Cometabolic degradation of TCE vapors in a foamed emulsion bioreactor

Effective cometabolic biodegradation of trichloroethylene (TCE) vapors in a novel gas-phase bioreactor called the foamed emulsion bioreactor (FEBR) was demonstrated. Toluene vapors were used as the primary growth substrate for Burkholderia cepacia G4 which cometabolically biodegraded TCE. Batch operation of the reactor with respect to the liquid feed showed a drastic decrease of TCE and toluene removal over time, consistent with a loss of metabolic activity caused by the exposure to TCE metabolites. Sustained TCE removal could be achieved when continuous feeding of mineral medium was implemented, which supported cell growth and compensated for the deactivation of cells. The FEBR exhibited its highest TCE removal efficiencies (82-96%) and elimination capacities (up to 28 gTCE m(-3) h(-1)) when TCE and toluene vapors were fed sequentially to circumvent the competitive inhibition by toluene. The TCE elimination capacity was 2-1000 times higher than reported in other gas-phase biotreatment reports. During the experiments, 85-101% of the degraded TCE chlorine was recovered as chloride. Overall, the results suggestthatthe FEBR can be a very effective system to treat TCE vapors cometabolically.     

Stable carbon isotope fractionation during aerobic biodegradation of chlorinated ethenes

Stable isotope analysis is recognized as a powerful tool for monitoring, assessing, and validating in-situ bioremediation processes. In this study, kinetic carbon isotope fractionation factors (epsilon) associated with the aerobic biodegradation of vinyl chloride (VC), cis-1,2-dichloroethylene (cDCE), and trichloroethylene (TCE) were examined. Of the three solvents, the largest fractionation effects were observed for biodegradation of VC. Both metabolic and cometabolic VC degradation were studied using Mycobacterium aurum L1 (grown on VC), Methylosinus trichosporium OB3b (grown on methane), Mycobacterium vaccae JOB5 (grown on propane), and two VC enrichment cultures seeded from contaminated soils of Alameda Point and Travis Air Force Base, CA. M. aurum L1 caused the greatest fractionation (epsilon = -5.7) while for the cometabolic cultures, epsilon values ranged from -3.2 to -4.8. VC fractionation patterns for the enrichment cultures were within the range of those observed for the metabolic and cometabolic cultures (epsilon = -4.5 to -5.5). The fractionation for cometabolic degradation of TCE by Me. trichosporium OB3b was low (epsilon = -1.1), while no quantifiable carbon isotopic fractionation was observed during the cometabolic degradation of cDCE. For all three of the tested chlorinated ethenes, isotopic fractionation measured during aerobic degradation was significantly smaller than that reported for anaerobic reductive dechlorination. This study suggests that analysis of compound-specific isotopic fractionation could assist in determining whether aerobic or anaerobic degradation of VC and cDCE predominates in field applications of in-situ bioremediation. In contrast, isotopic fractionation effects associated with metabolic and cometabolic reactions are not sufficiently dissimilar to distinguish these processes in the field.     

Quantification of bacterial chemotaxis in porous media using magnetic resonance imaging

Bacterial chemotaxis has the potential to enhance biodegradation of organic contaminants in polluted groundwater systems. However, studies of bacterial chemotaxis in porous media are scarce. In this study we use magnetic resonance imaging (MRI) for the noninvasive measurement of changes in bacterial-density distributions in a packed column at a spatial resolution of 330 microm as a function of time. We analyze both the diffusive and the chemotactic behavior of Pseudomonas putida F1 in the presence of the chemical stimulus trichloroethylene (TCE). The migration of motile bacteria in experiments without TCE was described using an effective motility coefficient, whereas the presence of TCE required addition of a nonzero chemotactic sensitivity coefficient, indicating a significant response to TCE. The need for a chemotactic sensitivity term was justified by a test for statistical significance. This study represents the first quantification of bacterial chemotactic parameters within a packed column. For conditions under which chemotaxis occurs in porous media, it may potentially be exploited to significantly improve rates of in situ pollutant biodegradation in the subsurface environment, particularlyfor pollutants dissolved in water trapped in low-permeability formations or lenses.     

A multitracer test proving the reliability of Rayleigh equation-based approach for assessing biodegradation in a BTEX contaminated aquifer

Compound-specific stable isotope analysis (CSIA) is one of the most important methods for assessing biodegradation activities in contaminated aquifers. Although the concept is straightforward, the proof that the method cannot be only used for a qualitative analysis but also to quantify biodegradation in the subsurface was missing. We therefore performed a multitracer test in the field with ring-deuterated (d5) and completely (d8) deuterium-labeled toluene isotopologues (400 g) as reactive tracers as well as bromide as a conservative tracer. The compounds were injected into the anoxic zone of a BTEX plume located down-gradient of the contaminant source. Over a period of 4.5 months the tracer concentrations were analyzed at two control planes located 24 and 35 m downgradient of the injection well. Deuterium-labeled benzylsuccinate was found in the aquifer, indicating the anaerobic biodegradation of deuterated toluene via the benzylsuccinate synthase pathway. Three independent methods were applied to quantify biodegradation of deuterated toluene. First, fractionation of toluene-d8 and toluene-d5 using the Rayleigh equation and an appropriate laboratory-derived isotope fractionation factor was used for the calculation of the microbial decomposition of deuterated toluene isotopologues (CSIA-method). Second, the biodegradation was quantified by the changes of the concentrations of deuterated toluene relative to bromide. Both methods gave similar results, implying that the CSIA-method is a reliable tool to quantify biodegradation in contaminated aquifers. The results of both methods yielded a biodegradation of deuterated toluene isotopologues of approximately 23-29% for the first and 44-51% for the second control plane. Third, the mineralization of deuterated toluene isotopologues was verified by determination of the enrichment of deuterium in the groundwater. This method indicated that parts of deuterium were assimilated into the biomass of toluene degrading microorganisms.     

Continuous treatment of gas-phase trichloroethylene by Burkholderia cepacia G4 in a two-stage continuous stirred tank reactor/trickling biofilter system

A two-stage continuous stirred tank reactor/trickling biofilter system was developed and operated for continuous treatment of gas-phase trichloroethylene (TCE) by Burkholderia cepacia. The maximum TCE elimination capacity was 28.0 mg TCE/l.d, and complete removal of TCE was obtained for inlet loading below 25.3 mg TCE/l.d. The reactor system was stably operated for more than 3 months.     

Cometabolism of cis-1,2-dichloroethene by aerobic cultures grown on vinyl chloride as the primary substrate

An aerobic enrichment culture was grown on vinyl chloride (VC) as the sole source of carbon and energy. In the absence of VC, the enrichment culture cometabolized cis-1,2-dichloroethene (cDCE) and, to a lesser extent, trans1,2-dichloroethene (tDCE), beginning with oxidation to the corresponding DCE-epoxides. When provided with VC (1.3 mM) and cDCE (0.2-0.3 mM), the enrichment culture cometabolized repeated additions of cDCE for over 85 days. Cometabolism of repeated additions of tDCE was also demonstrated but at a lower ratio of nongrowth substrate to VC. VC-grown Pseudomonas aeruginosa MF1 (previously isolated from the enrichment culture) also readily cometabolizes cDCE, with an observed transformation capacity (Tc,obs) of 0.82 micromol of cDCE/mg of total suspended solids (TSS). When provided with VC and cDCE, MF1 did not begin cometabolizing cDCE until nearly all of the VC was consumed. The presence of cDCE reduces the maximum specific rate of VC utilization. A kinetic model was developed that describes these phenomena via Monod parameters for substrate and nongrowth substrate, plus inactivation and inhibition coefficients. MF1 did not show any cometabolic activity on tDCE or trichloroethene and very limited activity on 1,1-DCE (Tc,obs = 2 x 10(-5) micromol/mg TSS). Above 40 microM, tDCE and TCE noticeably increased the maximum specific rate of VC utilization, even though neither compound was consumed during or after VC consumption. High concentrations of 1,1-DCE (950 microM) completely inhibited VC biodegradation. As there is currently no evidence for aerobic biodegradation of cDCE as a sole source of carbon and energy, the results of this study provide a potential explanation for in situ disappearance of cDCE when the only other significant substrate available is VC. It is fortuitous that the VC-grown cultures tested exhibit their highest cometabolic activity toward cDCE, because it is the predominant DCE isomer formed during anaerobic reductive dechlorination of trichloroethene and tetrachloroethene.     

Field evaluation of in situ source reduction of trichloroethylene in groundwater using bioenhanced in-well vapor stripping

Two technologies in combination, cometabolic bioremediation and in-well vapor stripping, were applied to reduce trichloroethylene (TCE) concentrations in groundwater at a contaminant source area without the need to pump contaminated groundwater to the surface for treatment. The vapor-stripping well reduced source TCE concentrations (as high as 6-9 mg/L) by over 95%. Effluent from the well then flowed to two bioremediation wells, where additional reductions of approximately 60% were achieved. TCE removal was extensively monitored (for research and not regulatory purposes) using an automated system that collected samples about every 45 min at 55 locations over an area of approximately 50 x 60 m2. During 4.5 months of system operation, total TCE mass removal was 8.1 kg, 7.1 kg of which resulted from in-well vapor stripping and 1.0 kg from biotreatment. The system reduced the average TCE concentration of about 3000 microg/L in the source-zone groundwater to about 250 microg/L in water leaving the treatment zone, effecting greater than 92% TCE removal. A 6 month rebound study after system operation ceased found TCE concentrations then increased significantly in the treatment zone due to diffusion from the fractured rock below and perhaps other processes, with mass increases of about 1.5 kg in the lower aquifer and 0.3 kg in the upper aquifer.     

Kinetics of 1,4-dioxane biodegradation by monooxygenase-expressing bacteria

1,4-Dioxane is a probable human carcinogen, and an important emerging water contaminant. In this study, the biodegradation of dioxane by 20 bacterial isolates was evaluated, and 13 were found to be capable of transforming dioxane. Dioxane served as a growth substrate for Pseudonocardia dioxanivorans CB1190 and Pseudonocardia benzenivorans B5, with yields of 0.09 g protein g dioxane(-1) and 0.03 g protein g dioxane(-1), respectively. Cometabolic transformation of dioxane was observed for monooxygenase-expressing strains that were induced with methane, propane, tetrahydrofuran, or toluene including Methylosinus trichosporium OB3b, Mycobacterium vaccae JOB5, Pseudonocardia K1, Pseudomonas mendocina KR1, Ralstonia pickettii PKO1, Burkholderia cepacia G4, and Rhodococcus RR1. Product toxicity resulted in incomplete dioxane degradation for many of the cometabolic reactions. Brief exposure to acetylene, a known monooxygenase inhibitor, prevented oxidation of dioxane in all cases, supporting the hypothesis that monooxygenase enzymes participated in the transformation of dioxane by these strains. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene-2- and toluene-4-monooxygenases of G4, KR1 and PKO1 were also capable of cometabolic dioxane transformation. Dioxane oxidation rates measured at 50 mg/L ranged from 0.01 to 0.19 mg hr(-1) mg protein(-1) for the metabolic processes, 0.1-0.38 mg hr(-1) mg protein(-1) for cometabolism by the monooxygenase-induced strains, and 0.17-0.60 mg hr(-1) mg protein(-1) for the recombinant strains. Dioxane was not degraded by M. trichosporium OB3b expressing particulate methane monooxygenase, Pseudomonas putida mt-2 expressing a toluene side-chain monooxygenase, and PseudomonasJS150 and Pseudomonas putida F1 expressing toluene-2,3-dioxygenases. This is the first study to definitively show the role of monooxygenases in dioxane degradation using several independent lines of evidence and to describe the kinetics of metabolic and cometabolic dioxane degradation.     

Monitoring biodegradation of methyl tert-butyl ether (MTBE) using compound-specific carbon isotope analysis

Methyl tert-butyl ether (MTBE), the most common gasoline oxygenate, is frequently detected in surface water and groundwater. The aim of this study was to evaluate the potential of compound-specific isotope analysis to assess in situ biodegradation of MTBE in groundwater. For that purpose, the effect of relevant physical and biological processes on carbon isotope ratios of MTBE was evaluated in laboratory studies. Carbon isotope fractionation during organic phase/gas-phase partitioning (0.50 +/- 0.15@1000), aqueous phase/gas-phase partitioning (0.17 +/- 0.05@1000), and organic phase/aqueous-phase partitioning (0.18 +/- 0.24@1000) was small in comparison to carbon isotope fractionation measured during biodegradation of MTBE in microcosms based on aquifer sediments of the Borden site. In experiments with MTBE as the only substrate and a cometabolic experiment with 3-methypentane as primary substrate, MTBE became enriched in 13C by 5.1 to 6.9@1000 after 95 to 97% degradation. For both experiments, similar isotopic enrichment factors were obtained (-1.52 +/- 0.06 to -1.97 +/- 0.05@1000). Biodegradation of TBA, which accumulated transiently in the cometabolic microcosms, was also accompanied by carbon isotope fractionation, with an isotopic enrichment factor of -4.21 +/- 0.07@1000. This study suggests that carbon isotope analysis is a potential tool to trace in situ biodegradation of MTBE and TBA and thus to better understand the fate of these contaminants in the environment.     

Stable isotope evidence for biodegradation of chlorinated ethenes at a fractured bedrock site

Stable carbon isotope analysis of chlorinated ethenes and ethene was performed at a site contaminated with trichloroethene (TCE), a dense non-aqueous phase liquid (DNAPL). The site is located in fractured bedrock and had variable groundwater hydraulic gradients during the study due to a local excavation project. Previous attempts to biostimulate a pilot treatment area at the site resulted in the production of cis-1,2-dichloroethene (cis-DCE), the first product of reductive dechlorination of TCE. Cis-DCE concentrations accumulated however, and there was no appreciable production of the breakdown products from further reductive dechlorination, vinyl chloride (VC) and ethene (ETH). Consequently, the pilot treatment area was bioaugmented with a culture of KB-1, a natural microbial consortium known to completely reduce TCE to nontoxic ETH. Due to ongoing dissolution of TCE from DNAPL in the fractured bedrock, and to variable hydraulic gradients, concentration profiles of dissolved TCE and its degradation products cis-DCE, VC, and ETH could not convincingly confirm biodegradation of the chlorinated ethenes. Isotopic analysis of cis-DCE and VC, however, demonstrated that biodegradation was occurring in the pilot treatment area. The isotope values of cis-DCE and VC became significantly more enriched in 13C over the last two sampling dates (in one well from -17.6%o to -12.8%o and from -22.5%o to -18.2%o for cis-DCE and VC, respectively). Quantification of the extent of biodegradation in the pilot treatment area using the Rayleigh model indicated that, depending on the well, between 21.3% and 40.7% of the decrease in cis-DCE and between 15.2% and 36.7% of the decrease in VC concentrations can be attributed to the effects of biodegradation during this time period. Within each well, the isotope profile of TCE remained relatively constant due to the continuous input of undegraded TCE due to DNAPL dissolution.     

Evaluation of isotopic enrichment factors for the biodegradation of chlorinated ethenes using a parameter estimation model: toward an improved quantification of biodegradation

A model was developed to predict the concentrations of chlorinated ethenes and ethene during sequential reductive dechlorination of tetrachloroethene (PCE) from stable carbon isotope values using Rayleigh model principles and specified isotopic enrichment factors for each step of dechlorination. The model was tested using three separate datasets of concentration and isotope values measured during three experiments involving the degradation of PCE to vinyl chloride (VC), trichloroethene (TCE) to ethene, and cis-1,2-dichloroethene (cDCE) to ethene. The model was then coupled to a parameter estimation method to estimate values for the isotopic enrichment factors of TCE, cDCE, and VC when they are intermediates in the dechlorination to ethene. The enrichment factors estimated for TCE and cDCE when they were intermediates in biodegradation experiments were close to or within the published range of enrichment factors determined from experiments where TCE or cDCE were the initial substrates. In contrast, the enrichment factors determined by parameter estimation for experiments in which VC was an intermediate in biodegradation experiments were consistently more negative (by approximately 10 per thousandth) than the most negative published enrichment factor determined from experiments where VC was the initial substrate. This finding suggests that the range of enrichment factors for VC dechlorination may not be as narrow as previously suggested (-21.5 per thousandth to -26.6 per thousandth) and that fractionation during VC dechlorination when VC is an intermediate compound may be significantly larger than when VC is the initial substrate. These findings have important implications both for the current practice of extrapolating laboratory-derived isotopic enrichment factors to quantify biodegradation of chlorinated ethenes in the field and for understanding the details of enzymatic reductive dechlorination.     

微生物处理甲苯废气的菌种选育

从土壤中分离筛选出以甲苯为惟一碳源的5种细菌菌株，测定茵体浓度随时间变化曲线，以较优菌种ZD5作为出发菌株进行紫外诱变，获得优势诱变菌株ZD5A．初步鉴定菌株ZD5A为假单胞菌，命名为Pseudomonas sp．ZD5A．得出甲苯降解和茵体浓度变化有相关性，且这种较简便降解挥发性有机废气的菌种评选方法是可行的．     

Trichloroethylene and tetrachloroethylene elimination from the air by means of a hybrid bioreactor with immobilized biomass

Two-phase bioreactors consisting of bacterial consortium in suspension and sorbents with immobilized biomass were used to treat waste air containing chlorinated ethenes, trichloroethylene (TCE) and tetrachloroethylene (PCE). Synthetic municipal sewage was used as the medium for bacterial growth. The system was operated with loadings in the range 1.48-4.76 gm(-3)h(-1) for TCE and 1.49-5.96 gm(-3)h(-1) for PCE. The efficiency of contaminant elimination was 55-86% in the bioreactor with wood chips and 33-89% in the bioreactor filled with zeolite. The best results were observed 1 week after the pollutant loading was increased. However, in these conditions, the stability of the process was not achieved. In the next 7 days the effectiveness of the system decreased. Contaminant removal efficiency, enzymatic activity and the biomass content were all diminished. The system was working without being supplied with additional hydrocarbons as the growth-supporting substrates. It is assumed that ammonia produced during the transformation of wastewater components induced enzymes for the cometabolic degradation of TCE and PCE. However, the evaluation of nitrogen compound transformations in the system is difficult due to the sorption on carriers and the combined processes of nitrification and the aerobic denitrification. An applied method of air treatment is advantageous from both economic and environmental point of views.     

Trichloroethylene removal from groundwater in flow-through columns simulating a permeable reactive barrier constructed with plant mulch

Groundwater contaminated with TCE is commonly treated with a permeable reactive barrier (PRB) constructed with zero-valence iron. The cost of iron has driven a search for less costly alternatives, and composted plant mulch has been used as an alternative at several sites. A column study was conducted that simulated conditions in a PRB at Altus Air Force Base, Oklahoma. The reactive matrix was 50% (v/v) shredded tree mulch, 10% cotton gin trash, and 40% sand. The mean residence time of groundwater in the columns was 17 days. The estimated retardation factor for TCE was 12. TCE was supplied at concentrations near 20 microM. Over 793 days of operation, concentrations of TCE in the column effluents varied from 0.1% to 2% of the column influents. Concentrations of cis-DCE, vinyl chloride, ethylene, ethane, and acetylene could account for 1% of the TCE that was removed; however, up to 56% of 13C added as [1,2-13C] TCE in the column influents was recovered as 13C in carbon dioxide. After 383 and 793 d of operation, approximately one-half of the TCE removal was associated with abiotic reactions with FeS that accumulated in the reactive matrix.     

In situ assessment of biodegradation potential using biotraps amended with 13C-labeled benzene or toluene

Stable isotope fractionation analysis of an aquifer heavily contaminated with benzene (up to 850 mg L(-1)) and toluene (up to 50 mg L(-1)) at a former hydrogenation plant in Zeitz (Saxonia, Germany) has suggested that significant biodegradation of toluene was occurring. However, clear evidence of benzene biodegradation has been lacking at this site. Determining the fate of benzene is often a determining factor in regulatory approval of a risk-based management strategy. The objective of the work described here was the demonstration of a new tool that can be used to provide proof of biodegradation of benzene or other organics by indigenous microorganisms under actual aquifer conditions. Unique in situ biotraps containing Bio-Sep beads, amended with 13C-labeled or 12C nonlabeled benzene and toluene, were deployed at the Zeitz site for 32 days in an existing groundwater monitoring well and used to collect and enrich microbial biofilms. Lipid biomarkers or remaining substrate was extracted from the beads and analyzed by mass spectrometry and molecular methods. Isotopic analysis of the remaining amounts of 13C-labeled contaminants (about 15-18% of the initial loading) showed no alteration of the 12C/13C ratio during incubation. Therefore, no measurable exchange of labeled compounds in the beads by the nonlabeled compounds in the aquifer materials occurred. Isotopic ratio analysis of microbial lipid fatty acids (as methyl ester derivatives) from labeled benzene- and toluene-amended biotraps showed 13C enrichment in several fatty acids of up to delta (13C) 13400%o, clearly verifying benzene and toluene biodegradation and the transformation of the labeled carbon into biomass by indigenous organisms under aquifer conditions. Fatty acid profiles of total lipid fatty acids and the phospholipid fatty acid fraction and their isotopic composition showed significant differences between benzene- and toluene-amended biotraps, suggesting that different microbial communities were involved in the biodegradation of the two compounds.     

Stable carbon isotope evidence for intrinsic bioremediation of tetrachloroethene and trichloroethene at area 6, Dover Air Force Base

Area 6 at Dover Air Force Base (Dover, DE) has been the location of an in-depth study by the RTDF (Remediation Technologies Development Forum Bioremediation of Chlorinated Solvents Action Team) to evaluate the effectiveness of natural attenuation of chlorinated ethene contamination in groundwater. Compound-specific stable carbon isotope measurements for dissolved PCE and TCE in wells distributed throughout the anaerobic portion of the plume confirm that stable carbon isotope values are isotopically enriched in 13C consistent with the effects of intrinsic biodegradation. During anaerobic microbial reductive dechlorination of chlorinated hydrocarbons, the light (12C) versus heavy isotope (13C) bonds are preferentially degraded, resulting in isotopic enrichment of the residual contaminant in 13C. To our knowledge, this study is the first to provide definitive evidence for reductive dechlorination of chlorinated hydrocarbons at a field site based on the delta13C values of the primary contaminants spilled at the site, PCE and TCE. For TCE, downgradient wells show delta13C values as enriched as -18.0/1000 as compared to delta13C values for TCE in the source zone of -25.0 to -26.0/1000. The most enriched delta13C value on the site was observed at well 236, which also contains the highest concentrations of cis-DCE, VC, and ethene, the daughter products of reductive dechlorination. Stable carbon isotope signatures are used to quantify the relative extent of biodegradation between zones of the contaminant plume. On the basis of this approach, it is estimated that TCE in downgradient well 236 is more than 40% biodegraded relative to TCE in the proposed source area.     

Pd-catalyzed TCE dechlorination in water: effect of [H2](aq) and H2-utilizing competitive solutes on the TCE dechlorination rate and product distribution

The aqueous-phase H2 concentration ([H2](aq)) and the presence of H2-utilizing competitive solutes affect TCE dechlorination efficiency in Pd-based in-well treatment reactors. The effect of [H2](aq) and H2-utilizing competing solutes (cis-DCE, trans-DCE, 1,1-DCE, dissolved oxygen (DO), nitrite, nitrate) on the TCE transformation rate and product distribution were evaluated using 100 mg/L of a powdered Pd-on-Al2O3 catalysts in batch reactors or 1.0 g of a 1.6-mm Pd-on-gamma-Al2O3 catalyst in column reactors. The TCE dechlorination rate constant decreased by 55% from 0.034 +/- 0.006 to 0.015 +/- 0.001 min-1 when the [H2](aq) decreased from 1000 to 100 microM and decreased sharply to 0.0007 +/- 0.0003 min-1 when the [H2](aq) decreased from 100 to 10 microM. Production of reactive chlorinated intermediates and C4-C6 radical coupling products increased with decreasing [H2](aq). At an [H2](aq) of 10 microM (P/Po = 0.01), DCE isomers and vinyl chloride accounted for as much as 9.8% of the TCE transformed at their maximum but disappeared thereafter, and C4-C6 radical coupling products accounted for as much as 18% of TCE transformed. The TCE transformation rate was unaffected by the presence of cis-DCE (202 microM), trans-DCE (89 microM), and 1,1-DCE (91 microM), indicating that these compounds do not compete with TCE for catalyst active sites. DO is twice as reactive as TCE but had no effect on TCE conversion in the column below a concentration of 370 microM (11.8 mg/L), indicating that DO and TCE will not compete for active catalyst sites at typical groundwater DO concentrations. TCE conversion in the column was reduced by as much as a factor of 10 at influent DO levels greater than 450 mM (14.3 mg/L) because the [H2](aq) fell below 100 microM due to H2 utilized in DO conversion. Nitrite reacts 2-5 times slower than TCE and reduced TCE conversion by less than 4% at a concentration of 6630 microM (305 mg/L). Nitrate was not reactive and did not effect TCE conversion at a concentration of 1290 microM (80 mg/L).     

Isotopic and geochemical assessment of in situ biodegradation of chlorinated hydrocarbons

Currently there is no in situ method to detect and quantify complete mineralization of chlorinated hydrocarbons (CHCs) to CO2. Combined isotopic measurements in conjunction with traditional chemical techniques were used to assess in situ biodegradation of trichloroethylene (TCE) and carbon tetrachloride (CT). Vadose zone CHC, ethene, ethane, methane, O2, and CO2 concentrations were analyzed using gas chromatography over 114 days at the Savannah River Site. delta13C of CHC and delta13C and 14C of vadose zone CO2, sediment organic matter, and groundwater dissolved inorganic carbon (DIC)were measured. Intermediate metabolites of TCE and CT accounted for < or = 10% of total CHCs. Delta13C of cis-1,2-dichloroethylene (DCE) was always heavier than TCE indicating substantial DCE biodegradation. 14C-CO2 values ranged from 84 to 128 percent modern carbon (pMC), suggesting that plant root-respired CO2 was dominant. 14C-CO2 values decreased over time (up to 12 pMC), and contaminated groundwater 14C-DIC (76 pMC) was substantially depleted relative to the control (121 pMC). 14C provided a direct measure of complete CHC mineralization in vadose zone and groundwater in situ and may improve remediation strategies.     

Bioreduction of trichloroethene using a hydrogen-based membrane biofilm reactor

A H2-based, denitrifying membrane-biofilm reactor (MBfR) was effective for removing trichloroethene (TCE) by reductive dechlorination. When TCE was first added to the MBfR, reductive dechlorination took place immediately and then increased over 18 weeks, and TCE was completely dechlorinated to ethene by about 120 days. These results indicate that TCE-dechlorinating bacteria were present naturally in the H2-based biofilm, and that enrichment for TCE-dechlorinating bacteria occurred. Dehalococcoides were documented in the MBfR biofilm before and after TCE feeding. Their proportion, quantified using the 16S rRNA gene, increased from 2.9 to 12% after TCE addition. This is the first report in which Dehalococcoides are proven to be present as part of an autotrophic biofilm community active in reductive dechlorination of TCE to ethene in a laboratory controlled experiment. Based on the complete reduction of TCE to ethene, the 16S rRNA clone libraries results, and the amount of tceA and bvcA, it appears that at least two Dehalococcoides strains were present in the enriched biofilm. One of them seems to be a new strain that is unique for having tceA and bvcA reductive dehalogenases.     

Laboratory study of treatment of trichloroethene by chemical oxidation followed by bioremediation

Studies were conducted with columns containing soil and emplaced trichloroethene (TCE) to investigate the potential for TCE source zone remediation with chemical oxidation followed by biologically mediated reductive dehalogenation. Following permanganate flushing of four columns, which resulted in rapid but incomplete removal of TCE DNAPL, no biological activity was observed following the addition of distilled water amended with ethanol and acetate, including two of the four columns that were bioaugmented with a TCE-dechlorinating microbial culture. Flushing with unsterilized site groundwater led to consumption of acetate and ethanol, accompanied by manganese reduction and methanogenesis. Reductive dechlorination of TCE to cis-1,2-dichloroethene (cis-DCE) followed the onset of ethanol and acetate biodegradation in bioaugmented columns only. Partial dechlorination of TCEto ethene was observed only in one of the bioaugmented columns after it was inoculated for a third time. At the end of the study (290 days), a trace amount of cis-DCE was observed in one of the two columns which was not bioaugmented. Reduced conditions created by biostimulation were also conducive to reduction of Mn(IV) from MnO2 in both bioaugmented and nonbioaugmented columns resulting in an increased dissolved manganese (Mn2+) concentration in groundwater.