The proposed study is based on the knowledge that many sites contaminated with xenobiotic/recalcitrant organic compounds are also contaminated with heavy metals. The hypothesis to be tested by this project is that the combined effect of toxic concentrations of metals and organics in soil can result in decreased bacterial biomass and activity, and inhibit bioremediation of that soil by indigenous organisms. The approach to be utilized herein is to utilize bacterial biomass and specific indicator bacteria (or a specific metabolic activity) as bioassays to assess the impact of toxic waste loads on soil viability. The influence of heavy metals on the metabolic and genetic potential of bacteria to degrade halohydrocarbons in soil will be addressed.
The specific aims for the proposed project period are: 1) To determine accurate values of total biomass, as well as selected indicator organisms and metabolic activities in metal/organic contaminated soils. 2) To measure the ability of contaminated soils in (1) to degrade an added organic compound which is normally biodegradable in soil. 3) To correlate the bioremediation potential of soils in (2) with the bacterial bioassay developed in (1). 4) To evaluate the influence of the heavy metals on the ability of specific bacteria to degrade organics in metal contaminated soils. 5) To use the biomarker assays developed in this project to document changes in the soil microflora following additions of contaminants to soil in field scale lysimeters. Bacterial biomass will be determined by direct extraction of DNA from soil samples followed by polymerase chain reaction (PCR) amplification of 16S ribosomal RNA encoding sequences. The amount of amplified DNA will be determined by HPLC analysis. PCR amplification will also be carried out utilizing primer pairs specific for particular bacterial genera or specific degradative pathway genes to determine if specific bacteria or catabolic capabilities are present in the soil. Arbitrary primed PCR will be used to generate fingerprints of soil communities to assess the diversity of the bacterial population present. Metabolic fingerprints of the bacterial communities will be obtained using Biology GN microplates. The test compound in the degradation studies will be 2,4-D, which will be added to microcosms of contaminated and noncontaminated soils at a rete of 1000 ppm. Degradation will be measured utilizing HPLC. 2,4-D degradation will then be measured in soil when added in combination with mercury. Alcaligenes eutrophus JMP134, which carries a plasmid specifying mercury resistance and 2,4-D degradation, will be utilized as a test organism. The effects of mercury and 2,4-D on the soil degrader population will be determined by a culture method as well as by PCR analysis utilizing primers specific for the tfdB gene. The procedures developed will ultimately be applied to an analysis of bacterial populations in a field scale lysimeter.
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