Summary and outlook
FIG. 3. Illustration of overlapping substrate specificities of five BVMOs; cyclohexanone monooxygenase (CHMO), cyclopentanone monooxygenase (CPMO), 4-hydroxy-acetophenone monooxygenase (HAPMO), phenylacetone monooxygenase (PAMO) and trifluoroacetophenone
The bacterial strains isolated and used in this study showed extensive potential for degradation of fluorinated environmental pollutants. Pure bacterial cultures that utilize fluorinated aromatic compounds are very useful for understanding the environmental fate of these compounds and make it possible to develop bioreactors for the removal of these toxicants. Industrial waste often contains mixtures of toxic compounds that often restrict bacterial degradation. For sanitation of polluted sites or wastewater, mixed bacterial strains like a consortium of Arthrobacter sp. strain G1 and Ralstonia sp. strain H1 could be a better choice.
Direct defluorination in productive pathways obviously is an attractive route for fluoroaromatic metabolism. The 4-fluorophenol monooxygenases described in this thesis are the first examples of such enzymes that act on fluorophenols. Fluorophenols are important environmental pollutants by themselves, and it is likely that they are also as products formed by cometabolic transformation of the more complex bioactive molecules mentioned elsewhere in this thesis.
During the course of this research, we have attempted to isolate a wider range of microbial strains to study the degradation of trifluoromethyl-containing compounds. We collected soil samples from the sites contaminated with insecticides, herbicides and other xenobiotic organohalogens. A mixture of these samples was incubated for a prolonged period in MMY medium with trifluoroacetate (TFA), trifluoroethanol (TFE), trifluorotoluene (TFT) and trifluoroacetophenone (TFAP). After incubation for two years, we isolated a Gordonia sp. strain SH2 that degrades trifluoroacetophenone. However, no defluorination occurred because TFAP was converted to phenol and trifluoroacetate, the latter remained present in the culture medium as dead-end metabolite (Fig. 4).
FIG. 4. First two steps in the degradation pathway of trifluoroacetophenone by Gordonia sp. strain SH2.
The fate of trifluorinated compounds thus is still unclear. The most common environmental product of these compounds probably is trifluoroacetate. Sequential reductive defluorination of TFA resulted in the release of one fluoride in sequential steps of transformation forming difluoroacetate, monofluoroacetate and acetic acid. Under aerobic conditions, fluoroform was formed (32). We have not yet found growth of a bacterial culture from the soil mixture that uses TFA as a carbon source, suggesting that TFA is highly recalcitrant to biodegradation under aerobic conditions. So far, this breeding experiment is going on for almost 3 years, but we could not isolate any pure or mixed culture that can grow on trifluoromethylated compounds, indicating the high recalcitrance of these compounds.
Overall, great challenges lie ahead for environmental microbiologists, as thousands of new chemical compounds are being produced and emitted and the possibility of biodegradation remains the major factor determining their behavior.
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