The Researches On Marine Microbial Emulsifier And Desulfurizing Thermophiles

It has been found some microbes can degrade some oil pollution in waters. The research of microbial degradation of oil pollution was about the theory of microbiology and molecular biology, and recently it is about the practical application mainly in biological restoration. Because traditional HDS hardly get rid of organic sulfur, just as DBT and its derivatives and its high cost, difficult operation, HDS hardly satisfies more and more rigid sulfur-free desire. However, biological desulphurization not only gets rid of organic sulfur efficiently, but also holds combustion value. Also due to the simple operation, low cost, and low lever greenhouse gas, so BDS can efficiently complements and even substitute HDS. This paper analyzes the active material of the emulsifier produced the microorganism from the deep-sea environment and the metabolic product produced by high-temperature desulphurization microorganisms.The first part analyzes the active material of emulsifiers produced by three Model Acinetobacter and three Acinetobacter strains isolated from our lab and connection of these different emulsifiers.Five strains of Acinetobacter were investigated for bioemulsifier by purification, components analysis and gene detection. Results showed that all the 5 bacteria could produce bioemulsifiers. Only from an isolate wp02421 from deep sediment, PCR assay detected the presence of the gene of esterase which located on the membrane and mediated Emuslan release from the cell to culture. Gas chromatography and mass spectrometry (GC/MS) analysis revealed the components of fatty acids as the constituents of bioemulsifier produced by isolate wpo2421, and identified as hexadecane and octacecanoic acids. The results of esterase and fatty acids indicated that glycolipid was most probably present in the bioemulsifier product of wpo2421 similar to Emulsan. In addition, from all the 5 strains by PCR, an outermembrane protein A gene (ompA) was detected. It showed varied homologies in range from 71.26%to 80.23% with the main emulsifying protein AlnA in the emulsifier Alasan produced by Acinetobacter radioresistens KA53. This suggests that OmpA may be the active component of the emulsifier produced by the 5 Acinetobacter strains. Therefore, both glycolipid and OmpA probably exist in isolate wp02421 as the bioemulsifers.Five Acinetobacter strains can code OmpA gene, in particularly, wp02421 OmpA gene which has the closet homology with Alasan gene has been expressed in E.coli. Primer OmpA is used to amplify wp02421 OmpA gene and then wp02421 OmpA whole gene sequence in ligated into E.coli. The result shows .OmpA protein can be expressed in E.coli abundantly in the form of inclusion body.Combined protein is still inclusion body although incubation time,temperature,IPTG concentration is reduced. We can get pure combined protein from the whole protein by renaturation and purification on Ni-Agarose medium. The purified protein has emulsifying activity to oil .We express OmpA in Yeast in order to get more protein excreted extra-cellular. Primer Omp is used to amplify wp02421 OmpA gene with endoenzyme point EcoRⅠand XbaⅠand then wp02421 OmpA whole gene sequence in transformed into Yeast which is induced by methanol. The combined protein has emulsifying activity to hexadecane. Recently, JCM6841,JCM6842,bme-3,bm-8 OmpA genes are ligated into plasmid whose is linearied into Yeast.The study about expression is in the process,Finally, we study the property of the strain wp02421.The second part is about the screen of normal temperature and high temperature desulfurizing strain in culture medium which the sulfur resource is DBT, and the domination of the strains are DBT-1,DBT-2,DBT-3,DBT-4(normal temperature) and DBT-601,DBT-602(high temperature).The typical DBT degradation pathway is 4S pathway which degrades DBT into 2-biphenyl.The research method of DBT degradation pathway is concentrating culture medium and then analyzing the metabolic products by GC-MS. Different from 4S pathway, the metabolism product of DBT is 3-OH-1,1′-Biphenyl.This pathway is catalyzed by dszA,dszB,dszC which are monooxygenases. According to conservative sequences, we design primers to amplify these monooxygenases in the high temperature strains DBT-601 and DBT-602,but we just amplified glycerin dehydrogenase. Finally,we determined the optimum growth temperature of the high temperature strain. Exclude DBT which is hardly degraded, there are other organic sulfur compound in oil. The degradation spectrum of organic sulfur is the standard of desulphurization ability. The high temperature strain 601 growth well or bad in the culture medium which thiophen,4-methyl DBT,2- formylthiophen and 2-aceticthiophen,which suggests the strain has broad application future in biodesulfurization.