13 



The C NMR analyses used to quantify the aromatics C/total C ratio 



failed to reveal any significant difference before (43.4 %) an after 

 (43.7 %) biodegradation. 



With respect to the resins, part of the polar compounds of this frac- 

 tion formed during biodegradation remained absorbed on the liquid chro- 

 matography column, and consequently the analyses performed on the eluted 

 resin fraction were not truly representative. This retention of polar 

 compounds of the liquid chromatography column was confirmed by elemental 

 analysis, showing oxygen to drop from 2.75 % (by weight) at the start 

 of the batch culture to 2.35 % at the end of the culture. 



The determination of molecular weights of the resins yielded the 

 following results: 690 at the start of the batch culture, 740 at the 

 end of the batch, namely very slightly differing molecular weights. 



1.2. Examination of oxidation products. 



Analyzing the aqueous phase sampled at the end of the batch culture 

 (volume sampled = 1 liter), centrifuged and filtered, we found a total 

 organic carbon concentration of 260 mg.1-1. 



We carried out an esterif ication (BF3-CHgOH) of the compounds of this 

 aqueous phase. The organic extract was evaporated and weighed. The 

 weight of the extracted compounds, related to one liter of culture, 

 was 120.2 mg. In the acidified residual aqueous phase, initial extrac- 

 tion with CHpClp, followed by a second extraction with benzene, yielded 

 a new organic phase that contained polar compounds such as alcohols, 

 ketones and phenols, which represented 7.58 mg/1 of aqueous phase after 

 evaporation. 



Identification by GC/MS coupling of compounds separated by gas phase 

 chromatography was difficult because of the presence of a strong 

 background of poorly resolved constituents, which could be hydrocarbons. 

 Despite these problems, we succeeded in identifying normal and iso 

 acidic compounds in the aqueous phase, in the form of their correspon- 

 ding esters, obtained after esterif ication of the aqueous phase. 



The GC/MS coupling enabled us to observe the masses m/e = 74 characte- 

 ristic of n-esters, and m/e = 88 characteristics of iso-esters. 



1.3. Changes in microbial flora with time. 



Three samples were taken during the batch culture, the first at the 

 start of growth, the second during the active biodegradation phase 

 (after 15 hours of culture), corresponding to consumption of the satu- 

 rated hydrocarbons, and the third after 25 hours, in the slowdown 

 period of the biodegradation process, corresponding to microbial 

 attack of the aromatics. 



In the three different stages investigated, different dominant strains 

 were found, belonging to two genera only, Pseudomonas and Moraxella , 

 confirming that changes in the crude oil during a biodegradation pro- 

 cess are accompanied automatically by changes in the microbial flora. 

 At the start and middle of the batch culture, we chiefly identified 



31 



