| Hydrocarbon uptake is key factor in the bioremediation of soil and water polluted by petroleum. Two modes of hydrocarbon transport to microbial cells are generally considered: (1) interaction of cells with "solubilized/pseudosolubilized" or "accommodated" hydrocarbon droplets much smaller than the cells, it means microbes uptake the hydrocarbon emulsified with the biosurfactant produced by the cells (2) direct contact of cells with large hydrocarbon drops, it means microbes uptake hydrocarbon by the direct contact. It was found that major microbes in natural uptake hydrocarbon with the first mode. As to the second mode, the research of it was little and the existence of it was not confirmed. The existence of two modes of hydrocarbon uptake will be verified in this paper, the main study results are as follows:1. A hydrophilic bacterium and a hydrophobic bacterium were screened from soil polluted by petroleum according to the distribution character in oil-water system. They both belonged to Pseudomonas aeruginosa identified by morphological, biological tests and 16S rDNA. The hydrophilic bacterium was named as P.aeruginosa S; the hydrophobic bacterium was named as P.aeruginosa Y.2. The flask culture conditions for growth of P. aeruginosa in kerosene medium were investigated and optimized. The factors affected the growth of P.aeruginosa Y from major to minor were: kerosene volume, natural organism, nitrogen source, inoculation. The optimal kerosene medium for growth of P.aeruginosa Y was: CO(NH2)2 1.5g/L, kerosene 14%, peptone 0.1g/L, pH7.0 in mineral salt medium. The optimal culture conditions of P.aeruginosa Y were: inoculation 10%, shaking speed 200 r/min in 50mL/250mL flask, growth temperature 37℃. The factors affected the growth of P.aeruginosa S from major to minor were: temperature, kerosene volume, natural organism, inoculation. The optimal kerosene medium for growth of P.aeruginosa S was: kerosene 6%, beef extracts 0.1g/L, pH7.0 in mineral salt medium. The optimal culture conditions of s were: inoculation size 7%, shaking speed 200 r/min in 30mL/250mL flask, growth temperature 32℃. After optimum, the dry cell weight of P.aeruginosa Y increased from 1.9g/L to 3.0g/L and the dry cell weight of P.aeruginosa S increased from 3.3g/L to 4.34g/L.3. The biodegradation of hydrocarbon by two bacteria was studied. The mixed hydrocarbon was better biodegraded than single hydrocarbon by two bacteria. The biodegradation process of hexadecane was analyzed, the intracellular hexadecane and hexadecanoic acid were checked by GC, the extracellular fatty acid was checked by GC, and the extracellular organic acids were checked by HPLC. It could be known that hexadecane turned to hexadecanoic alcohol by momoterminal, hexadecanoic alcohol was oxygenated to hexadecanoic aldehyde and hexadecanoic acid, hexadecanoic acid was β-oxygenated to short-chain fatty acids, then the fatty acids were degraded into AcCoA, which switched to TCA,and was degraded to carbon dioxide and water in the end.4. According to the former experiments, the different modes of hydrocarbon uptake were studied in two bacteria. Growth: they both grew well in LB medium and the value of A600 was almost same in stationary phase, however, the growth velocity of them was different in kerosene medium, the growth of P.aeruginosa S was better than P.aeruginosa Y. Hydrophilicity: P.aeruginosa Y owned good hydrophobic character, the value of MATH was 0.994, P.aeruginosa S owned good hydrophilic character, the value of MATH was 0.007. Emulsification: the decrease of surface tension in P.aeruginosa Y kerosene medium was less 14 mN m-1 than that in P.aeruginosa S kerosene medium; the main cells of P.aeruginosa Y were aggregated in oil phase, there was no obvious emulsification in water phase, the main cells of P.aeruginosa S were aggregated in oil-water phase, there was apparent emulsification in water phase; the emulsified ability was measured, the value of P.aeruginosa Y was nearly zero and the value of P.aeruginosa S was about 25%; the growth ability of... |
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