DEVELPMENT AND APPLICATION OF GENE PROBES TO DETERMINE DEGRADATION POTENTIAL AND TO IMPROVE IN-SITU BIROMEDIATION EFFORTS

THE GOALS OF THIS PROJECT ARE TO DEVELOP A METHOD TO RAPIDLY GENERATE GENE PROBES TO IDENTIFY AND MONITOR INDIGENOUS DEGRADATIVE MICROORGANISMS AT POLLUTED SITES AND DEGRADATIVE ORGANISMS ADDED TO AUGMENT NATURAL POPULATIONS, AND TO USE THE PROBES AS A PREDICTIVE TOOL FOR DETERMINING DEGRADATIVE POTENTIAL FOR SELECTING APPROPRIATE TREATMENT CONDITIONS. THE PROBES WILL ALSO BE USED TO ASSESS THE PROGRESS OF TREATMENT SO TIMELY ADJUSTMENTS CAN BE MADE TO MAINTAIN OPTIMUM TREAMENT PERFORMANCE. THE PROBES WILL BE GENERATED BY USING A NOVEL ARBITRARILY PRIMED POLYMERASE CHAIN REACTION (AP-PCR) PROTOCOL THAT GENERATES A "FINGERPRINT" OF DEGRADATIVE STRAINS, AND ALLOWS THE SELECTION OF UNIQUE DNA FRAGMENTS WHICH WILL FUNCTION AS SUB-STRAIN SPECIFIC PROBES. THE AP-PCR PROBES ALLOW THE DETECTION OF INDIVIDUAL STRAINS WITHOUT RELYING ON HOMOLOGY TO PROBES BASED ON ALREADY-CLONED DEGRADATIVE GENES, THEREBY ALLOWING DETECTION OF STRAINS WITH NOVEL DEGRADATIVE GENES OR STRAINS NOT DETECTED BY "STANDARD PROBES". GENE PROBING WILL BE PERFORMED IN CONJUNCTION WITH THE USE OF NOVEL DNA EXTRACTION AND FRACTIONATION PROTOCOLS WHICH SEPARATE THE COMMUNITY DNA BASED ON ITS % G+C. THESE PROTOCOLS ALLOW THE ANALYSIS OF THE TOTAL MICROBIAL COMMUNITY INCLUDING NON-CULTURABLE CELLS, AND IMPROVE THE SENSITIVITY OF DETECTING SPECIFIC ORGANISMS. THEY ALSO PROVIDE A MECHANISM FOR MONITORING GROSS CHANGES IN THE MICRBIAL COMMUNITY IN RESPONSE TO ENVIRONMENTAL CHANGES, THERBY ALLOWING ADJUSTMENT OF TREATMENT CONDITIONS TO OPTIMIZE PERFORMANCE.