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|a Ten bacterial strains previously isolated from petrochemical wastes were selected for the screening of biosurfactant producer(s), via four different methods; (i) surface tension measurements, (ii) blood hemolysis test, (iii) drop-collapsing test, and (iv) bacterial adherence to hydrocarbon (BATH) test. Two isolates coded AB-Cr1 and ETL-Cr1 identified as Actinobacillus sp. and Aeromonas sp., respectively were chosen to be the best candidates for biosurfactant production. Biosurfactant productions by both isolates were found to be growth-associated in all conditions tested. Biosurfactant production in glucose/crude oil medium (7.18-8.26 g/L) was found similar to that observed in crude oil-free medium (6.33-8.76 g/L). The production of biosurfactant was also studied in a fermentor using isolate AB-Cr1, as a factor of temperature, initial glucose concentration, pH and initial nitrogen concentration. The highest production of 12.45 g/L was obtained with AB-Cr1 grown in medium (pH 7) supplemented with 25mM NH4NO3 as nitrogen source and 3mM glucose as carbon source, incubated at 37°C under non-pH controlled strategy. TLC and FTIR characterization of crude biosurfactant produced by both isolates in medium supplemented or not with crude oil indicated the presence of lipoprotein and non-aromatic glycolipid types of biosurfactant. GC-MS analysis of fatty acid metyl esters indicated the presence of pentadecanoic acid in crude biosurfactant from both isolates as well as octadecanoic and heptadecanoic acid in the biosurfactant produced by AB-Cr1 and ETL-Cr1, respectively. The CMC of the biosurfactant produced in the presence and absence of crude oil were approximately (g/L) 1.0 and 0.1 for ABCr1, and 1.2 and 0.2 for ETL-Cr1, respectively. The biosurfactants were found capable of producing a relatively stable emulsion with hydrocarbon at pH 10. It was also found stable at various pHs (3.0-13.0 and 5.0-9.0) for AB-Cr1 and ETL-Cr1, respectively and thermostable for 1 hour at 100°C, based on the value of surface tension
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