Date of Award

8-2015

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Environmental Engineering and Science

Advisor

Freedman, David L.

Committee Member

Finneran, Kevin T.

Committee Member

Carraway, Elizabeth

Abstract

Halogenated methanes are among the most frequently encountered contaminants at hazardous waste sites. One such site in California is contaminated with chloroform (CF), carbon tetrachloride (CT), and trichlorofluoromethane (CFC-11). CF, CT and CFC-11 are the main focus of this research; they are present at the California site at concentrations in the mg per liter range and a plume of contaminated groundwater emanating from a source zone is approaching a property boundary, necessitating remediation. Anaerobic bioremediation is one of the technologies under consideration for addressing the source zone and the less concentrated downgradient plume. A microcosm study was performed to evaluate natural attenuation, biostimulation and bioaugmentation. Treatment approaches using abiotic processes such as zero valent iron (ZVI), followed by bioremediation, were evaluated in additional experiments. The first objective of this laboratory study was to determine the effectiveness of biostimulation at removing halogenated methanes in the downgradient and upgradient locations. Three types of electron donors were tested; two relatively fast acting (corn syrup and lactate) and one longer lasting (Slow Release Substrate (SRSTM¬), from Terra Systems). The effect of cyanocobalamin (B12) was also evaluated with corn syrup, since the benefits of adding B12 are better documented with corn syrup versus lactate. Addition of B12 was also be evaluated with SRS™, to determine if the same advantages observed with corn syrup can be achieved with a longer-lasting electron donor. For each location, the concentration of major competing electron acceptors (e.g., nitrate and sulfate) was monitored. The second objective was to determine the effectiveness of bioaugmentation for improving the biodegradation rate for halogenated methanes in the downgradient and upgradient locations. KB-1© Plus (containing the required mixture of Dehalobacter spp.) was evaluated for the downgradient and upgradient locations, while DHM-1 was tested with only the higher concentration upgradient location. KB-1© Plus is advantageous from the perspective that it does not require B12 and the downgradient location has low concentrations of potentially inhibitory compounds (e.g., CT). On the other hand, the upgradient location was more likely to have inhibitory compounds and a prior study indicated DHM-1 can be effective in that type of environment. The third objective was to determine the effectiveness of pH adjustment for accomplishing biostimulation and bioaugmentation at the downgradient location. Since groundwater at the downgradient location has a pH greater than 6, bioremediation may be feasible without pH adjustment, although the buffering capacity in this area is not well established. All treatments for the upgradient location were pH-adjusted, since the current pH is already below 6 and dechlorination of higher concentrations of CF will drive the pH even lower. Sodium bicarbonate was used for pH adjustment. The fourth objective was to determine the effectiveness of abiotic and combined abiotic/biotic processes for treating the upgradient location. These processes included ZVI followed by bioaugmentation with KB-1© Plus, sodium dithionite, calcium polysulfide, and a combination of titanium(III) citrate + B12 + corn syrup. The final objective of this study was to perform a mass balance for CF during biodegradation by the KB-1© Plus culture, using 14C-CF. Although previous studies have suggested that the end products are non-hazardous, a complete mass balance to confirm this has not yet been performed. For objectives 1 through 4, five sets of microcosms (i.e., Sets I through V) were prepared with soil and groundwater from the industrial site. For objective 5, only the KB-1© Plus culture was used. Based on results from the microcosms, all of the donor-amended treatments removed CT, in the low and high concentration microcosms. Although the rates differed, removal of CT occurred consistently and is not a major factor in selection of a bioremediation strategy. For the downgradient portion of the plume, the most effective bioremediation strategy for CF and dichloromethane (DCM) is bioaugmentation with KB-1© Plus amended with lactate. pH control does not appear to be necessary; the no pH control (Set II) and microcosms with pH control (Set III) performed similarly. Somewhat unexpected was the robust performance of the pH adjusted microcosms that received lactate as an electron donor. These results suggest that the site contains indigenous Dehalobacter spp. that are capable of organohalide respiration of CF to DCM and fermentation of DCM to nonchlorinated products. Although bioaugmentation with KB-1© Plus amended with lactate was effective for CF and DCM in the downgradient portion of the plume, CFC-11 persisted until most of the CF was consumed, at which point a relatively fast reduction to CHCl2F occurred. For the upgradient portion of the plume, the most effective bioremediation strategy for CF and DCM is bioaugmentation with DHM-1 amended with SRS and B12. This treatment achieved complete removal of CF and significant removal of CFC-11 and DCM. Removal of the DCM suggests that the site contains an indigenous population of anaerobic microbes capable of fermenting DCM as a sole substrate. Bioaugmentation with the KB-1© Plus amended with lactate also showed promise for the upgradient zone, although additional incubation is needed to confirm consumption of DCM formed from CF. Also, KB-1© Plus was less effective in removing CFC-11; follow-up bioaugmentation with DHM-1 should be feasible to address the CFC-11. The high level of sulfate in the downgradient and upgradient groundwater created a high demand for electron donor. Significant improvement in the rate of CFC-11, CF, and DCM biodegradation ensued following an increase in the frequency and amount of electron donor additions after 300 days of incubation, commensurate with the onset of sulfate reduction. This lag could have been shortened significantly by following a much more aggressive schedule of electron donor additions earlier in the incubation period. Lactate and corn syrup were more effective in establishing sulfate reducing conditions than SRS. It is likely that the initially slow rate of electron donor additions resulted in wasteful additions of bioaugmentation culture and B12; these additions should have been made only after sulfate reduction was well established. The sulfide generated from sulfate reduction likely created the low redox conditions that are conducive to halomethane degradation. The most effective abiotic treatment involved the use of ZVI; after approximately three months of incubation, the CT, CF, and CFC-11 were removed. However, DCM and CHCl2F persisted; an attempt to biodegrade DCM by augmenting with KB-1© Plus was ineffective, for reasons that are not yet known. Also, unknown volatile compounds accumulated in the ZVI-amended microcosms. While ZVI was faster than the biotic treatments in this study, this advantage would likely diminish if the biotic-only treatments had received more aggressive additions of electron donor from the outset;. Using 14C-CF, it was shown that the principal product from anaerobic biodegradation of CF by KB-1© Plus is CO2. This is the first report to demonstrate a complete mass balance on the products from CF biodegradation by KB-1© Plus, and to show that these products are nonhazardous. CO2 accumulated in treatments with and without 2-bromoethanesulfonate added (to inhibit methanogenesis), and significant amounts accumulated even when CF was consumed but some DCM remained. This suggests that the pathway for DCM metabolism in the KB-1© Plus culture is different from that reported for Dehalobacterium formi coaceticum, which directly incorporates the carbon in DCM into formate and acetate. The disposition of reducing equivalents gained during fermentation of DCM is not yet known, although a preliminary experiment confirmed that hydrogen accumulates transiently as DCM is consumed. It seems likely that the reducing equivalents formed from DCM are consumed for methane production and/or organic acid formation. These products are not 14C-labeled most likely because the 14CO2 that is generated enters a large pool of bicarbonate in the mineral medium, such that any bicarbonate used for methane or organic acid formation has a low percentage of labeled compound (i.e., the 14CO2 is diluted out).

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