Date of Award


Document Type


Degree Name

Master of Science (MS)

Legacy Department

Environmental Engineering and Science

Committee Chair/Advisor

Freedman, David L

Committee Member

Lee , Cindy M

Committee Member

Kurtz , Harry D


Tetrachloroethene and trichloroethene are among the most prevalent groundwater contaminants found at hazardous waste sites throughout the United States. Since implementing some of the common treatment methods to remediate these hazardous waste sites would exceed hundreds of billions of dollars, there is considerable interest in reducing costs while achieving remediation regulations. Under anaerobic conditions, these compounds can undergo reduction reactions known as reductive dechlorination. This occurs when an electron donor provides the reducing equivalents needed to replace the chlorine atoms with hydrogen atoms. The daughter products from higher chlorinated ethenes are ethene and ethane which can be used to document the process with a simple mass balance.
However, at many hazardous waste sites, the sum of the daughter products often does not account for the amount of tetrachloroethene and trichloroethene consumed. At least two explanations have been offered for this phenomenon. First, downgradient sampling of plumes may not be accurately representing where the plume is. Second, it is possible that the lesser chlorinated products, in particular vinyl chloride (VC) and ethene, could be undergoing anaerobic or aerobic oxidation. If this occurs, CO2 and Cl- are the major daughter products. While both are nonhazardous, it is far more difficult to show that these compounds were formed from VC and ethene, rather than other compounds.
Bio-oxidation of ethene and the aerobic biodegradation of VC at levels that are too low to be accurately measured in the field are possible explanations for the lack of mass balances. Additionally, the pathways for catabolism of both compounds have been proposed and they share considerable overlap. The first step involves insertion of an oxygen atom from O2 via an alkene monooxygenase, forming an epoxide followed by a series of reactions forming acetyl coenzyme A, a central intermediate in metabolic processes. Since an oxygenase is required to initiate aerobic oxidation of VC, oxygen must be available as a reactant. Substitution of nitrate for oxygen as the electron acceptor would decrease the amount of oxygen required for growth on VC.
The overall purpose of this project was to improve the methods needed to document the fate of VC and ethene in situ. The specific objectives were 1) to document in microcosms the anaerobic oxidation of VC, using groundwater from a hazardous waste site where the field evidence suggests that bio-oxidation is occurring; 2) to document in microcosms the anaerobic oxidation of ethene, using uncontaminated soil as a source of inoculum, under a variety of anaerobic conditions; 3) to determine if microbes subjected to anaerobic conditions for longer than one year are then able to aerobically oxidize VC; and 4) to determine if nitrate can replace oxygen as a terminal electron acceptor under oxygen-limited conditions that still permit the use of oxygen as a reactant for the alkene monooxygenase.
In approximately eight of the 83 microcosms prepared with first flush groundwater from a hazardous waste site, significant levels of VC bio-oxidation occurred. However, there was considerable uncertainty whether the process observed was anaerobic or aerobic, as a consequence of oxygen leaking into the bottles. Lines of evidence in support of each scenario were reviewed. Overall, the weight of evidence suggests the activity was aerobic. Some of the considerations include the detection of low levels of oxygen by analysis of headspace samples (coupled with controls that indicated these measurements were not false positives), the pink color of resazurin during most of the time when VC was consumed, consumption of methane during or after the VC was consumed (methane is generally recalcitrant under anaerobic conditions), and the ability of microbes that grow aerobically on VC to use oxygen to the point when it is no longer detectable. Incubation of five of the active microcosms is on-going, to further assess the question of aerobic versus anaerobic activity.
Anaerobic oxidation of ethene was observed in microcosms amended with Fe(III) and Fe(III) chelated with ethylenediaminetetraacetic acid (EDTA), and in a chlororespiring enrichment culture amended with sulfate. Ethene oxidation occurred when the concentration of ethene in the headspace was increased to 1% (v/v) or higher. Prior to that, ethene was stoichiometrically reduced to ethane. Oxidation was not observed in unamended microcosms and ones with nitrate and glucose added. Unlike the results with VC, there was a high level of confidence that oxidation occurred under anaerobic conditions. Compared to VC, the rate of ethene oxidation was considerably slower. Also, oxidation activity often slowed down after 10% or less of the ethene was consumed. This type of pattern suggested the process may be cometabolic, although additional studies are needed to verify this. Also, confirmation of oxidation is needed using [14C]ethene.
Microcosms were prepared with soil and groundwater from a hazardous waste site and incubated under anaerobic conditions for 1.7 years, with no evidence for biodegradation of VC. When oxygen was added to the microcosms, only two of the 12 exhibited biodegradation of VC, along with methane. The results of this study indicate that aerobic biodegradation of VC may not always occur when soil and groundwater have been subjected to anaerobic conditions for an extended period, in this case 1.7 years.
The results of this study indicated that nitrate did not substitute for oxygen as a terminal electron acceptor under conditions with limited oxygen (but enough to allow functioning of the alkene monooxygenase) and an excess of nitrate. However, the results may be specific to the enrichment culture that was tested, which was able to biodegrade VC aerobically and also use acetate as a substrate under denitrifying conditions. If this process is possible, it would decrease the stoichiometric amount of oxygen needed for VC oxidation.
The inconsistencies in observations of anaerobic bio-oxidation of VC leave many questions unanswered. Considerable uncertainties also remain for the occurrence of anaerobic oxidation of ethene. As promising as previous studies appear, the hallmark of advances in science is reproducibility. Until the results of others can be replicated under conditions that are unquestionably anaerobic, the uncertainties surrounding anaerobic oxidation of VC and ethene will persist. As a consequence, so too, will the uncertainties associated with explaining the lack of mass balances in situ for remediation of PCE and TCE.



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