Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Environmental Engineering and Earth Science


Freedman, David L

Committee Member

Lee , Cindy M

Committee Member

Cao , Weiguo

Committee Member

Kurtz, Jr. , Harry D


Groundwater at an industrial site is contaminated with γ -hexachlorocyclohexane (HCH) and α-HCH (i.e., lindane) (0.3-0.5 ppm). Other contaminants in the 1-15 ppm range include 1,2,4-trichlorobenezene (TCB), 1,2-dichlorobenzene (DCB), 1,3-DCB, 1,4-DCB, chlorobenzene (CB), benzene, trichloroethene (TCE) and cis-1,2-dichloroethene (cDCE). The aquifer consists of a shallow layer of soil over fractured dolomite, where most of the contaminant mass resides. The objective of this study was to compare 1) anaerobic reductive dechlorination of the polychlorinated contaminants, followed by aerobic biodegradation of the daughter products (mainly DCBs, CB, and benzene); and 2) aerobic biodegradation of α- and γ-HCH, TCB, DCBs, CB and benzene, followed by anaerobic reduction of TCE and cDCE to ethene. Conventional wisdom suggests that sequential anaerobic and aerobic conditions are desirable for bioremediating sites contaminated by mixtures of polychlorinated organics. The results of this microcosm study suggest that a sequential aerobic and anaerobic approach may be more successful, although implementing this in the field presents some major challenges. In the dolomite microcosms incubated initially under aerobic conditions (59 days), α- and γ-HCH were biodegraded close to the maximum contaminant level for lindane; all of the aromatic compounds were consumed; and there was partial removal of TCE and cDCE (presumptively via cometabolism). The subsequent switch to anaerobic conditions (day 101) yielded reductive dechlorination of the remaining TCE; a significant level of ethene was produced, although some cDCE and VC persisted. In contrast, sequential anaerobic (393 days) and aerobic treatment (498 days) for the dolomite microcosms was ineffective in completely removing the aromatic compounds, α-HCH, cDCE and VC. For the soil microcosms, both treatment sequences were effective, most likely reflecting a greater abundance of the necessary microbes and electron donor in this part of the site.
In the microcosm study for an industrial site contaminated with HCH isomers (predominantly γ-HCH), we observed rapid anaerobic biodegradation of γ-HCH to benzene and chlorobenzene. The pattern and rate of activity suggested that γ-HCH may be used as a terminal electron acceptor. Using inoculum from microcosms that exhibited high rates of γ-HCH reduction, enrichment cultures were developed in groundwater from the industrial site and subsequently transferred to an anaerobic mineral medium without loss of γ-HCH dechlorination. Analysis of the enrichment culture by denaturant gradient gel electrophoresis and sequencing revealed the presence of a Desulfomicrobiumsp. and several uncultured bacteria. The absence of a Dehalobacter sp. (previously reported to anaerobically biodegrade γ-HCH) was verified by DNA amplification with Dehalobacter specific primers. The culture was further enriched in a sulfate free media with two different types of buffers (bicarbonate and HEPES) and γ-HCH as the only terminal electron acceptor and hydrogen as electron donor. Electron balance calculations in the bicarbonate buffered enrichment cultures revealed that only a small fraction of the hydrogen was involved in γ-HCH dechlorination; most was consumed for acetogenesis. Based on the fraction of electron equivalents used for γ-HCH dechlorination in HEPES-buffered enrichment culture and the ability to transfer this culture with γ-HCH as the sole terminal electron acceptor, this study is the first to demonstrate chlororespiration of γ-HCH. Molecular analysis of enrichment cultures (in bicarbonate and HEPES buffered medium) in this study did not provide sufficient information to associate a specific microbe with chlororespiration of γ-HCH. The development of γ-HCH dechlorinating culture in this study will improve our understanding of remediation of γ-HCH by natural attenuation and engineered approaches. However, γ-HCH dechlorination will have to be coupled with improvements in anaerobic bioremediation of the terminal dechlorination products, benzene and chlorobenzene, or aerobic treatment of dechlorination products that would ensure that the endpoint is environmentally acceptable.
Using inoculum from a the microcosms study that exhibited aerobic transformation of cis-dichloroethene (cDCE) and trichloroethene (TCE) commensurate with biodegradation of the monoaromatic compounds, enrichment cultures were developed in groundwater by providing benzene, chlorobenzene, dichlorobenzene isomers and 1,2,4-trichlorobenzene as carbon and energy sources. These enrichments were subsequently transferred to a mineral salt medium and were grown on each of the monoaromatic compounds separately and were successfully maintained through several transfers. Isolates growing on benzene, chlorobenzene, 1,2-dichlorobenzene and 1,3-dichlorobenzene were identified as Rhodococcus, Ralstonia, Variovorax and Ralstonia(/italic>, respectively, by 16S rRNA gene sequence. The yield measured on the isolates growing on corresponding substrates ranged from 0.36-0.45 mg biomass/mg substrate, with highest yield on benzene and lowest yield on 1,3-dichlorobenzene. Cometabolic transformation of cDCE and TCE evaluated based on pseudo-first order cometabolic degradation rate constant, transformation capacity and transformation yield for resting cells were observed to be on the low end of the reported values on phenol or toluene grown isolates. Cometabolic transformation of cDCE and TCE was also evaluated with growing cells of each isolate and were observed to be on low end of reported values for TCE and this is the first study to report for cDCE. In general, the cometabolic transformation parameters observed for cDCE were greater than TCE. The results of this study confirm the potential for cometabolism of cDCE and TCE during aerobic growth on benzene, CB, 1,2-DCB and 1,3-DCB. This is especially relevant for natural attenuation scenarios when these compounds occur as co-contaminants and may be transported from an anaerobic to an aerobic environment. Although it may seem reasonable to assume that all aerobically biodegradable monoaromatic compounds may serve as primary substrates for cDCE and TCE cometabolism, the wide range of dioxygenases involved indicates this is not necessarily the case.
The major findings from this dissertation are; (i) aerobic/anaerobic treatment should be considered as an alternative to anaerobic/aerobic treatment for bioremediation of complex mixtures of chlorinated contaminants, (ii) chlororespiration of γ-HCH was demonstrated for the first time and this improves the opportunities for in situ bioremediation, and (iii) aerobic cometabolism of cDCE and TCE with aromatic contaminants helps to explain the potential pathways for natural attenuation of complex mixtures.