by 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 in 20 randomly 

 selected fields and the mean concentration determined. 



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 " 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 L 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 ' 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). 



