Biodegradation and bioremediation of hexachlorocyclohexane isomers, chlorinated ethenes, chlorinated benzenes and benzene.

Groundwater at an industrial site is contaminated with alpha-hexachlorocyclohexane (HCH) and gamma-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 alpha- and gamma-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 microcosm study for an industrial site contaminated with HCH isomers (predominantly gamma-HCH), we observed rapid anaerobic biodegradation of gamma-HCH to benzene and chlorobenzene. The pattern and rate of activity suggested that gamma-HCH may be used as a terminal electron acceptor. Using inoculum from microcosms that exhibited high rates of gamma-HCH reduction, enrichment cultures were developed in groundwater from the industrial site and subsequently transferred to an anaerobic mineral medium without loss of gamma-HCH dechlorination. The culture was further enriched in a sulfate free media with two different types of buffers (bicarbonate and HEPES) and gamma-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 gamma-HCH dechlorination; most was consumed for acetogenesis. Based on the fraction of electron equivalents used for gamma-HCH dechlorination in HEPES-buffered enrichment culture and the ability to transfer this culture with gamma-HCH as the sole terminal electron acceptor, this study is the first to demonstrate chlororespiration of gamma-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 gamma-HCH. The development of gamma-HCH dechlorinating culture in this study will improve our understanding of remediation of gamma-HCH by natural attenuation and engineered approaches. Using inoculum from a microcosm 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, respectively, by their 16S rRNA gene sequences. The yield measured on the isolates growing on corresponding substrates ranged from 0.36-0.45 mg biomass/mg substrate, with highest yield...