Toxic effects of halogenated-aliphatic compounds: Competition for carbon, and recoverability of perturbed populations

Observations characterizing biotransformation of chlorinated aliphatics under both aerobic and anaerobic conditions by pure and mixed cultures of bacteria have revealed biological dehalogenations under methanogenic, sulfate reducing, and denitrifying conditions. The purpose of this study is to determine the effect of halogenated compounds as a perturbation to bacterial populations present in naturally occurring microbial communities. In this study trichlorofluormethane (CFC-11) dichlorofluoromethane (HCFC-21), chloroform (CF), and dichloromethane (DCM) are analyzed for their ability to impact bacterial community structure and function. In addition, the toxicity to prokaryotic and eukaryotic organisms are compared for these four pollutants.; Methods used to determine the perturbation effect on community function and community structure include metabolic, genetic and culture based techniques to determine the metabolic activity (community function) and identification (community structure) of anaerobic microbial populations incubated in the presence and absence of chlorinated aliphatics. The metabolic activity of methanogenic bacteria and sulfate-reducing bacteria were measured by a combination of colorimetric and gas chromatographic tests. Genetic determination of relevant populations of sulfate-reducers and methanogens was determined using 16S based rRNA probing procedures. Most Probable Number (MPN) determinations were done for methanogens and sulfate reducers were also done. Toxicological analysis of eukaryotic organisms were performed with Ceriodaphnia dubia according to procedures outlined by the United States Environmental Protection Agency, and toxicological effects to prokaryotes were based upon inhibition to methanogenesis.; In minus-pollutant control incubations, methanogenesis was the dominant anaerobic activity. However, in the presence of 5 ppm pollutant, methanogenesis was inhibited (nearly 100% in CF-incubated microcosms) and sulfidogenesis was stimulated (by approximately 80% in CF and CFC-11 incubated microcosms. Therefore, community function was greatly impacted by these pollutants. Hybridizaiton of 16S rRNA probes specific for methanogenic and sulfate-reducing bacteria indicated very little difference between control and pollutant exposed microcosms suggesting very little impact on community structure. Most probable number data also suggested little impact on community structure. These data also imply that populations that are enzymatically inhibited are able to recover when pollutants are removed. This hypothesis was tested with the development of flow-through columns allowing the removal of pollutant after exposure. However, when pollutant was removed methanogenesis never recovered after exposure to 5 ppm CF. Toxicity to methanogenic bacteria was CF > CFC-11 > HCFC-21 > DCM, in contrast to Ceriodaphnia dubia where CF > HCFC-21 > DCM > CFC-11. The dechlorination of CFC-11 to HCF-21 significantly increased the toxicity to C. dubia.; These studies have revealed how sensitive enzyme activity is to the presence of one carbon halogenated compounds and yet how resilient the populations are. The data also confirm the need to perform toxicological assays to test if biodegradation indeed reduces the toxicity. By analyzing the effect of halogenated aliphatics to enzymatic activity of bacteria and performing detailed toxicological assays better strategies for the remediation of contaminated sites may be developed.