Characterization And Metabolic Pathway For Biodesulfurizing Organic Sulfur Compounds By Newly Isolated Strain

The emission of sulfur-oxides during fossil fuels combustion has caused serious air pollutions, such as acid rain. With the economic development, the demand of clean fuel is expanded, but actually sulfur content of the crude oil reserved is increasing. More and more stringent regulations demand developing deeper desulfurization technologies. In comparison with hydrodesulfurization (HDS), biodesulfurization (BDS) has the potential benefits due to the mild operational conditions, lower capital and operational costs, and selective removal of sulfur from some sulfur heterocyclics, such as dibenzothiophene (DBT) and substituted DBTs, which are recalcitrant to HDS. Therefore, BDS technology may provide an attractive alternative or be used complementarily to HDS for deeper desulfurization. This thesis investigated the characterization and metabolic pathway for biodesulfurization of organic sulfur compounds by newly isolation Mycobacterium sp. ZD-19. Moreover, resting cells of ZD-19 were investigated as biocatalyst for model fuel oil desulfurization.First work is to isolate and select bacterial strains with high and specific activity of removal sulfur from organic sulfur compounds. Seven bacteria capable of converting DBT into 2-hydroxybiphenyl (2-HBP) by cleaving the carbon-sulfur bonds were isolated from different soil samples. Among them, strain 19~# was chosen for further study because of high desulfurization activity and especial characterization. By 16S rDNA amplification and sequencing, 19~# was identified as Mycobacterium sp. and named Mycobacterium sp. ZD-19.Secondly, the desulfurization ability of newly isolated strain Mycobacterium sp. ZD-19 was studied. This strain could use DBT and other organic sulfur compounds, such as thiophene (TH) benzothiophene (BTH) 4,6-dimethyldibenzothiophene (4,6-DMDBT) and diphenylsulfld (DPS) as sole sulfur source to cultivate, showing a potential application to the desulfurization of fossil fuels. Moreover, desulfurization ability of resting cells for various organic sulfur compounds was performed, and the result is: TH > BTH > DPS > DBT > 4,6-DMDBT.In addition, when DBT and 4,6-DMDBT in a mixture as sulfur substrate, the desulfurization of ZD-19 proceeded simultaneously without preference for either, which showed the degradation rate of total sulfur related to the composition and structure of sulfur substrates.Thirdly, the metabolic pathway for biodesulfurization of organic sulfurcompounds by Mycobacterium sp. ZD-19 was studied. The final product of DBT biodesulfurization was determined by GC-MS, and identified as 2-methoxybiphenyl (2-MBP). It was possibly that 2-HBP, as the final product previously reported in sulfur-specific pathway (i.e. 4S pathway), was further converted to 2-MBP. The metabolites of 4,6-DMDBT had a similarly to DBT, which showed that metabolic pathway for Mycobacterium sp. ZD-19 desulfurzation of DBT and its derivate 4,6-DMDBT was different from the traditional 4S pathway, but a correspondingly extended 4S pathway.The final metabolite of biodesulfurizing DBT via 4S pathway, i.e. 2-HBP, would inhibit the microbial growth and DBT degradation. When initial concentration of 2-HBP was 0.5 mmol/L, the cells almost stopped growing and the desulfurization activity of resting cells was repressed by 1/3 than that without 2-HBP added to the fresh aqueous media. And at 2-HBP concentration of 2.0 mmol/L, the strain lost its desulfurization activity. Therefore, it was proposed that production of 2-MBP by Mycobacterium sp. ZD-19 have a potential advantage of partially eliminating the inhibition effect of 2-HBP.Fourthly, effects of the volume ratio of oil-to-aqueous, cell concentration and DBT concentration on desulfurization activity of resting cells were investigated in model oil system. The results showed that the presence of n-hexadecane would enhance the activity of desulfurization, but over-abundance hexadecane also would poison the cells. The resting cells of ZD-19 had highest desulfurization activity at the volume ratio of oil-to-aqueous of 1.0, and cell concentration of 10-20 g/L. In addition, at less than 5 mmol/L DBT initial concentration, the kinetics of DBT desulfurization by enzyme catalysis could be represented as the Michaelis-Menten Equation, and the parameters of vmax and Km were 0.45 mmol/(L-h) and 1.58 mmol/L, respectively. When DBT concentration was more than 10 mmol/L, substrate inhibition would appear and desulfurization ability of resting cells was repressed. At DBT concentration of 20 mmol/L, the resting cells of Mycobacterium sp. ZD-19 lost desulfurization activity.