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y silica gel column chromatography, weight determination, glass
capillary gas chromatography and mass spectrometry.
Enumeration of Microbial Populations
Total numbers of microorganisms per gram dry weight of sediment
were determined by direct count procedures. Portions of collected
sediment samples were preserved with formalin. Microorgansims in the
preserved samples were collected on a 0.2 mm pore size Nuclepore filter
which had been stained with irgalan black. The microorganisms were
stained with acridine orange and viewed using an Olympus epif luorescence
microscope. Cells staining orange or green were counted
selected fields and the mean concentration determined.
in 20 randomly
Hydrocarbon utilizing microorganisms were enumerated using a three
tube Most Probable Number (MPN) procedure. Serial dilutions of sediment
samples, prepared using Rila marine salts solutions, were inoculated
into sealed serum vials containing 10 ml BushnjeAl Haas broth (Difco) and
50 ml of Arabian crude oil spiked with C hexadecane (sp. act.
1 mCi/ml) . After 14 days incubation at 15°C, the C0 2 (if any) in the
head space was collected by flushing and trapping in oxifluor CO .and
quantitated by liquid scintillation counting. Vials showing CO
production (counts significantly above background) were scored as
positive and the Most Probable Number of hydrocarbon utilizers per gram
dry weight calculated from standard MPN tables.
Biodegradation Potentials
Portions of sediment samples were placed into serum vials
containing 10 ml Bushnell Haas broth. and 50 ml light Arabian crude oil
spiked with either., C hexadecane, C pristane, C naphthalene, C
benzanthracene or C 9-methylanthracene. After 14 days incubation,
microbial hydrocarbon degrading activities were stopped by addition of
KOH. The
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C0„ produced from mineralization of the radiolabelled
hydrocarbon was determined by acidifying the solution, flushing the
headspace, trapping the CO in oxifluor C0„ and quantitating the C
by liquid scintillation counting. The residual undegraded hydrocarbons
and biodegradation products were recovered by extraction with hexane.
The
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C in each solvent extract was determined and fractionated, using
silica gel column chromatography, into undegraded hydrocarbon fractions
(hexane + benzene eluates) and degradation product fractions (methanol
eluate + residual non-eluted counts). A 0.75 cm diameter X 10 cm column
packed with 70-230 mesh silca gel 60 was used. Radiolabelled material
in each fraction was quantitated by liquid scintillation counting.
Sterile controls were used to correct for efficiency of recovery and
fractionation. Triplicate determinations were made for each sample and
radiolabelled hydrocarbon substrate combination. The percent
hydrocarbon mineralization was calculated as C0 2 produced (above
sterile control)/ C hydrocarbon, added. The .percent hydrocarbon
biodegradation was calculated as
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C0„ produced +, C methanol fraction
+ C residual (all above sterile control)/ C hydrocarbon added.
Carbon balances generally accounted for approximately 90% of the
radiolabelled carbon added to the sediment (except for naphthalene where
volatility losses prevented efficient recovery).