Characterization of 1, 2-DCA degrading Ancylobacter aquaticus strains isolated in South Africa.

Abstract

1,2-Dichloroethane (1,2-DCA), a highly toxic and recalcitrant compound, is produced anthropogenically in larger quantities than any other chlorinated compound. It is regarded as a mutagen and carcinogen, thus making it a priority target molecule for biological degradation. In addition, the intermediates of 1,2-DCA degradation are highly reactive and toxic, due to the electrophilic nature of the carbonyl groups in these compounds. Aerobic biodegradation of 1,2-DCA, resulting in complete mineralization, has previously been reported in Xanthobacter autotrophicus GJ10 and some Ancylobacter aquaticus strains. X. autotrophicus GJ10 has been found to possess chloroacetaldehyde (CAA) dehydrogenase and haloacid (HA) dehalogenase enzymes, both of which play a crucial role in 1,2-DCA degradation. Five strains of Ancylobacter aquaticus capable of utilizing 1,2-DCA as a sole carbon and energy source have recently been isolated in our laboratory. The degradation potential and specific dehalogenase activities of these bacterial isolates against 1,2-DCA and other halogenated compounds as a carbon source were investigated and compared to previously characterized organisms, viz., X. autotrophicus GJ10 and Ancylobacter aquaticus strains AD25 and AD27. Furthermore, this study proposed to detect the presence of the CAA dehydrogenase (aldB) and HA dehalogenase (dhlB) encoding genes in these isolates. Growth of all strains in the presence of 1,2-DCA as a carbon source was monitored over an 84 h period, in minimal medium supplemented with either vitamins or yeast extract. Dehalogenase activities were measured colorimetrically by monitoring halide release by crude cell extracts of the isolates. In order to detect the presence of dhlB and aldB genes, genomic DNA of the isolates was digested with individual restriction endonucleases, viz., EcoRI, PstI, HindIII and BamHI, and then subjected to Southern hybridization experiments. All isolates demonstrated significant growth rates in both vitamin and yeast extract supplemented media, with the former having a greater overall growth effect. Ancylobacter aquaticus DH5 demonstrated the highest growth rate of 0.147.h-1 in the presence of vitamins while Ancylobacter aquaticus DH12 displayed the highest growth rate of 0.118.h-1 with yeast extract. Optimum haloalkane dehalogenase activities of these bacterial isolates were confirmed at pH 8, similar to the activity in X. autotrophicus GJ10, while haloaciddehalogenase activity had a broader pH range. Hydrolytic dehalogenase activity of the bacterial isolates using a range of halogenated aliphatic compounds was also determined. Results demonstrated a wide substrate range with activity being observed on 1,3- dibromopropane, 1,2-dibromoethane and 1,3-dichoropropene, for all isolates. Southern Hybridization experiments confirmed the presence of both aldB and dhlB genes in X. autotrophicus GJ10. The dhlB probe produced a positive signal for an EcoRI fragment in Ancylobacter aquaticus DH12 while the aldB probe hybridized and produced a single positive signal on similar sized PstI fragments for all organisms except A. aquaticus AD25 which produced two positive signals. The results in this study demonstrate the potential application of the newly isolated strains of Ancylobacter aquaticus. in future bioremediation strategies. The detection of the genes involved in 1,2-DCA degradation further support the use of these isolates and/or their enzymes for the degradation of 1,2- DCA as well as other halogenated compounds. Future work need to determine sequence similarity of these genes detected in A. aquaticus strains to the genes in Xanthobacter autotrophicus GJ10 and other previously reported genes. It may also be important to investigate the activity of the enzymes under various environmental conditions and to determine enzyme structure and the catalytic sites, so as to gain knowledge of their degradation potential on site. Characterization of enzymes at both the molecular and protein levels may be necessary and beneficial for implementation in strategies involving bioremediation for the biological degradation of a wide range of halogenated aliphatic hydrocarbons.