Identification Of A Biosurfactant Producing Bacterium And Optimization Of Fermention Conditions

Biosurfactants (Biosurfactant, BS) is a surface-active substance which produced during microbial growth and metabolism and with many incomparable advantages that chemical surfactants don’t have:low toxicity, sensitization is generally not digestible, can be used in the field of cosmetics and as a food additive; available for environmental governance; good stability; structural diversity, may have some usefulness in specific areas. With the development of biotechnology, bio-surfactants are increasingly concerned by the academic community. Therefore, finding out more biosurfactant producing strains, improving their production in the fermentation broth will make a wide range of biological and economic significance.In the experiment we have separated a surfactant producing bacteria in the oil contaminated soil before. Through analysis of its morphology and its 16S rDNA sequence, determined its taxonomic status and identified it as Escherichia coli 1098-3.Conducted a series of optimization of fermentation conditions to benefict the growth of bacterial and synthesis of surfactant in the fermentation broth. The single factor variable experimental results showed that the most beneficial conditions for Escherichia coli 1098-3 growth and biosurfactants production is:37℃, initial pH 7.0, speed 200 r/min, with medium ratio of 2.5% soluble starch,1.5% tryptone,0.3% NaCl,0.5% potassium dihydrogen phosphate,0.004% calcium chloride,0.6% ammonium sulfate, magnesium sulfate 0.07%, yeast extract 0.06%,50 OmL flask containing 200 mL medium. Under this condition, the surface tension of the liquid dropped from 69.3 mN/m down to 34.3 mN/m, relative biosurfactant concentration was 500 in the fermentation broth. By measuring the relation between the cell concentration and the surface tension, the surfactant was found largely accumulate in the stable period of the growth curve. In order to induce more biosurfactant and increase biosurfactant production, added atolin, kerosene, glycine, ethylenediaminetetraacetic acid (EDTA) and FeSO4 respectively into the medium after a 12 h fermentation. The results showed:when added 0.3% glycine and 0.015% EDTA to the culture after 12 h fermentation, the broth tension declined from 69.2 mN/m to 32.1 mN/m in the fermentation broth. Maintaining carbon and nitrogen concentrations constant, conducted a multivariate orthogonal of salt concentration and the inducer concentration for further optimize the medium composition. The results showed that:shaking speed 200 r/min, culture temperature 37℃, initial pH 7.0,500 mL flask containing 200 mL medium, (2.5% soluble starch,1.5% tryptone,0.5% NaCl,0.7% potassium dihydrogen phosphate,0.004% calcium chloride,0.7% ammonium sulfate,0.07% magnesium sulfate,0.06% of yeast extract,0.03% glycine) was the theoretical optimal ratio for the medium. After verification of orthogonal experiment showed that, in this culture condition, the fermentation broth surface tension decreased from 69.3 mN/m to 30.2 mN/m, compared with the first optimal value of the orthogonal experiment 31 mN/m, the surface tension reduced by 2.5%. The orthogonal experimental design was realistic, so choosed it as the optimum fermentation condition of the bacteria Escherichia coli 1098-3.