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. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/6189 |
| Date | January 2011 |
| Creators | Pillay, Thiloshini. |
| Contributors | Pillay, Basil., Olaniran, Ademola O. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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