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    Bioremediation of Atrazine- and BTX-contaminated soils : insights through molecular/physiological characterization.
    (2001) Ralebitso, Theresia Komang.; Van Verseveld, Henk W.
    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.