Diversity Analysis Of Oil-Degrading Bacteria In The Indian Ocean And Optimization Of Biosurfactant Production

Marine microorganisms play an important role in elimination and bioremediation of marine oil pollution. Biosurfactant play a role of promoting dissolution and degradation in hydrophobic substrate. In this study, we have analyzed the diversity of oil-degrading bacteria acrossing the Indian Ocean surface seawater during the 19th scientific expedition of "DAYANG YIHAO" in 2007. In addition, we optimized the production of a biosurfactant from the oil-degrading bacteria Alcanivorax dieselolei B-5.The main contents and findings are as following:(1) In order to investigate the diversity of the oil-degrading bacteria in the surface seawater across the India Ocean, and obtain new oil-degrading bacteria. Potential oil-degrading bacteria were selected out via 1:1 mixture of diesel and crude oil as sole carbon source. Meanwhile, the community structure of 13 enrichments was analyzed by polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE). We obtained 51 unique strains of 29 genera after screening via morphological, physiological, biochemical and 16S rRNA analyses. They mainly belonged to a andγ-Proteobacteria. The four genera Alcanivorax (accounting for 18%), Novosphingobium (10%), Marinobacter (6%) and Thalassospira (6%) were the most predominant bacteria. Ecological analyses showed that the bacteria had high diversity with Shannon-Winner index (H) of 4.57968, and distributed even with Evenness index (E) as 0.8664771. Then Further experiments revealed oil-degrading capability of 49 strains. In addition, our investigation revealed oil-degrading ability of genera Sinomonas, Knoellia and Mesoflavibacter for the first time. DGGE fingerprint patterns indicated that the genus Alcanivorax was an important oil-degrading bacteria in the surface seawater across the India Ocean. This study demonstrated a high diversity of the oil-degradation bacteria in the surface seawater of Indian Ocean, these bacteria are of potential in bioremediation of marine oil pollution.(2) Quantification of the biosurfactants produced by a variety of microorganisms is a time taking and dif fi cult task due to the lack of rapid, ef fi cient and accurate methods. This work presents a simple turbidometric method forquanti fi cation of crude biosurfactants based on their property to become insoluble at low pH values. Biosurfactants obtained from A. dieselolei B-5 using different carbon substrates showed a good linear correlation(R2>0.95) between biosurfactant concentrations and turbidity in the range of 2 to 10 g/L of crude biosurfactants. The MSM, MMSM, DMSM developed in this work effectively predicted the amount of crude biosurfactant produced in different sets of fermentation experiments validating the method.The biosurfactant produced in the fermentation medium was quantified directly by cell-free solution turbidity corresponding standard curve. The nutritional medium requirement for biosurfactant production by A. dieselolei B-5 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were liquid paraffin, TWEEN-80 and SrCl2. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were:liquid paraffin, 33.18 ml/L; TWEEN-80,4.15 ml/L; SrCl2,0.22 g/L respectively. Using this statistical optimization method, the biosurfactant yield was increased from 2.2 g/L to 4.31 g/L, which is 95.91 percent higher than the non-optimized rich medium.