this inherent complexity at different spatial scales. Future research must address key dynamics which link supply of limiting reactants, residence times and microbial ecophysiology to better understand the natural attenuation capacity of hyporheic systems.

Cometabolic degradation of dichloroethenes by Comamonas testosteroni RF2.

An environmental isolate Comamonas testosteroni strain RF2, which has been found to cometabolize trichloroethene (TCE) in the presence of phenol and sodium lactate as growth substrates, was tested to investigate its capacity for degrading 1,2-cis-dichloroethene (cDCE), 1,2-trans-dichlorothene (tDCE), and 1,1-dichloroethene (1,1DCE). Degradation assays were performed for single DCEs, as well as for a mixture of DCEs with TCE, which resembled contaminated plume in groundwater. RF2 was capable of efficiently removing all three dichloroethenes (DCEs) at the initial aqueous concentrations of 6.01 mg L-1 for cDCE, 3.80 mg L-1 for tDCE and 0.65 mg L-1 for 1,1DCE, with a removal efficiency of 100% for cDCE, 65.8% for tDCE, and 46.8% for 1,1DCE. Furthermore, complete removal of TCE, cDCE and 1,1DCE (122.5 µg L-1, 84.3 µg L-1 and 51.4 µg L-1, respectively) was observed in a mixture sample that also contained 72.33 µg L-1 of tDCE, which was removed to the amount of 72.3%. Moreover, degradation of cDCE (6.01 mg L-1) led to a 93.8% release of inorganic chloride, and 2,2-dichloroacetaldehyde was determined as the first intermediate of cDCE transformation. The findings of this study suggest that the strain RF2 exhibits the potential to remediate groundwater contaminated with less chlorinated ethenes.

Secondary compound hypothesis revisited: Selected plant secondary metabolites promote bacterial degradation of cis-1,2-dichloroethylene (cDCE).

Cis-1,2-dichloroethylene (cDCE), which is a common hazardous compound, often accumulates during incomplete reductive dechlorination of higher chlorinated ethenes (CEs) at contaminated sites. Simple monoaromatics, such as toluene and phenol, have been proven to induce biotransformation of cDCE in microbial communities incapable of cDCE degradation in the absence of other carbon sources. The goal of this microcosm-based laboratory study was to discover non-toxic natural monoaromatic secondary plant metabolites (SPMEs) that could enhance cDCE degradation in a similar manner to toluene and phenol. Eight SPMEs were selected on the basis of their monoaromatic molecular structure and widespread occurrence in nature. The suitability of the SPMEs chosen to support bacterial growth and to promote cDCE degradation was evaluated in aerobic microbial cultures enriched from cDCE-contaminated soil in the presence of each SPME tested and cDCE. Significant cDCE depletions were achieved in cultures enriched on acetophenone, phenethyl alcohol, p-hydroxybenzoic acid and trans-cinnamic acid. 16S rRNA gene sequence analysis of each microbial community revealed ubiquitous enrichment of bacteria affiliated with the genera Cupriavidus, Rhodococcus, Burkholderia, Acinetobacter and Pseudomonas. Our results provide further confirmation of the previously stated secondary compound hypothesis that plant metabolites released into the rhizosphere can trigger biodegradation of environmental pollutants, including cDCE.

Reutilization of waste scrap tyre as the immobilization matrix for the enhanced bioremoval of a monoaromatic hydrocarbons, methyl tert-butyl ether, and chlorinated ethenes mixture from water.

BTEX (benzene, toluene, ethylbenzene, ortho-, meta-, and para-xylenes), methyl tert-butyl ether (MTBE), cis-1,2-dichloroethylene (cis-DCE), and trichloroethylene (TCE) are among the major soil and groundwater contaminants frequently co-existing, as a result of their widespread uses. Pseudomonas plecoglossicida was immobilized on waste scrap tyre to remove these contaminants mixture from synthetic contaminated water. The microbial activity was enhanced in the immobilized system, shown by the higher colony forming units (CFUs) (40%), while BTEX were used as growth substrates. The adsorption capacity of tyres toward contaminants reached a maximum within one day, with BTEX (76.3%) and TCE (64.3%) showing the highest sorption removal capacities, followed by cis-DCE (30.0%) and MTBE (11.0%). The adsorption data fitted the Freundlich isotherm with a good linear correlation (0.989-0.999) for the initial contaminants concentration range applied (25-125mg/L). The monoaromatic hydrocarbons were almost completely removed in the immobilized system and the favourable removal efficiencies of 78% and 90% were obtained for cis-DCE and TCE, respectively. The hybrid (biological, immobilization/physical, sorption) system was further evaluated with the contaminants spiked intermittently for the stable performance. The addition of mineral salt medium further enhanced the bioremoval of contaminants by stimulating the microbial growth to some extent.

The corrinoid cofactor of reductive dehalogenases affects dechlorination rates and extents in organohalide-respiring Dehalococcoides mccartyi.

Corrinoid auxotrophic organohalide-respiring Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of toxic and carcinogenic chloroorganic contaminants. We demonstrate that the lower base attached to the essential corrinoid cofactor of reductive dehalogenase (RDase) enzyme systems modulates dechlorination activity and affects the vinyl chloride (VC) RDases BvcA and VcrA differently. Amendment of 5,6-dimethylbenzimidazolyl-cobamide (DMB-Cba) to Dhc strain BAV1 and strain GT cultures supported cis-1,2-dichloroethene-to-ethene reductive dechlorination at rates of 107.0 (±12.0) µM and 67.4 (±1.4) µM Cl(-) released per day, respectively. Strain BAV1, expressing the BvcA RDase, reductively dechlorinated VC to ethene, although at up to fivefold lower rates in cultures amended with cobamides carrying 5-methylbenzimidazole (5-MeBza), 5-methoxybenzimidazole (5-OMeBza) or benzimidazole (Bza) as the lower base. In contrast, strain GT harboring the VcrA RDase failed to grow and dechlorinate VC to ethene in medium amended with 5-OMeBza-Cba or Bza-Cba. The amendment with DMB to inactive strain GT cultures restored the VC-to-ethene-dechlorinating phenotype and intracellular DMB-Cba was produced, demonstrating cobamide uptake and remodeling. The distinct responses of Dhc strains with BvcA versus VcrA RDases to different cobamides implicate that the lower base exerts control over Dhc reductive dechlorination rates and extents (that is, detoxification), and therefore the dynamics of Dhc strains with discrete reductive dechlorination capabilities. These findings emphasize that the role of the corrinoid/lower base synthesizing community must be understood to predict strain-specific Dhc activity and achieve efficacious contaminated site cleanup.

Biodegradation of cis-1,2-Dichloroethene in Simulated Underground Thermal Energy Storage Systems.

Underground thermal energy storage (UTES) use has showed a sharp rise in numbers in the last decades, with aquifer thermal energy storage (ATES) and borehole thermal energy storage (BTES) most widely used. In many urban areas with contaminated aquifers, there exists a desire for sustainable heating and cooling with UTES and a need for remediation. We investigated the potential synergy between UTES and bioremediation with batch experiments to simulate the effects of changing temperature and liquid exchange that occur in ATES systems, and of only temperature change occurring in BTES systems on cis-DCE reductive dechlorination. Compared to the natural situation (NS) at a constant temperature of 10 °C, both UTES systems with 25/5 °C for warm and cold well performed significantly better in cis-DCE (cis-1,2-dichloroethene) removal. The overall removal efficiency under mimicked ATES and BTES conditions were respectively 13 and 8.6 times higher than in NS. Inoculation with Dehalococcoides revealed that their initial presence is a determining factor for the dechlorination process. Temperature was the dominating factor when Dehalococcoides abundance was sufficient. Stimulated biodegradation was shown to be most effective in the mimicked ATES warm well because of the combined effect of suitable temperature, sustaining biomass growth, and regular cis-DCE supply.

Spatial and temporal dynamics of organohalide-respiring bacteria in a heterogeneous PCE-DNAPL source zone.

Effective treatment of sites contaminated with dense non-aqueous phase liquids (DNAPLs) requires detailed understanding of the microbial community responses to changes in source zone strength and architecture. Changes in the spatial and temporal distributions of the organohalide-respiring Dehalococcoides mccartyi (Dhc) strains and Geobacter lovleyi strain SZ (GeoSZ) were examined in a heterogeneous tetrachloroethene- (PCE-) DNAPL source zone within a two-dimensional laboratory-scale aquifer flow cell. As part of a combined remedy approach, flushing with 2.3 pore volumes (PVs) of 4% (w/w) solution of the nonionic, biodegradable surfactant Tween® 80 removed 55% of the initial contaminant mass, and resulted in a PCE-DNAPL distribution that contained 51% discrete ganglia and 49% pools (ganglia-to-pool ratio of 1.06). Subsequent bioaugmentation with the PCE-to-ethene-dechlorinating consortium BDI-SZ resulted in cis-1,2-dichloroethene (cis-DCE) formation after 1 PV (ca. 7 days), while vinyl chloride (VC) and ethene were detected 10 PVs after bioaugmentation. Maximum ethene yields (ca. 90 µM) within DNAPL pool and ganglia regions coincided with the detection of the vcrA reductive dehalogenase (RDase) gene that exceeded the Dhc 16S rRNA genes by 2.0±1.3 and 4.0±1.7 fold in the pool and ganglia regions, respectively. Dhc and GeoSZ cell abundance increased by up to 4 orders-of-magnitude after 28 PVs of steady-state operation, with 1 to 2 orders-of-magnitude increases observed in close proximity to residual PCE-DNAPL. These observations suggest the involvement of these dechlorinators the in observed PCE dissolution enhancements of up to 2.3 and 6.0-fold within pool and ganglia regions, respectively. Analysis of the solid and aqueous samples at the conclusion of the experiment revealed that the highest VC (≥155 µM) and ethene (≥65 µM) concentrations were measured in zones where Dhc and GeoSZ were predominately attached to the solids. These findings demonstrate dynamic responses of organohalide-respiring bacteria in a heterogeneous DNAPL source zone, and emphasize the influence of source zone architecture on bioremediation performance.

Effect of toluene concentration and hydrogen peroxide on Pseudomonas plecoglossicida cometabolizing mixture of cis-DCE and TCE in soil slurry.

An indigenous Pseudomonas sp., isolated from the regional contaminated soil and identified as P. plecoglossicida, was evaluated for its aerobic cometabolic removal of cis-1,2-dichloroethylene (cis-DCE) and trichloroethylene (TCE) using toluene as growth substrate in a laboratory-scale soil slurry. The aerobic simultaneous bioremoval of the cis-DCE/TCE/toluene mixture was studied under different conditions. Results showed that an increase in toluene concentration level from 300 to 900 mg/kg prolonged the lag phase for the bacterial growth, while the bioremoval extent for cis-DCE, TCE, and toluene declined as the initial toluene concentration increased. In addition, the cometabolic bioremoval of cis-DCE and TCE was inhibited by the presence of hydrogen peroxide as the additional oxygen source, while the bioremoval of toluene (900 mg/kg) was enhanced after 9 days of incubation. The subsequent addition of toluene did not improve the cometabolic bioremoval of cis-DCE and TCE. The obtained results would help to enhance the applicability of bioremediation technology to the mixed waste contaminated sites.

Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene.

A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium.

Influence of nitrate and sulfate reduction in the bioelectrochemically assisted dechlorination of cis-DCE.

This paper investigated the reductive dechlorination (RD) of cis-dichloroethylene (cis-DCE) (average influent 14.2±0.7 µM) by a bioelectrochemical system (BES), in the presence of real contaminated groundwater containing high levels of nitrate and sulfate. The BES enhanced both the RD and competing reactions, such as nitrate and sulfate reductions, which occurred with neither an external organic carbon source nor any inoculum other than the indigenous microbial consortia in the real groundwater. In preliminary batch tests, RD and full nitrate removal occurred after a short lag phase, whereas sulfate reduction occurred slowly and alongside the RD. Under continuous flow conditions (hydraulic retention time, HRT, 1.4 d), the competition of different electron acceptors was strongly affected by the cathodic potential in the range -550 to -750 mV vs. standard hydrogen electrode (SHE). Nitrate reduction was driven to completion at all tested cathodic potentials, whereas sulfate reduction and the RD rate increased as the cathodic potential became more negative. At -750 mV vs. SHE, strong methanogenesis was also observed and became the most important sink of electrons. The overall coulombic efficiency decreased while the potential became more negative. The RD contribution was always less than 1%. Hence, greater energy consumption was required to obtain higher RD rate and better conversion. Anodic oxidation was only observed at -750 mV vs. SHE where almost 39% of residual vinyl chloride (VC) was oxidized and the sulfate was formed back from sulfide (further contributing to electric waste).

Microbial ecology of chlorinated solvent biodegradation.

This study focused on the microbial ecology of tetrachloroethene (PCE) degradation to trichloroethene, cis-1,2-dichloroethene and vinyl chloride to evaluate the relationship between the microbial community and the potential accumulation or degradation of these toxic metabolites. Multiple soil microcosms supplied with different organic substrates were artificially contaminated with PCE. A thymidine analogue, bromodeoxyuridine (BrdU), was added to the microcosms and incorporated into the DNA of actively replicating cells. We compared the total and active bacterial communities during the 50-day incubations by using phylogenic microarrays and 454 pyrosequencing to identify microorganisms and functional genes associated with PCE degradation to ethene. By use of this integrative approach, both the key community members and the ecological functions concomitant with complete PCE degradation could be determined, including the presence and activity of microbial community members responsible for producing hydrogen and acetate, which are critical for Dehalococcoides-mediated PCE degradation. In addition, by correlation of chemical data and phylogenic microarray data, we identified several bacteria that could potentially oxidize hydrogen. These results demonstrate that PCE degradation is dependent on some microbial community members for production of appropriate metabolites, while other members of the community compete for hydrogen in soil at low redox potentials.

Degradation kinetics of chlorinated aliphatic hydrocarbons by methane oxidizers naturally-associated with wetland plant roots.

Chlorinated aliphatic hydrocarbons (CAHs) are common groundwater contaminants that can be removed from the environment by natural attenuation processes. CAH biodegradation can occur in wetland environments by reductive dechlorination as well as oxidation pathways. In particular, CAH oxidation may occur in vegetated wetlands, by microorganisms that are naturally associated with the roots of wetland plants. The main objective of this study was to evaluate the cometabolic degradation kinetics of the CAHs, cis-1,2-dichloroethene (cisDCE), trichloroethene (TCE), and 1,1,1-trichloroethane (1,1,1TCA), by methane-oxidizing bacteria associated with the roots of a typical wetland plant in soil-free system. Laboratory microcosms with washed live roots investigated aerobic, cometabolic degradation of CAHs by the root-associated methane-oxidizing bacteria at initial aqueous [CH4] ~1.9mgL(-1), and initial aqueous [CAH] ~150µgL(-1); cisDCE and TCE (in the presence of 1,1,1TCA) degraded significantly, with a removal efficiency of approximately 90% and 46%, respectively. 1,1,1TCA degradation was not observed in the presence of active methane oxidizers. The pseudo first-order degradation rate-constants of TCE and cisDCE were 0.12±0.01 and 0.59±0.07d(-1), respectively, which are comparable to published values. However, their biomass-normalized degradation rate constants obtained in this study were significantly smaller than pure-culture studies, yet they were comparable to values reported for biofilm systems. The study suggests that CAH removal in wetland plant roots may be comparable to processes within biofilms. This has led us to speculate that the active biomass may be on the root surface as a biofilm. The cisDCE and TCE mass losses due to methane oxidizers in this study offer insight into the role of shallow, vegetated wetlands as an environmental sink for such xenobiotic compounds.

Biodegradation of cis-dichloroethene and vinyl chloride in the capillary fringe.

Volatile chlorinated compounds are major pollutants in groundwater, and they pose a risk of vapor intrusion into buildings. Vapor intrusion can be prevented by natural attenuation in the vadose zone if biodegradation mechanisms can be established. In this study, we tested the hypothesis that bacteria can use cis-dichloroethene (cis-DCE) or vinyl chloride (VC) as an electron donor in the vadose zone. Anoxic water containing cis-DCE or VC was pumped continuously beneath laboratory columns that represented the vadose zone. Columns were inoculated with Polaromonas sp. strain JS666, which grows aerobically on cis-DCE, or with Mycobacterium sp. JS60 and Nocardiodes sp. JS614 that grow on VC. Complete biodegradation with fluxes of 84 ± 15 µmol of cis-DCE · m(-2) · hr(-1) and 218 ± 25 µmole VC·m(-2) · h(-1) within the 23 cm column indicated that microbial activities can prevent the migration of cis-DCE and VC vapors. Oxygen and volatile compound profiles along with enumeration of bacterial populations indicated that most of the biodegradation took place in the first 10 cm above the saturated zone within the capillary fringe. The results revealed that cis-DCE and VC can be biodegraded readily at the oxic/anoxic interfaces in the vadose zone if appropriate microbes are present.

Chlorine isotope effects from isotope ratio mass spectrometry suggest intramolecular C-Cl bond competition in trichloroethene (TCE) reductive dehalogenation.

Chlorinated ethenes are prevalent groundwater contaminants. To better constrain (bio)chemical reaction mechanisms of reductive dechlorination, the position-specificity of reductive trichloroethene (TCE) dehalogenation was investigated. Selective biotransformation reactions (i) of tetrachloroethene (PCE) to TCE in cultures of Desulfitobacterium sp. strain Viet1; and (ii) of TCE to cis-1,2-dichloroethene (cis-DCE) in cultures of Geobacter lovleyi strain SZ were investigated. Compound-average carbon isotope effects were -19.0‰ ± 0.9‰ (PCE) and -12.2‰ ± 1.0‰ (TCE) (95% confidence intervals). Using instrumental advances in chlorine isotope analysis by continuous flow isotope ratio mass spectrometry, compound-average chorine isotope effects were measured for PCE (-5.0‰ ± 0.1‰) and TCE (-3.6‰ ± 0.2‰). In addition, position-specific kinetic chlorine isotope effects were determined from fits of reactant and product isotope ratios. In PCE biodegradation, primary chlorine isotope effects were substantially larger (by -16.3‰ ± 1.4‰ (standard error)) than secondary. In TCE biodegradation, in contrast, the product cis-DCE reflected an average isotope effect of -2.4‰ ± 0.3‰ and the product chloride an isotope effect of -6.5‰ ± 2.5‰, in the original positions of TCE from which the products were formed (95% confidence intervals). A greater difference would be expected for a position-specific reaction (chloride would exclusively reflect a primary isotope effect). These results therefore suggest that both vicinal chlorine substituents of TCE were reactive (intramolecular competition). This finding puts new constraints on mechanistic scenarios and favours either nucleophilic addition by Co(I) or single electron transfer as reductive dehalogenation mechanisms.

Kinetics of the aerobic co-metabolism of 1,1-dichloroethylene by Achromobacter sp.: a novel benzene-grown culture.

Batch experiments were performed for the aerobic co-metabolism of 1,1-dichloroethylene (1,1-DCE) by Achromobacter sp., identified by gene sequencing of 16S rRNA and grown on benzene. Kinetic models were employed to simulate the co-metabolic degradation of 1,1-DCE, and relevant parameters were obtained by non-linear least squares regression. Benzene at 90 mg L(-1) non-competitively inhibited degradation of 1,1-DCE (from 125 to 1,200 µg L(-1)). The maximum specific utilization (kc) rate and the half-saturation constant (Kc) for 1,1-DCE were 54 ± 0.85 µg h(-1) and 220 ± 6.8 µg L(-1), respectively; the kb and Kb for benzene were 13 ± 0.18 mg h(-1) and 28 ± 0.42 mg L(-1), respectively. This study provides a theoretical basis to predict the natural attenuation when benzene and 1,1-DCE occur as co-contaminants.