use of molecular techniques to investigate microbial populations in

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USE OF MOLECULAR TECHNIQUES TO INVESTIGATE MICROBIAL POPULATIONS IN SOIL
IMPACTED BY HYDROCARBONS AND BRINE
1
K L. Sublette, 1Lorrie Houston, 1Steve Harmon, 2K. Duncan, and 3Ravi Kolhatkar
1
Center for Environmental Research & Technology, University of Tulsa, 600 S. College Ave. Tulsa, OK
74104; 2University of Oklahoma, Norman, OK; 3BP Amoco, Naperville, IL.
An investigation of the impact of a hydrocarbon spill or leak on soil ecology or the potential or
progress of bioremediation of contaminated soil typically requires an enumeration and characterization of
microbial populations in the soil. Most conventional methods for enumeration require culturing on a solid
medium (viable plate counts) or in liquid medium (most probable number counts). However, it has been
estimated that 90% of the microorganisms in soil are non-culturable. Molecular microbial techniques which
do not depend on culturing give a more complete picture of the soil microbiology of an impacted site. For
example, phospholipid fatty acids (PFLA) from microbial cell membranes and cell walls may be extracted
with an organic solvent and characterized by GC/MS to yield information about the total viable biomass
present, community structure, metabolic status and environmental stress. Specific PLFA biomarkers can
indicate the presence and numbers of sulfate reducing bacteria (SRB), methane-oxidizing bacteria, fungi,
eucaryotes, etc. DNA extracted from soils can be probed for specific genes such as those specific for
aerobic degradation of aromatic or aliphatic hydrocarbons, ammonium oxidation, or sulfide reduction of
SRB. Amplification of 16s ribosomal DNA by PCR and separation of the products using degrading gradient
gel electrophoresis (DGGE) can allow determination of the number and relative abundance of eubacteria in
soil samples. Subsequent sequencing of individual bands from the gel can identify dominant organisms.
Use of these techniques to characterize the microbial community in soil impacted by crude oil will be
discussed.
Key words: microbial populations, biomass, hydrocarbon spill
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