Molecular Monitoring of Microbial Populations During Bioremediation of Contaminated SoilsEPA Grant Number: U915621
Title: Molecular Monitoring of Microbial Populations During Bioremediation of Contaminated Soils
Investigators: Mills, DeEtta K.
Institution: George Mason University
EPA Project Officer: Bishop, Linda
Project Period: August 1, 1999 through August 1, 2003
Project Amount: $46,780
RFA: STAR Graduate Fellowships (1999) RFA Text | Recipients Lists
Research Category: Fellowship - Life Sciences , Academic Fellowships , Biology/Life Sciences
The objective of this research project is to use molecular techniques to examine the microbial population dynamics (changes) during the bioremediation of petroleum- contaminated soils in laboratory bioreactor microcosms and the impact nutrient enhancement has on the microbial community structure.
Natural-occurring base substitutions, deletions, or insertions within the nucleic acid sequences provide molecular markers that can be used to distinguish between different genomes. Microbial community profiles based on the restriction enzyme site polymorphisms or natural- occurring sequence differences in the 16S ribosomal DNA (rDNA) genes produce different sized DNA fragments or fingerprints. Therefore, this research will use microbial community DNA fingerprints to monitor the temporal microbial dynamics and the impact of nutritional amendment during the bioremediation process. Briefly, whole-soil community genomic DNA will be extracted from bioreactor slurry soil samples using a modified bead beating protocol. The polymerase chain reaction (PCR) will be used to amplify 16S rDNA with fluorescent-labeled nucleotides or fluorescent-labeled universal primers. The purified PCR products will be either: (1) subjected to restriction enzyme digests (restriction fragment length polymorphisms and fluorescent terminal length polymorphisms); or (2) directly loaded onto a gel (amplicon length heterogeneity). Resolution of the fingerprint patterns will be performed on the Perkin Elmer ABI Prism 377 fluorescent DNA sequencing instrument. These three fluorescent-based methods will be assessed for their ability to screen the complexities of the microbial community. To probe the microbial diversity of the samples, cloning of the PCR products and subsequent DNA sequencing of these clones will determine the species that are present at any given time.
These molecular techniques, utilizing direct nucleic acid analyses, will contribute information about the biotic processes that is not readily obtained by traditional microbiological methods, will provide data about the impact nutrient enhancement has on the microbial community structure, and can be useful as a remediation monitoring tool.