Most natural products and xenobiotic molecules, irrespective of their molecular or structural complexity, are degradable by some microbial species/associations within
particular environments. Atrazine- and selected petroleum hydrocarbon (benzene,
toluen~ and 0-, m- and p-xylene (BTX))-degrading associations were enriched and
isolated"trom atrazine- and petroleum hydrocarbon (PHC)-contaminated KwaZuluNatal
loamy and sandy soils, respectively. In total, eight pesticide- and forty BTXcatabolizing
associations were isolated. Electron microscopy revealed that,
numerically, rods constituted the majority of the populations responsible for both
atrazine and PHC catabolism. Cocci and, possibly, spores or fungal reproductive
bodies were observed also. For the BTX-catabolizing associations, the population
profiles appeared to be dependent on the enrichment pH and the molecule
concentration.
After combining selected associations, to ensure that all the isolated species were
present, batch cultures were made to determine the optimum pH and temperature for
growth; With an atrazine concentration of 30 mgr1, the highest specific growth rates,
as determined by biomass (OD) changes, were recorded at 30DC and pH 4 although
the rate§ at 25DC and pH 5 were comparable. For the BTX (50 mgr1)-catabolizing
associations, the highest growth rates were recorded at pH 4 for the four temperatures
(15, 20, 25 and 30DC) examined. The sole exception was p-xylene with the highest
specific growth rate recorded at pH 5 and 30De.
Batch and continuous (retentostat) cultivations in the presence/absence of methanol
and under C- and N-limited conditions were used to investigate the impacts of the
solvent and the catabolic potentials of a combined atrazine-catabolizing culture
(KRA30). In general, different degradation rates were recorded for the culture in
response to element limitation. Addition of citrate as the primary carbon source /
effected atrazine (100 mg!"l) degradation rates comparable to that of Pseudomonas sp.
strain ADP while succinate addition effected herbicide co-metabolism. Carbon
supplementation may, therefore, be considered for site amelioration practices.
To complement conventional culture-based microbiological procedures, molecular
techniques were employed to explore the diversities and analyze the structures of the
microbial communities. In parallel, anaerobic microbial associations which targeted
atrazine were also characterized. The soil DNA isolation/characterization protocol
adopted consisted of a clean-up step followed by the polymerase chain reaction (peR)
and 16S rDNA fingerprinting by denaturing-gradient gel electrophoresis (DGGE).
The preliminary results suggested that despite different, but chemically similar,
petroleum hydrocarbon molecules, the common selection pressures of the primary
enrichments effected the isolation of similar and complex aerobic microbial
associations. Some similar numerically-dominant bands characterized the aerobic and
anaerobic atrazine-catabolizing associations although distinct differences were also
recorded on the basis of the enrichment/isolation pH value and the concentration of the herbicide. Cloning and sequencing were then used to identify some of the
numerically-dominant and non-dominant association members.
Community-level physiological profiling (CLPP) for physiological fingerprinting was
made with Biolog EcoPlates and highlighted the differences in the isolated aerobic
atrazine-catabolizing associations depending on the enrichment pH and molecule
concentration.
Logarithmic-phase cultures of the combined atrazine- and BTX-catabolizing
associations were used to explore the association profiles following pH and
temperaiure optImIzation. Although some common numerically-dominant
components were maintained, differences in numerical and, possibly, activity
dominance were observed in the 16S rDNA profiles in response to changes in pH and
temperature. This indicated that environmental parameter optimization and
characterization of catabolic association structure must precede bioaugmentation so
that control of key variables will facilitate maintenance of the dominant site-specific
species.
Following KRA30 cultivation in the presence/absence of methanol and under carbon and
nitrogen-limited conditions, the population fingerprints showed that the presence
of methanol effected shifts in species numerical dominance and, possibly, changes in
atrazine catabolic capacity. Also, Coulter counter results, optical density readings and
16S rDNA characterization by DGGE indicated that degradation rate changes were
accompanied by shifts in species numerical/activity dominance within the association.
Although N-limitation effected the highest rates of herbicide catabolism, a potential
versatility of the combined association for bioaugmented and/or biosupplemented
remediation with acceptable rates regardless of any elemental limitation was recorded.
To determine if the contaminated and pristine source soils contained comparable
catabolic populations and, thus, offered potential for intrinsic bioremediation, PCRDGGE
was used to characterize the populations in comparison with the
enriched/isolated associations. Some similar dominant bands characterized the
contaminated soils and the enriched/isolated associations. The significance of this, in
relation to a possible correlation between numerical and activity dominance in the
component species, is discussed with respect to the use of PCR-DGGE to identify
natural attenuation potential and monitor sustained intrinsic and enhanced
(bioaugmented and biosupplemented) bioremediation. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2001.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/4909 |
| Date | January 2001 |
| Creators | Ralebitso, Theresia Komang. |
| Contributors | Van Verseveld, Henk W. |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
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