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Screening of Biosurfactant Producing Microorganisms and Structural Characterization of the Biosurfactants

Author: ChengBinBin
Tutor: YangShiZhong
School: East China University of Science and Technology
Course: Physical and chemical
Keywords: lipopeptide glycolipid screening composition structure
CLC: O647
Type: Master's thesis
Year: 2013
Downloads: 89
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Abstract

Biosurfactants are a diverse group of surface-active biomolecules produced by several organisms. Lipopeptide and glycolipid are the two groups of the most important biosurfactants. This work presents an efficient, rapid and accurate screening method for the screening potential biosurfactants-producing bacteria. Biosurfactants-producing bacteria were isolated from the production water of several oil fields. The isolates were screened by developed methods. The biosurfactants-producing bacteria were identified through molecular biological method and the chemical structure of biosurfactants were analysed.The biosurfactants are insoluble at low pH value. We examined the change of absorbance of lipopeptides fermentation broth by Microplate Reader and obtained optimal experiment conditions:trichloroacetic acid with the amount of100μL; the volume of fermentation broth is50μL, and the reaction time is30min.307strains were isolated from oil field production water and examined by the estabolished method. TLC analysis showed that there were8strains producing lipopeptide and10strains producing glycolipid. The types of biosurfactants produced by the selected bacteria were analyzed through the ESI-MS method. The bacteria were identified by16S rDNA analysis. The result showed that lipopeptide producing bacteria are closely related to Bacillus subtilis and Ochrobactrum sp. and those producing glycolipid were are relatives of Pseudomonas aeruginosa.The chemical structures of biosurfactants were determined by GC-MS and MS/MS. For the structure of lipopeptide produced by the Ochrobactrum sp., the lipid part was composed of iso C13, anteiso C13, iso C14, n C14, iso C15, anteiso C15, iso C16, and n C16β-hydroxy fatty acids, whereas all the lipopeptides had peptide parts with the same amino acid composition of Val, Leu, Asp, Glu, and the sequence of the lipopeptide with iso C13, anteiso C13, iso C14, n C14, iso C15, anteiso C15β-hydroxy fatty acids was determined to be N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C. The lipopeptide produced by the Bacillus subtilis had similar structure as the lipopeptide from the Ochrobactrum sp.. Glycolipids produced by Pseudomonas aeruginosa were Monorhamnolipid Rha C10C8, Rha C10C10and dirhamnolipid RhaRha C10C10.The established method screening biosurfactants-producing microbes is feasible. It is first reported that Ochrobactrum sp. can produce lipopeptide, and the result provide a new information about the species of lipopeptide producers. Lipopeptide producers and glycolipid producers were both found in the high temperature oil field Shengli zhan-3, which enriched the knowledge about the diversity of biosurfactants-producing microbes in high temperature oil field reservoir microbial.

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