Date of Award


Document Type


Degree Name

Master of Science (MS)

Legacy Department

Environmental Engineering

Committee Chair/Advisor


Committee Member


Committee Member



Removal of the chlorinated methanes, carbon tetrachloride (CT), chloroform (CF) and dichloromethane (DCM), and the halogenated methane, trichlorofluoromethane (commonly known as CFC-11 or Freon-11) from contaminated soil and groundwater remains challenging. Releases to the environment resulted from historically poor disposal practices, as well as spills and leaks from underground storage tanks. Among these three compounds, CF is considered the benchmark for evaluating the feasibility of bioremediation, due to its typically high level of toxicity towards most anaerobic prokaryotes. However, inhibitory effects of low concentrations of CFC-11 on anaerobes have been reported as well. Bioremediation is an attractive option for clean-up, but requires developments in the technology, especially when the concentrations present are in the tens or hundreds of milligrams per liter. The focus of this thesis is on developing improved methods to bioremediate high concentrations of CFC-11, CF, and DCM. In particular, the research focused on further characterizing two enrichment cultures previously developed at Clemson University. The DHM-1 enrichment culture cometabolically degrades high concentrations of CT, CF and CFC-11 with corn syrup as the electron donor and vitamin B12 as an essential cofactor. The DCM enrichment culture uses DCM as its sole source of carbon and energy via fermentation, releasing formate and acetate as major products.
The objectives of this research were 1) to determine a mass balance for fluoride release during biodegradation of CFC-11 by DHM-1, in the presence of corn syrup and B12; 2) to evaluate the effect of the dose of DHM-1 on the rate biodegradation of 500 mg/L of CF; 3) to evaluate the tolerance of the Clemson DCM enrichment culture to DCM concentrations greater than 500 mg/L; 4) to determine the effect of CF on DCM biodegradation by Clemson's DCM enrichment culture, including the potential for acclimation to increasing concentrations of CF; 5) to confirm the accumulation of hydrogen during DCM biodegradation by Clemson's DCM enrichment culture; 6) to evaluate the effect of hydrogen concentration on DCM biodegradation by Clemson's DCM enrichment culture; and 7) to further characterize the identity of the microbe responsible for DCM biodegradation by Clemson's DCM enrichment culture.
Experiments performed with the DHM-1 enrichment culture proved that in the presence of corn syrup and vitamin B12, the culture completely biodegraded 26 mg/L of CFC-11. On a molar basis, less than 6% of the CFC-11 consumed accumulated as dichlorofluoromethane (HCFC-21) and chlorofluoromethane (HCFC-31). The balance of the CFC-11 was defluorinated, based on stoichiometric release of fluoride. These results confirm the ability of the DHM-1 enrichment culture to convert high concentrations of CFC-11 into nonhazardous products. This result is consistent with previous research that demonstrates the same ability of DHM-1 with CT and CF. The DHM-1 enrichment culture was also able to biodegrade approximately 500 mg/L of CF when the inoculum dose was as low as 10-8 percent by volume. These results demonstrate the potential to use the culture for bioaugmentation of source areas, since the dose required is minor relative to the volume of groundwater that could be treated.
Experiments performed with the DCM enrichment culture demonstrated an ability to biodegrade at least 740 mg/L of DCM, indicating its potential usefulness in treating groundwater located near source areas containing dense, nonaqueous phase liquid (DNAPL). The addition of up to 6% H2 (by volume) to the headspace did not affect the degradation of DCM. With experiments designed to analyze the effect of CF on DCM degradation, addition of 1 mg/L of CF to the enrichment culture completely inhibited DCM biodegradation. CF concentrations of 0.1 to 0.8 mg/L slowed the rate of DCM utilization but did not prevent it. Preliminary efforts suggest it will be possible to gradually acclimate the DCM enrichment culture to CF concentrations above 1 mg/L. Efforts to isolate the DCM degrading microbe from this culture by sequential dilution in agar did not yield a pure culture; however, the process narrowed down the microbial community and facilitated better microscopic observation. The dominant morphology consisted of gram positive thick rods, thin rods and filaments. The rods were similar in appearance and gram stain to Dehalobacterium, one of two genera known to grow on DCM as a sole carbon and energy source via fermentation.
The results of this study provide definitive evidence in support of the ability of the DHM-1 enrichment culture to degrade CFC-11 to non-hazardous end products, and degrade high concentrations of CF with a volumetric inoculum dose as low as 10-8%. Characterization of the DCM enrichment culture proved its ability to degrade high DCM concentrations. Even though 1 mg/L of CF inhibited DCM degradation, preliminary experiments set up to acclimate the culture to CF concentrations above 1 mg/L have shown promise. In order to identify the microbe that grows on DCM, additional effort is warranted to obtain a pure culture by making additional transfers in sequential agar dilutions.



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