Effects of the nonionic surfactant tween 80 on microbial reductive dechlorination of chlorinated ethenes

Recent field studies have indicated synergistic effects of coupling microbial reductive dechlorination with physicochemical remediation (e.g., surfactant flushing) of dense nonaqueous phase liquid (DNAPL) source zones. This study explored chlorinated ethene (e.g., tetrachloroethene [PCE]) dechlorination in the presence of 50-5000 mg/L Tween 80, a nonionic surfactant employed in source zone remediation. Tween 80 did not inhibit dechlorination by four pure PCE-to-cis-1,2-dichloroethene (cis-DCE) or PCE-to-trichloroethene (TCE) dechlorinating cultures. In contrast, cis-DCE-dechlorinating Dehalococcoides isolates (strain BAV1 and strain FL2) failed to dechlorinate in the presence of Tween 80. Bio-Dechlor INOCULUM (BDI), a PCE-to-ethene dechlorinating consortium, produced cis-DCE in the presence of Tween 80, further suggesting that Tween 80 inhibits dechlorination by Dehalococcoides organisms. Quantitative real-time PCR analysis applied to BDI revealed that the number of Dehalococcoides cells decayed exponentially (R(2) = 0.85) according to the Chick-Watson disinfection model (pseudo first-order decay rate of 0.13+/-0.02 day(-1)) from an initial value of 6.6 +/-1.5 x 10(8) to 1.3+/-0.8 x 10(5) per mL of culture after 58 days of exposure to 250 mg/L Tween 80. Although Tween 80 exposure prevented ethene formation and reduced Dehalococcoides cell numbers, Dehalococcoides organisms remained viable, and dechlorination activity pist cis-DCE was recovered following the removal of Tween 80. These findings suggest that sequential Tween 80 flushing followed by microbial reductive dechlorination is a promising strategy for remediation of chlorinated ethene-impacted source zones.     

Comparative analysis of three tetrachloroethene to ethene halorespiring consortia suggests functional redundancy

Three anaerobic, dechlorinating consortia were enriched from different sites using methanol and tetrachloroethene (PCE) and maintained for approximately 3 years. These consortia were evaluated using chemical species analysis including distribution of dechlorination products, production of organic acids and methane, and using qualitative and quantitative PCR (qPCR), terminal restriction fragment length polymorphism (TRFLP), and denaturing gradient gel electrophoresis (DGGE) with primers specific to Dehalococcoides 16S rRNA gene sequences. TRFLP and analysis of organic acids revealed differing fermentative populations in each consortium, which were dominated by acetogens. Monitoring methane production combined with qPCR for archaea showed that complete dechlorination of PCE-to-ethene occurred in the presence and absence of methanogens. The 16S rRNA gene-based analyses demonstrated that enrichment with PCE resulted in dechlorinating communities dominated by Dehalococcoides and Dehalobacter, and that up to four different PCE-dechlorinating organisms coexisted in one consortium. Further, the DGGE analysis suggested that at least one consortium contained multiple Dehalococcoides strains. The combined analysis of 16S rRNA and reductive dehalogenase genes suggested that one consortium contained a member of the Dehalococcoides "Cornell" group with the ability to respire VC.     

Stable carbon isotope enrichment factors for cis-1,2-dichloroethene and vinyl chloride reductive dechlorination by Dehalococcoides

Compound-specific stable isotope analysis (CSIA) is a promising tool for monitoring in situ microbial activity, and enrichment factors (ε values) determined using CSIA can be employed to estimate compound transformation. Although ε values for some dechlorination reactions catalyzed by Dehalococcoides (Dhc) have been reported, reproducibility between independent experiments, variability between different Dhc strains, and congruency between pure and mixed cultures are unknown. In experiments conducted with pure cultures of Dhc sp. strain BAV1, ε values for 1,1-DCE, cis-DCE, trans-DCE, and VC were -5.1, -14.9, -20.8, and -23.2‰, respectively. The ε value for 1,1-DCE dechlorination was 48.9% higher than the value reported in a previous study, but ε values for other chlorinated ethenes were equal between independent experiments. For the dechlorination of cis-DCE and VC by Dhc strains BAV1, FL2, GT, and VS, average ε values were -18.4 and -23.2‰, respectively. cis-DCE and VC ε values determined in pure Dhc cultures with different reductive dehalogenase genes (e.g., vcrA vs bvcA) varied by less than 36.8 and 8.3%, respectively. In the BDI consortium, ε values for cis-DCE and VC dechlorination were -25.3‰ and -19.9‰, 31.6% higher and 15.3% lower, respectively, compared to the average ε value for Dhc pure cultures. As cis-DCE and VC ε values are all within the same order-of-magnitude and fractionation is always measured during Dhc dechlorination, CSIA may be a valuable approach for monitoring in situ cis-DCE and VC reductive dechlorination.     

Quantifying the effects of 1,1,1-trichloroethane and 1,1-dichloroethane on chlorinated ethene reductive dehalogenases

Mixtures of chlorinated ethenes and ethanes are often found at contaminated sites. In this study, we undertook a systematic investigation of the inhibitory effects of 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA) on chlorinated ethene dechlorination in three distinct Dehalococcoides-containing consortia. To focus on inhibition acting directly on the reductive dehalogenases, dechlorination assays used cell-free extracts prepared from cultures actively dechlorinating trichloroethene (TCE) to ethene. The dechlorination assays were initiated with TCE, cis-1,2-dichloroethene (cDCE), or vinyl chloride (VC) as substrates and either 1,1,1-TCA or 1,1-DCA as potential inhibitors. 1,1,1-TCA inhibited VC dechlorination similarly in cell suspension and cell-free extract assays, implicating an effect on the VC reductases associated with the dechlorination of VC to nontoxic ethene. Concentrations of 1,1,1-TCA in the range of 30-270 μg/L reduced VC dechlorination rates by approximately 50% relative to conditions without 1,1,1-TCA. 1,1,1-TCA also inhibited reductive dehalogenases involved in TCE and cDCE dechlorination. In contrast, 1,1-DCA had no pronounced inhibitory effects on chlorinated ethene reductive dehalogenases, indicating that removal of 1,1,1-TCA via reductive dechlorination to 1,1-DCA is a viable strategy to relieve inhibition.     

Growth of Dehalococcoides strains with chlorophenols as electron acceptors

Dehalococcoides strains reductively dechlorinate a wide variety of halogenated compounds including chlorinated benzenes, biphenyls, naphthalenes, dioxins, and ethenes. Recent genome sequencing of the two Dehalococcoides strains CBDB1 and 195 revealed the presence of 32 and 18 reductive dehalogenase homologous genes, respectively, and therefore suggested an even higher dechlorinating potential than previously anticipated. Here, we demonstrate reductive dehalogenation of chlorophenol congeners by Dehalococcoides strains CBDB1 and 195. Strain CBDB1 completely converted 2,3-dichlorophenol, all six trichlorophenols, all three tetrachlorophenols, and pentachlorophenol to lower chlorinated phenols. Observed dechlorination rates in batch cultures with cell numbers of 10(7) mL(-1) amounted up to 35 microM day(-1). Chlorophenols were preferentially dechlorinated in the ortho position, but also doubly flanked and singly flanked meta- or para-chlorine substituents were removed. We used a newly designed computer-assisted direct cell counting protocol and quantitative PCR to demonstrate that strain CBDB1 uses chlorophenols as electron acceptors for respiratory growth. The growth yield of strain CBDB1 with 2,3-dichlorophenol was 7.6 x 10(13) cells per mol of Cl- released, and the growth rate was 0.41 day(-1). For strain 195, fast ortho dechlorination of 2,3-dichlorophenol, 2,3,4-trichlorophenol, and 2,3,6-trichlorophenol was detected, with only the ortho chlorine removed. Because chlorinated phenolic compounds are widely distributed as natural components in anaerobic environments, our results reveal one mode in which the Dehalococcoides species could have survived through earth history.     

Vinyl bromide as a surrogate for determining vinyl chloride reductive dechlorination potential

Site evaluation for bioremediation of chlorinated ethenes may need treatability studies to assess the reductive dechlorination potential of vinyl chloride (VC). Dehalogenation of vinyl bromide (VB) was investigated as a surrogate measurement for the dechlorination potential of VC. VB dehalogenation rates and kinetics were studied and compared with those of VC by a methanogenic reductive dechlorinating enrichment culture that was dominated by Dehalococcoides species and by microcosms from two chloroethene-contaminated sites. The enrichment culture dehalogenated VB to ethene at higher rates than VC at similar concentrations. VB was dehalogenated with a higher enzyme affinity than was VC, as indicated by their half-velocity constants, 240 +/- 150 and 21 +/- 8 microM, for VC and VB, respectively. Cross-inhibition study exhibited some evidence for competitive inhibition between VC and VB, suggesting that their degradation might be catalyzed by the same enzyme in the culture. Laboratory microcosm studies using subsurface soil and groundwater from two contaminated sites demonstrated that the production of the reductive dehalogenation product (ethene) could be detected faster with VB as a substrate than with VC. As a result, a substantially shorter (up to 5-10 times) incubation time would be required to detect the same level of reductive dehalogenation activity using VB as a surrogate for VC in treatability assessments.     

Growth and yields of dechlorinators, acetogens, and methanogens during reductive dechlorination of chlorinated ethenes and dihaloelimination of 1 ,2-dichloroethane

The population dynamics of a mixed microbial culture dechlorinating trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), 1,2-dichloroethane (1,2-DCA), and vinyl chloride (VC) to ethene were studied. Quantitative PCR revealed that Dehalococcoides, Geobacter, Sporomusa, Spirochaetes, and Methanomicrobiales phylotypes grew in short-term experiments. Both Geobacter and Dehalococcoides populations grew during TCE dechlorination to cDCE, but only Dehalococcoides populations grew during further dechlorination to ethene. The cell yields for Dehalococcoides determined in this study were similar on an electron equivalent basis regardless of the chlorinated compound transformed: (0.9+/-0.3) x 10(8)16S rRNA gene copies/microelectron equivalent (microeeq) ethene produced during cDCE dechlorination, (1.5 +/-0.3) x 10(8) copies/microeeq ethene produced during VC dechlorination, and (1.6+/-0.8) x 10(8) copies/ u,eeq ethene produced during 1,2-DCA dihaloelimination. The yield for the Geobacter population on TCE was estimated to be (1+/-0.5) x 10(8) copies/microeeq cDCE produced. Calculations showed that the Geobacter population was likely responsible for approximately 80% of the TCE dechlorinated to cDCE in this experiment. Acetogenesis by a Sporomusa population was the main competition to dechlorination for reducing equivalents. Sporomusa did not transform any chlorinated substrates tested, but was capable of converting methanol to acetate and hydrogen for dechlorination. Understanding the functions of various populations in mixed communities may explain why Dehalococcoides spp. are active at some sites and not others, and may also assist in optimizing the growth of bioaugmentation cultures, both in the laboratory and in the field.     

Inoculation of a DNAPL source zone to initiate reductive dechlorination of PCE

The ability to inoculate a PCE-NAPL source zone with no prior dechlorinating activity was examined using a near field-scale simulated aquifer. A known mass of PCE was added to establish a source zone, and the groundwater was depleted of oxygen using acetate and lactate prior to culture addition. An active and stable dechlorinating culture was used as an inoculum, and dechlorination activity was observed within 2 weeks following culture transfer. PCE reduction to TCE and cis-DCE was observed initially, and the formation of these compounds was accelerated by the addition of a long-term source of hydrogen (Hydrogen Releasing Compound). cis-DCE was the predominant chlorinated ethene present in the effluent after 225 days of operation, and production of VC and ethene lagged the formation of TCE and cis-OCE. However, dechlorination extent continued to improve over time, and VC eventually became a major product, suggesting that reinoculation was unnecessary. The detection of Dehalococcoides species in the source culture and in the simulated aquifer postinoculation indicated that the metabolic capability to dechlorinate beyond cis-DCE (t = 86 days and t = 245 days) was present. Elevated levels of TCE and cis-DCE were present in the source zone, but neither VC nor ethene were detected in the vicinity of NAPL. The results of this research indicated that adding dechlorinating cultures may be useful in the application of source zone bioremediation but that dechlorination beyond cis-DCE may be limited to regions downgradient of the source zone.     

Assessment of indigenous reductive dechlorinating potential at a TCE-contaminated site using microcosms, polymerase chain reaction analysis, and site data

A combination of microcosm studies, polymerase chain reaction (PCR) analysis, and site data was used to assess the indigenous reductive dechlorinating potential in a trichloroethene (TCE)-contaminated aquifer at Cape Canaveral Air Station, Florida. Sediment and groundwater were obtained from two distinct locations approximately 10 m apart. Microcosm studies were performed to assess dechlorinating activity under a variety of nutrient and electron donor amendment conditions. Most live microcosms constructed using material from the first location, near well 9 (W09), were negative for dechlorination. All live microcosms constructed using material from the second location (W06) exhibited dechlorination of TCE to vinyl chloride (VC) and ethene (ETH). DNA encoding 16S ribosomal RNA (rDNA) with a sequence nearly identical with that from Dehalococcoides ethenogenes strain 195 was detected in the active microcosms and in the sediment from W06 with polymerase chain reaction (PCR) using primers targeted to unique regions of Dehalococcoides 16S rDNA. Dehalococcoides was not detected in the autoclaved microcosms from W06, nor in sediment and most microcosms from W09. The results of the microcosm studies and PCR analysis were supported by field data, which indicated significant accumulation of cis-1,2-dichloroethene (cisDCE) and VC at W06, but not at W09. The different microcosm results obtained for the two locations and the spatial variation of positive PCR results indicates heterogeneous distribution of dechlorinating activity and a specific dechlorinating organism, Dehalococcoides, at the site. As both Dehalococcoides and dechlorination activity were similarly, heterogeneously distributed, this suggests that molecular-probing (which could and should be extended in the future to include virtually all known dechlorinators and/or dehalogenases) can provide a relatively quick and facile method for investigating spatial distributions of dechlorinators on-site.     

Bacterial cultures preferentially removing singly flanked chlorine substituents from chlorobenzenes

The wide though not ubiquitous distribution of chlorobenzene-dechlorinating bacteria in anaerobic sludge from German sewage plants is demonstrated. The model substrates 1,2,3- and 1,2,4-trichlorobenzene (TCB) were dechlorinated to dichlorobenzenes (DCBs) and monochlorobenzene (MCB) via distinct pathways. For easy visualization and differentiation of the pathways, a novel plotting method was developed. While many of the cultures showed a dechlorination pattern similar to that previously found for Dehalococcoides species, removing doubly flanked rather than singly flanked chlorine substituents from TCBs, some cultures formed 1,2-DCB from 1,2,3-TCB and/or 1,3-DCB from 1,2,4-TCB. Stable cultures preferentially catalyzing the removal of singly flanked chlorines were obtained by repeated subcultivation in sediment-free synthetic medium. This dechlorination pattern is potentially of great benefit for remediation as the accumulation of persistent intermediates such as 1,3,5-TCB from highly chlorinated compounds can be avoided. In addition, the cultures dechlorinated 1,3,5-TCB, pentachlorobenzene (PeCB), and hexachlorobenzene (HCB). Nested PCR demonstrated the presence of low numbers of Dehalococcoides species. However, the observed insensitivity of the dechlorinating bacteria in our cultures to oxygen and sensitivity to vancomycin is not in accordance with the reported properties of Dehalococcoides species, suggesting that other bacteria than Dehalococcoides catalyzed the removal of singly flanked chlorines from TCB.     

Reductive dechlorination of cis-1,2-dichloroethene and vinyl chloride by "Dehalococcoides ethenogenes"

cis-Dichloroethene (DCE) and vinyl chloride (VC) often accumulate in contaminated aquifers in which tetrachloroethene (PCE) or trichloroethene (TCE) undergo reductive dechlorination. "Dehalococcoides ethenogenes" strain 195 is the first isolate capable of dechlorinating chloroethenes past cis-DCE. Strain 195 could utilize commercially synthesized cis-DCE as an electron acceptor, but doses greater than 0.2 mmol/L were inhibitory, especially to PCE utilization. To test whether the cis-DCE itself was toxic, or whether the toxicity was due to impurities in the commercial preparation (97% nominal purity), we produced cis-DCE biologically from PCE using a Desulfitobacterium sp. culture. The biogenic cis-DCE was readily utilized at high concentrations by strain 195 indicating that cis-DCE was not intrinsically inhibitory. Analysis of the commercially synthesized cis-DCE by GC/mass spectrometry indicated the presence of approximately 0.4% mol/mol chloroform. Chloroform was found to be inhibitory to chloroethene utilization by strain 195 and at least partially accounts for the inhibitory activity of the synthetic cis-DCE. VC, a human carcinogen that accumulates to a large extent in cultures of strain 195, was not utilized as a growth substrate, and cultures inoculated into medium with VC required a growth substrate, such as PCE, for substantial VC dechlorination. However, high concentrations of PCE or TCE inhibited VC dechlorination. Use of a hexadecane phase to keep the aqueous PCE concentration low in cultures allowed simultaneous utilization of PCE and VC. At contaminated sites in which "D. ethenogenes" or similar organisms are present, biogenic cis-DCE should be readily dechlorinated, chloroform as a co-contaminant may be inhibitory, and concentrations of PCE and TCE, except perhaps those near the source zone, should allow substantial VC dechlorination.     

Acetate versus hydrogen as direct electron donors to stimulate the microbial reductive dechlorination process at chloroethene-contaminated sites

A study to evaluate the dechlorination end points and the most promising electron donors to stimulate the reductive dechlorination process at the chloroethene-contaminated Bachman Road site in Oscoda, MI, was conducted. Aquifer materials were collected from inside the plume and used to establish microcosms under a variety of electron donor conditions using chlorinated ethenes as electron acceptors. All microcosms that received an electron donor showed dechlorination activity, but the end points depended on the sampling location, indicating a heterogeneous distribution of the dechlorinating populations in the aquifer. Interestingly, several microcosms that received acetate as the only electron donor completely dechlorinated PCE to ethene. All acetate-amended microcosms rapidly converted PCE to cis-DCE, whereas PCE dechlorination in H2-fed microcosms only occurred after a pronounced lag time and after acetate had accumulated by H2/CO2 acetogenic activity. The microcosm experiments were corroborated by defined co-culture experiments, which demonstrated that H2 sustained PCE to cis-DCE dechlorination by acetotrophic populations in the presence of H2/CO2 acetogens. In sediment-free nonmethanogenic enrichment cultures derived from ethene-producing microcosms, acetate alone supported complete reductive dechlorination of chloroethenes to ethene, although the addition of H2 resulted in higher cis-DCE and VC dechlorination rates. Measurements of H2 production and consumption suggested that syntrophic acetate-oxidizing population(s) were active in the enrichment cultures. These findings demonstrated that either acetate or H2 alone can be sufficient to promote complete     

Kinetics and inhibition of reductive dechlorination of chlorinated ethylenes by two different mixed cultures

Kinetic studies with two different anaerobic mixed cultures (the PM and the EV cultures) were conducted to evaluate inhibition between chlorinated ethylenes. The more chlorinated ethylenes inhibited the reductive dechlorination of the less chlorinated ethylenes, while the less chlorinated ethylenes weakly inhibited the dechlorination of the more chlorinated ethylenes. Tetrachloroethylene (PCE) inhibited reductive trichloroethylene (TCE) dechlorination but not cis-dichloroethylene (c-DCE) dechlorination, while TCE strongly inhibited c-DCE and VC dechlorination. c-DCE also inhibited vinyl chloride (VC) transformation to ethylene (ETH). When a competitive inhibition model was applied, the inhibition constant (K(I)) for the more chlorinated ethylene was comparable to its respective Michaelis-Menten half-velocity coefficient, K(S). Model simulations using independently derived kinetic parameters matched the experimental results well. k(max) and K(S) values required for model simulations of anaerobic dechlorination reactions were obtained using a multiple equilibration method conducted in a single reactor. The method provided precise kinetic values for each step of the dechlorination process. The greatest difference in kinetic parameters was for the VC transformation step. VC was transformed more slowly by the PM culture (k(max) and K(S) values of 2.4+/-0.4 micromol/mg of protein/day and 602+/-7 microM, respectively) compared to the EV culture (8.1+/-0.9 micromol/mg of protein/day and 62.6+/-2.4 microM). Experimental results and model simulations both illustrate how low K(S) values corresponded to efficient reductive dechlorination for the more highly chlorinated ethylenes but caused strong inhibition of the transformation of the less chlorinated products. Thus, obtaining accurate K(S) values is important for modeling both transformation rates of parent compounds and their inhibition on daughter product transformation.     

Experimental evidence for a lack of thermodynamic control on hydrogen concentrations during anaerobic degradation of chlorinated ethenes

Hydrogen (H2) concentrations during reductive dechlorination of cis-dichloroethene (cDCE) and vinyl chloride (VC) were investigated with respectto the influence of parameters entering the Gibbs free energy expression of the reactions. A series of laboratory experiments was conducted employing a mixed, Dehalococcoides-containing enrichment culture capable of complete dechlorination of chlorinated ethenes. The objective was to investigate whether a constant energy gain controls H2 levels in dechlorinating systems, thereby evaluating the applicability of the partial equilibrium approach to microbial dechlorination at contaminated sites. Variations in the temperature between 10 and 30 degrees C did not affect the H2 concentration in a fashion that suggested thermodynamic control through a constant energy gain. In another set of experiments, H2 levels at constant ionic strength were independent of the chloride concentration between 10 and 110 mmol chloride per liter. These findings demonstrate that the partial equilibrium approach is not directly applicable to the interpretation of reductive degradation of chlorinated ethenes. We also present recalculated thermodynamic properties of aqueous chlorinated ethene species that allow for calculation of in-situ Gibbs free energy of dechlorination reactions at different temperatures.     

Experimental evaluation and mathematical modeling of microbially enhanced tetrachloroethene (PCE) dissolution

Experiments to assess metabolic reductive dechlorination (chlororespiration) at high concentration levels consistent with the presence of free-phase tetrachloroethene (PCE) were performed using three PCE-to-cis-1,2-dichloroethene (cis-DCE) dechlorinating pure cultures (Sulfurospirillum multivorans, Desulfuromonas michiganensis strain BB1, and Geobacter lovleyi strain SZ) and Desulfitobacterium sp. strain Viet1, a PCE-to-trichloroethene (TCE) dechlorinating isolate. Despite recent evidence suggesting bacterial PCE-to-cis-DCE dechlorination occurs at or near PCE saturation (0.9-1.2 mM), all cultures tested ceased dechlorinating at approximately 0.54 mM PCE. In the presence of PCE dense nonaqueous phase liquid (DNAPL), strains BB1 and SZ initially dechlorinated, but TCE and cis-DCE production ceased when aqueous PCE concentrations reached inhibitory levels. For S. multivorans, dechlorination proceeded at a rate sufficient to maintain PCE concentrations below inhibitory levels, resulting in continuous cis-DCE production and complete dissolution of the PCE DNAPL. A novel mathematical model, which accounts for loss of dechlorinating activity at inhibitory PCE concentrations, was developed to simultaneously describe PCE-DNAPL dissolution and reductive dechlorination kinetics. The model predicted that conditions corresponding to a bioavailability number (Bn) less than 1.25 x 10(-2) will lead to dissolution enhancement with the tested cultures, while conditions corresponding to a Bn greater than this threshold value can result in accumulation of PCE to inhibitory dissolved-phase levels, limiting PCE transformation and dissolution enhancement. These results suggest that microorganisms incapable of dechlorinating at high PCE concentrations can enhance the dissolution and transformation of PCE from free-phase DNAPL.     

Natural attenuation processes during in situ capping

Chlorinated solvents are common groundwater contaminants that threaten surface water quality and benthic health when present in groundwater seeps. Aquatic sediments can act as natural biobarriers to detoxify chlorinated solvent plumes via reductive dechlorination. In situ sediment capping, a remedial technique in which clean material is placed at the sediment-water interface, may alter sedimentary natural attenuation processes. This research explores the potential of Anacostia River sediment to naturally attenuate chlorinated solvents under simulated capping conditions. Results of microcosm studies demonstrated that intrinsic dechlorination of dissolved-phase PCE to ethene was possible, with electron donor availability controlling microbial activity. A diverse microbial community was present in the sediment, including multiple Dehalococcoides strains indicated by the amplification of the reductive dehalogenases tceA, vcrA, and bvcA. An upflow column simulating a capped sediment bed subject to PCE-contaminated groundwater seepage lost dechlorination activity with time and only achieved complete dechlorination when microorganisms present in the sediment were provided electron donor. Increases in effluent chloroethene concentrations during the period of biostimulation were attributed to biologically enhanced desorption and the formation of less sorptive dechlorination products. These findings suggest that in situ caps should be designed to account for reductions in natural biobarrier reactivity and for the potential breakthrough of groundwater contaminants.     

Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene

A laboratory microcosm study and a pilot scale field test were conducted to evaluate biostimulation and bioaugmentation to dechlorinate tetrachloroethene (PCE) to ethene at Kelly Air Force Base. The site groundwater contained about 1 mg/L of PCE and lower amounts of trichloroethene (TCE) and cis-1,2-dichloroethene (cDCE). Laboratory microcosms inoculated with soil and groundwater from the site exhibited partial dechlorination of TCE to cDCE when amended with lactate or methanol. Following the addition of a dechlorinating enrichment culture, KB-1, the chlorinated ethenes in the microcosms were completely converted to ethene. The KB-1 culture is a natural dechlorinating microbial consortium that contains phylogenetic relatives of Dehalococcoides ethenogenes. The ability of KB-1 to stimulate biodegradation of chlorinated ethenes in situ was explored using a closed loop recirculation cell with a pore volume of approximately 64,000 L The pilot test area (PTA) groundwater was first amended with methanol and acetate to establish reducing conditions. Under these conditions, dechlorination of PCE to cDCE was observed. Thirteen liters of the KB-1 culture were then injected into the subsurface. Within 200 days, the concentrations of PCE, TCE, and cis-1,2-DCE within the PTA were all below 5 microg/L, and ethene production accounted for the observed mass loss. The maximum rates of dechlorination estimated from field date were rapid (half-lives of a few hours). Throughout the pilot test period, groundwater samples were assayed for the presence of Dehalococcoides using both a Dehalococcoides-specific PCR assay and 16S rDNA sequence information. The sequences detected in the PTA after bioaugmentation were specific to the Dehalococcoides species in the KB-1 culture. These sequences were observed to progressively increase in abundance and spread downgradient within the PTA. These results confirm that organisms in the KB-1 culture populated the PTA aquifer and contributed to the stimulation of dechlorination beyond cDCE to ethene.     

Biological enhancement of tetrachloroethene dissolution and associated microbial community changes

A bench-scale study was performed to evaluate the enhancement of tetrachloroethene (PCE) dissolution from a dense nonaqueous phase liquid (DNAPL) source zone due to reductive dechlorination. The study was conducted in a pair of two-dimensional bench-scale aquifer systems using soil and groundwater from Dover Air Force Base, DE. After establishment of PCE source zones in each aquifer system, one was biostimulated (addition of electron donor) while the other was biostimulated and then bioaugmented with the KB1 dechlorinating culture. Biostimulation resulted in the growth of iron-reducing bacteria (Geobacter) in both systems as a result of the high iron content of the Dover soil. After prolonged electron donor addition methanogenesis dominated, but no dechlorination was observed. Following bioaugmentation of one system, dechlorination to ethene was achieved, coincident with growth of introduced Dehalococcoides and other microbes in the vicinity and downgradient of the PCE DNAPL (detected using DGGE and qPCR). Dechlorination was not detected in the nonbioaugmented system over the course of the study, indicating that the native microbial community, although containing a member of the Dehalococcoides group, was not able to dechlorinate PCE. Over 890 days, 65% of the initial emplaced PCE was removed in the bioaugmented, dechlorinating system, in comparison to 39% removal by dissolution from the nondechlorinating system. The maximum total ethenes concentration (3 mM) in the bioaugmented system occurred approximately 100 days after bioaugmentation, indicating that there was at least a 3-fold enhancement of PCE dissolution atthis time. Removal rates decreased substantially beyond this time, particularly during the last 200 days of the study, when the maximum concentrations of total ethenes were only about 0.5 mM. However, PCE removal rates in the dechlorinating system remained more than twice the removal rates of the nondechlorinating system. The reductions in removal rates over time are attributed to both a shrinking DNAPL source area, and reduced flow through the DNAPL source area due to bioclogging and pore blockage from methane gas generation.     

Microbial diversity and changes in the distribution of dehalogenase genes during dechlorination with different concentrations of cis-DCE

A dechlorinating consortium (designated as TES-1 culture) able to convert trichloroethene (TCE) to ethene was established from TCE-contaminated groundwater. This culture had the ability of complete dechlorination of TCE within about one month. From the clone library analysis of 16S rRNA gene, this culture was mainly composed of fermentation bacteria, such as Clostridium spp., and Desulfitobacterium spp. known as facultative dechlorinator. PCR using specific primers for Dehalococcoides spp. and the dehalogenase genes confirmed that the culture contained the Dehalococcoides spp. 16S rRNA gene and three dehalogenase genes, tceA, vcrA and bvcA. Dechlorination experiments using cis-dichloroethene (cis-DCE) at concentrations of 37-146 μM, revealed that the gene copy numbers of tceA, vcrA, and bvcA increased up to 10? copy/mL, indicating that Dehalococcoides spp. containing these three dehalogenase genes were involved in cis-DCE dechlorination. However, in the culture to which 292 μM of cis-DCE was added, only the tceA gene and the Dehalococcoides spp. 16S rRNA gene increased up to 10? copy/mL. The culture containing 292 μM of cis-DCE also exhibited about one tenth slower ethene production rate compared to the other cultures.