• residual phosphorus, 



• residual ammoniacal nitrogen and the intracellular nitrogen concen- 

 tration (Kjeldahl's method); these two analyses served to determine 

 the quantity of biomass formed. 



The residual hydrocarbons were extracted from a second liquid sample. 

 Asphaltenes were precipitated from the hydrocarbon residue using hot 

 heptane for one hour, dried and weighed. 



The residue obtained after evaporation of the heptane was processed to 

 separate the three main families of hydrocarbons in crude oil: 

 saturates, aromatics and resins, by thin layer chromatography (50 mg 

 samples) or liquid chromatography (samples weighing about 1 g) . 



The sum of the weights of the three fractions thus recovered, using 

 liquid chromatography, compared with the initial rate of the hydrocar- 

 bons deposited on the column, always accounted for a proportion 

 between 90 and 100 %. 



The loss percentage increased when the test samples were taken at 

 increasingly long culture times, hence with samples that underwent the 

 longest biodegradation times. These losses are likely to be due 

 largely to the retention of polar compounds of the resins on the 

 column, compounds that are formed during oxydation reactions, or pos- 

 sibly by biochemical co-oxydation, and whose concentration increases 

 with biodegradation time. 



Using the different fractions obtained (saturates, aromatics, resins), 

 we performed more detailed analyses by gas phase chromatography 

 (Varian 3700 chromatograph) equipped with "Splitless" injection and 

 flamme ionization detector), a combination of gas phase chromatography 

 and mass spectrometry (Varian CH5DF spectrometer), proton NMR that 

 yielded the fraction of hydrogen belonging to methyl groups in the sa- 

 turates family, 13 C NMR, which yields the percentage of aromatic carbon 

 in comparison with total carbon in the aromatic fraction, and by infra- 

 red spectrometry on the resins. 



1. BATCH CULTURES 



1.1. Biodegradation of hydrocarbon families and sub-families in ALC 

 240 + . 



We selected a mixed culture of bacteria from samples of muds and slud- 

 ges collected on places hit by crude oil spills. 



The experiment was conducted with ALC 240 in an initial concentration 

 of 2.65 g.l -1 , over a period of 48 hours. 



Of the 2.65 g.l -1 of initial hydrocarbons, 1.08 g.l -1 were consumed, 

 representing 41 % degradation. It appears clearly that the saturates 

 fraction is most sensitive to biodegradation, because 67 % of this 

 fraction were consumed, whereas only 27 % of the aromatics fraction 

 were degraded. The quantity of hydrocarbons evaporated was negligible. 



From the standpoint of reproducibility of results, a previous experi- 

 ment yielded the following results: hydrocarbons consumed 44 %, satu- 

 rates degraded 63.1 %, aromatics disappeared 48.6 %. 



29 



