1ANALYSIS OF FUNGAL DIVERSITY IN A PETROLEUM LAND TREATMENT UNIT USING TERMINAL RESTRICTION FRAGMENT PATTERNS (TRFPS)

The genetic composition of the fungal community in a petroleum remediation pilot scale land treatment unit (LTU) was analyzed using terminal restriction fragment patterns (TRFPs). The objective of the LTU was to create a suitable environment in order to promote the biodegradation of petroleum-contaminated sand. The LTU consisted of four separate cells treated with different soil amendments. Cell 2, amended with corn steep, was the cell of interest here. The 18S ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR) using primers that were previously designed and which proved to give the expected fragment sizes for Aspergillus genomic DNA. The primers yielded bands of approximately 550 base pairs (bp). One primer was a fluorescent-labeled primer. The tagged rDNA product was digested using either MspI, HaeIII, or DpnII restriction enzymes. The fragments were separated by capillary electrophoresis on an ABI310 Genetic Analyzer ® to observe the terminal restriction fragments (TRFs) generated by restriction digestion. The TRF patterns changed over time for each of the enzymes used. Both predicted and unpredicted peaks were observed for all three enzymes. Accurate predictions of diversity were not accomplished by the TRFs produced. TRF patterns for all enzymes showed a decrease in number of peaks from day 14 to day 49. By day 98