The goal of this study was to use molecular biology techniques to access and monitor the efficacy of bioremediation on a trichloroethene (TCE) polluted site. We added emulsified hydrogen releasing materials to stimulate onsite microbial growth and the biodegradation of TCE. This process was known as enhanced bioremediation. In this study, there were two bioremediation sites had been treated anaerobically. Groundwater samples were taken periodically for microbial analysis. Denaturing gradient gel electrophoresis (DGGE) was used to evaluate the variations in microbial community structures during the in situ groundwater remediation. The DGGE DNA bandings were sequenced to determine the 16S rRNA gene sequences and identify the dominate bacterial species. In addition, we used Dehalococcoides spp. 16S rRNA genes as the targets to do real-time PCR. Results show that the emulsified hydrogen releasing materials could enhance anaerobic reductive dechlorination. After addition of emulsified hydrogen releasing materials, we found that the volatile organic compounds concentrations (i.e., TCE, 1, 1-DCE and VC) were decreased. In microbial analysis, the diversities of the microbial community were increased after nutrient supplement. According to the DNA sequencing results, there were 31 bacterial species had been found that related to TCE degradation (i.e., Acidovorax sp., Burkholderiales, Pseudomonas sp., £]-proteobacterium, Comamonadaceae, Iron-reducing bacterium, Hydrogenophilaceae, Clostridium sp., Geobacter sp., Rhodoferax ferrireducens, Dehalospirillum multivorans and Dehalococcoides spp.). Dehalococcoides spp. can be used as a biomarker to evaluate the efficacy of anaerobic bioremediation on a TCE contaminated site. Therefore, we quantified Dehalococcoides populations to explain the capacity of bioremediation after addition of emulsified hydrogen releasing materials to groundwater. Results reveal that Dehalococcoides cell numbers of site A were 4.47¡Ñ103-8.26¡Ñ104 CFU/liter, site B were 4.60¡Ñ102-9.31¡Ñ107 CFU/liter. This data indicated that the addition of emulsified substrate would increase the growth of total Dehalococcoides population under anaerobic conditions. Overall, results from this study demonstrated that the microbial analysis and quantities of Dehalococcoides at different time points can provide useful information to proceed with bioremediation methods.

Abstract The industrial solvents tetrachloroethylene (PCE) and trichloroethylene (TCE) are among the most ubiquitous chlorinated compounds found in groundwater contamination. One potential method for managing PCE/TCE contaminated sites is the intrinsic bioremediation. Recent regulations adopted by U.S. Environmental Protection Agency allow intrinsic bioremediation to be considered as an alternative during development of corrective action plans. In some remediation cases, enhanced bioremediation are performed to accelerate the contaminant biodegradation rate. The main objective of this study was to evaluate the potential of using intrinsic and enhanced bioremediation technologies to clean up PCE/TCE contaminated aquifers. PCE/TCE bioavailability was evaluated by laboratory microcosms under four reduction/oxidation (redox) conditions including aerobic cometabolism, methanogenesis, iron reduction, and reductive dechlorination. Acclimated bacteria, activated sludge, and aquifer sediments from a pentachlorophenol contaminated site were used as the inocula in this study. Methane, toluene, phenol, sludge cake, and cane molasses were used as the primary substrates (carbon sources) in the cometabolism and reductive dechlorination microcosms. Results from this study show that PCE and TCE can be significantly biodegraded under reductive dechlorination and aerobic cometabolism conditions, respectively. All five carbon sources evaluated in this study can be applied as the primary substrates by microbial consortia to enhance the aerobic cometabolism of TCE. The highest TCE degradation rate [Up to 100% of TCE removal (with an initial concentration of 3.6µM)] was observed in the microcosms with toluene enrichment bacteria as the microbial inocula and toluene as the primary substrate. Under reductive dechlorination conditions, both sludge cake and cane molasses could be used as the primary substrates by microbial consortia (from activated sludge and aquifer sediments) and enhanced the biodegradation of PCE. The highest PCE degradation rate [Up to 100% of PCE removal (with an initial concentration of 17µM)] was observed in the microcosms with anaerobic activated sludge as the microbial inocula and sludge cake as the primary substrate. Except for reductive dechlorination microcosms, no significant PCE removal was observed in the microcosms prepared under iron reduction conditions. Results from this feasibility study would be useful in designing a scale-up in situ (e.g., in situ biobarrier system) or on-site bioremediation system (e.g., bioslurry reactor) for field application. Moreover, the application of non-toxic organic waste to enhance PCE/TCE biodegradation has the potential to become an environmentally and economically acceptable technology for the bioremediation of chlorinated-solvent contaminated groundwater.

The purpose of this research was to investigate the microorganisms, genetics and biochemistry of anaerobic dechlorination of chlorinated ethanes, which are common groundwater contaminants. Specifically, this project used mixed microbial cultures to study the dechlorination of 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,1-trichloroethane (1,1,1-TCA). A mixed microbial culture enriched from a contaminated multilayered aquifer in West Louisiana dechlorinated 1,2-DCA, 1,1,2-TCA, tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride (VC) to non-toxic ethene when amended with ethanol as the electron donor. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organisms Dehalobacter and Dehalococcoides spp. Denaturing gradient gel electrophoresis analysis and quantitative PCR (qPCR) with species-specific primers demonstrated that both organisms grew during the dichloroelimination of 1,2-DCA to ethene. Conversely, during the dichloroelimination of 1,1,2-TCA to VC only Dehalobacter grew, while during the reductive dechlorination of VC to ethene only Dehalococcoides grew. Further enrichment with 1,2-DCA, H2 and acetate yielded a co-culture of Dehalobacter and Acetobacterium spp. that did not dechlorinate other chlorinated ethanes or ethenes. Dehalobacter grew in the presence but not in the absence of 1,2-DCA, while Acetobacterium growth was not affected by 1,2-DCA. A novel putative Dehalobacter-associated 1,2-DCA reductive dehalogenase gene was identified and was shown to be transcribed only in the presence of 1,2-DCA. An enrichment microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States was also studied. This culture, referred to as MS, reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) when amended with methanol, ethanol, acetate and lactate. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organism Dehalobacter sp., whose growth during 1,1,1-TCA and 1,1-DCA dechlorination was confirmed by qPCR. In the presence of chlorinated ethenes, dechlorination 1,1,1-TCA by the culture MS was slowed, while dechlorination of 1,1-DCA was completely inhibited. Experiments with cell-free extracts and whole cell suspensions of culture MS suggested that chlorinated ethenes have direct inhibitory effects on 1,1,1-TCA reductive dehalogenase(s), while the inhibition of 1,1-DCA dechlorination may be due to effects on non-dehalogenase components of Dehalobacter sp. cells. Additionally, two novel reductive dehalogenase genes associated with 1,1,1-TCA reductive dechlorination were identified.

abstract: Contamination by chlorinated ethenes is widespread in groundwater aquifers, sediment, and soils worldwide. The overarching objectives of my research were to understand how the bacterial genus Dehalococcoides function optimally to carry out reductive dechlorination of chlorinated ethenes in a mixed microbial community and then apply this knowledge to manage dechlorinating communities in the hydrogen-based membrane biofilm reactor (MBfR). The MBfR is used for the biological reduction of oxidized contaminants in water using hydrogen supplied as the electron donor by diffusion through gas-transfer fibers. First, I characterized a new anaerobic dechlorinating community developed in our laboratory, named DehaloR^2, in terms of chlorinated ethene turnover rates and assessed its microbial community composition. I then carried out an experiment to correlate performance and community structure for trichloroethene (TCE)-fed microbial consortia. Fill-and-draw reactors inoculated with DehaloR^2 demonstrated a direct correlation between microbial community function and structure as the TCE-pulsing rate was increased. An electron-balance analysis predicted the community structure based on measured concentrations of products and constant net yields for each microorganism. The predictions corresponded to trends in the community structure based on pyrosequencing and quantitative PCR up to the highest TCE pulsing rate, where deviations to the trend resulted from stress by the chlorinated ethenes. Next, I optimized a method for simultaneous detection of chlorinated ethenes and ethene at or below the Environmental Protection Agency maximum contaminant levels for groundwater using solid phase microextraction in a gas chromatograph with a flame ionization detector. This method is ideal for monitoring biological reductive dechlorination in groundwater, where ethene is the ultimate end product. The major advantage of this method is that it uses a small sample volume of 1 mL, making it ideally suited for bench-scale feasibility studies, such as the MBfR. Last, I developed a reliable start-up and operation strategy for TCE reduction in the MBfR. Successful operation relied on controlling the pH-increase effects of methanogenesis and homoacetogenesis, along with creating hydrogen limitation during start-up to allow dechlorinators to compete against other microorgansims. Methanogens were additionally minimized during continuous flow operation by a limitation in bicarbonate resulting from strong homoacetogenic activity. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2012

Microcosms were constructed from aquifer sediment samples taken from an actively degrading chlorinated solvent contaminated site located in Virginia Beach, Virginia. The objective of this study was to determine if and how the addition of cyclodextrin (CD) affects reductive dechlorination of chlorinated ethenes. After chlorinated solvent degradation rates were established in anaerobic and aerobic microcosms, 100 mg/L of CD solution was added for a period of 21 days. CD was then removed after 26 days to simulate the degradation response of the aquifer in a post CD injection environment. Degradation rates were determined by analyzing PCE, TCE, and cis-DCE concentration data over the various phases of the experiment. Results from this study indicated that chlorinated solvent degradation could be either impaired or facilitated by the addition of CD. CD appeared to stimulate one anaerobic microcosm (IY-2c) where daughter production had not previously occurred. The activity of this microcosm was greatly enhanced by the addition of CD (0 uM/day to 13.89 uM/day). However, biotransformation of PCE in another anaerobic microcosm in which reductive dechlorination was occurring, ceased after the addition of CD (IY-1a). In a third group of microcosms the rate and extent of reductive dechlorination was greatly enhanced by the addition of CD. The effect of adding CD was also found to be highly dependent on the redox conditions in the microcosm, specifically if the conditions were strongly reducing. The most active microcosms, found in the Aerobic Group, also had the lowest ferrous iron concentrations (3.57 mg/L for BY-1a, 2.25 mg/L for BY-1b, and 0.41 mg/L for BY-1c). The microcosm (IY-2b) that showed no daughter production had the highest level of ferrous iron (44.22 mg/L). This study presents a qualitative approach to the affect of CD on MNA. / Master of Science

Laboratory microcosm experiments were conducted to assess the potential for biostimulation and bioaugmentation as source reduction measures in support of a monitored natural attenuation remedial strategy at Naval Amphibious Base (NAB) Little Creek. Previous work with laboratory microcosms conducted under simulated natural (unamended) conditions has demonstrated that indigenous dehalorespirators were capable of partial dechlorination of tetrachloroethene (PCE) to cis-dichloroethene (cis-DCE). This study attempts to achieve complete reductive dechlorination with amendments to static microcosms to test the hypotheses that nutrient-limited or microorganism-limited conditions exist in aquifer sediments obtained from the site. The enhanced bioremediation experiments were comprised of nutrient-amended microcosms receiving additions of electron donors, mineral medium, or anaerobic digester supernatant, and dechlorinating culture-amended microcosms were inoculated with a culture capable of transforming PCE to ethene. Reductive dechlorination in the nutrient-amended microcosms proceeded to cis-DCE over a 260-day study period, at slightly higher rates than in experiments conducted with aquifer sediments from the same location under natural conditions. Inoculation of aquifer sediments with a small amount of dechlorinating culture initiated rapid transformation of PCE to vinyl chloride (VC) by day 18 of the study. Zero-order rates of PCE dechlorination in unamended, propionate-, formate-, mineral medium-, digester supernatant-, and dechlorinating culture-amended microcosms were 0.24, 0.750, 1.30, 0.339, 0.177, and 1.75 µM/day, respectively. The results of this study suggest that an engineered biostimulation approach alone may not be as beneficial for PCE source reduction at NAB Little Creek, than bioaugmentation with competent dehalorespirators, along with the inclusion of supplemental nutrients which would be available to stimulate dechlorination activity of both indigenous and introduced microorganisms. / Master of Science

The entire stretch of the Ohio River is under fish consumption advisories due to contamination with polychlorinated biphenyls (PCBs). In this study, natural attenuation and biostimulation of PCBs and microbial communities responsible for PCB transformations were investigated in Ohio River sediments. Natural attenuation of PCBs was negligible in sediments, which was likely attributed to low temperature conditions during most of the year, as well as low amounts of available nitrogen, phosphorus, and organic carbon. Moreover, surface sediments were relatively oxidized, as indicated by the prevalence of aerobic bacteria such as beta- Proteobacteria, alpha-Proteobacteria, Sphingobacteria, and Nitrospira in 16S rRNA sediment clone libraries. On the other hand, several reductive dechlorinators were detected in sediments, including Dehalococcoides, Desulfitobacterium spp. which suggested that reductive dechlorination might be possible in sediments under certain biogeochemical conditions. Considerable amounts of PCBs were transformed by reductive dechlorination (80% in 177 days by pattern N) when sediments were maintained under anaerobic conditions, amended with nutrients and organic carbon, and incubated at 25 ºC in lab microcosms. Analysis of 16S rRNA clone libraries from these treatments revealed that Bacteroidetes, Chloroflexi and Firmicutes were enriched and Proteobacteria were depleted compared to clone libraries from treatment without organic amendments. Reductive dechlorination was decreased in sediments incubated at 10 and 40 ºC, and was not affected by FeSO4 amendments compared to unamended sediments incubated at 25 ºC. Transformations of PCB-153 were investigated in sediments under anaerobic, aerobic and sequential anaerobic and aerobic conditions. Transformations were only observed in treatments with an anaerobic phase, which occurred by reductive dechlorination by pattern N. Neither PCB-153 nor dechlorination products PCB-99 or PCB-47 were transformed under aerobic conditions. Analysis of 16S rRNA clone libraries revealed that Bacteoridetes, Chloroflexi, and Firmicutes were enriched under anaerobic conditions and Proteobacteria were enriched under aerobic conditions. Results from this study revealed that natural attenuation and biostimulation were not effective at removing PCBs from Ohio River sediments. Hence, other remediation methods will need to be employed to decrease PCB levels in this ecosystem.

For this study, we revisited whether the common iron Fe mineral, magnetite Fe3O4 (s), can reduce tetrachloroethylene (PCE) and trichloroethylene (TCE) as discrepancies exist in the literature regarding rates and extent of reduction. We measured PCE and TCE reduction in batch reactors as a function of magnetite stoichiometry (x = Fe2+/Fe3+ ratio), solids loading, pH, and Fe(II) concentration. Our results show that magnetite reacts only slowly with TCE (t1/2 = 7.6 years) and is not reactive with PCE over 150 days. The addition of aqueous Fe(II) to magnetite suspensions, however, results in slow, but measurable PCE and TCE reduction under some conditions. The solubility of ferrous hydroxide, Fe(OH)2(s), appears to play an important role in whether magnetite reduces PCE and TCE. In addition, we found that Fe(OH)2(s) reduces PCE and TCE at high Fe(II) concentrations as well. At certain conditions degradation of the PCE and TCE is enhanced by an unexplored synergistic response from magnetite and ferrous hydroxide iron phases. Our work suggests that measuring dissolved Fe(II) concentration and pH may be used as indicators to predict whether PCE and TCE will be abiotically degraded by groundwater aquifer solids containing magnetite.

No description available.

Iron-based degradative solidification/stabilization (Fe(II)-DS/S) is a treatment method that could be economically applied to smaller DNAPL-contaminated sites and to those sites with impermeable soils. Reductive dechlorination is achieved by compounds that are formed by reaction of ferrous iron with components of Portland cement or with defined chemicals (FeCl3 + Ca(OH)2). These dechlorinating agents can effectively degrade chlorinated hydrocarbons (PCE, TCE, and 1,1,1-TCA) that are dissolved in aqueous solution. This research investigated the application of Fe(II)-DS/S to remove chlorinated hydrocarbons that are present as DNAPLs in source zones and to compared the reactivity of ferrous iron in different mixtures, including the conventional mixture with cement (Fe(II)+C) and an iron-solid mixture (ISM) that was synthesized without the addition of cement. The modified first-order model, which the rate was proportional to the concentration of target in the aqueous phase and it was also nearly constant when DNAPL was present, was developed to describe dechlorination kinetics. The modified second-order model assumed that the rate was proportional to the product of the concentration of target in the aqueous phase and the concentration of reductive capacity of the solid reductant. The modified first-order model was used to describe degradation of target compounds with ISM, and the modified second-order model was used to describe removals for TCE and 1,1,1-TCA with Fe(II)+C. Results of experiments on PCE dechlorination with ISM indicated that the increase of Fe(II) in ISM increased rate constants and decreased the solubility of targets. The half-life was increased with increasing total PCE concentration. The product analysis implied that degradation of PCE with ISM was via a combination of the hydrogenolysis and β-elimination pathways. A comparison of the types of targets and reductants indicated that Fe(II)+C had better reactivity for chlorinated ethenes (PCE and TCE) than ISM. However, ISM could dechlorinate a chlorinated ethane (1,1,1-TCA) as rapidly as Fe(II)+10%C. The ratio of [RC]o/[Fe(II)]o implied that Fe(II) in Fe(II)+C was more involved in reducing chlorinated ethenes than was Fe(II) in ISM. Dechlorination of a DNAPL mixture followed the same order of reactivity as with individual DNAPLs with both reductants.