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Fossil fuels, including oil, coal, natural gas is the most widely used energy substances in the world today. Especially oil, industrial production is closely related to and closely linked with the economic lifeline of the world's industrial and therefore known as \Fossil fuels in various fields of life are inseparable. Almost universally due to special causes and structure of fossil fuels containing sulfur element, the majority of the inorganic sulfur to elemental sulfur in the form of pyrite FeS2, organic sulfur thiol, thioether, thiophene, and other more complex structure of heterocyclic compounds. Fossil fuels in the process of using the sulfur contained in various forms of emissions into the environment, causing serious pollution and damage. Therefore, the high-sulfur fuel after desulfurization treatment can be used for fuel desulfurization processing certain depth, is a major problem facing today's fossil fuel application process research and development of cost-effective desulfurization technology has become the fossil fuel industry is the most urgent One of the tasks it to improve fuel efficiency and reduce processing costs, improve the ecological environment and so has an extremely important significance. Since the summer of 2000, China began to implement new fuel standards. The provisions of the new standard, the sulfur content in gasoline should not exceed 800 mg / L, respectively. Such a standard would be first implemented in the three cities of Beijing, Guangzhou and Shanghai since July 1, 2000, to January 1, 2003, and other parts of the country began to perform. The new standard is more stringent compared with the old standard of 1200 mg / L sulfur content value. Of sulfides in the diesel combustion generates sulfur oxides cause acid rain and other series of environmental problems, has attracted the attention of the governments of the world, many countries struggling through technological innovation, process improvement to reduce the sulfur content in the fuel. Typical crude oil sulfur content between 1.0 × 103 ~ 3.0 × 103 mg / L, the the typical diesel sulfur content of approximately 5 × 103 mg / L. After technical improvements of the last few decades, the the finished diesel sulfur content has been reduced from 5 × 103 mg / L to 500 mg / L, between 2005 and 2007, the sulfur content of diesel fuel will be reduced to 10 of 15 mg / L. Conventional hydrodesulfurization (hydrodesulfurization, referred to as HDS) technology can effectively reduce the sulfur content in the distillate. HDS technology is a desulphurization carried out under high temperature (GT; 300 ° C), high pressure (>; 100 ATM) technology, are equally effective in the removal of the sulfur sulfides, mercaptans, thioethers, eventually sulfur atoms converted to H2S and / or structure of more complex organic sulfides. HDS to be removed dibenzo thiophene (dibenzothiophene, referred to as DBT), benzothienyl (benzothiophene, referred to as BT), thiophene (thiophene, abbreviated T) and the corresponding derivatives of sulfur need to strengthen the operating conditions, is not available from the operating costs line, and will reduce the value of fuel combustion. In recent years, made great progress in the research and development of biocatalytic desulfurization (biocatalysis desulfurization, referred to as BDS) technology. BDS technology is to use at room temperature and pressure for the particular specificity produced by a microorganism enzyme catalyzed heterocycle \The BDS technology meet desulfurization requirements at the same time, do not reduce the calorific value of the fuel, hydrogen, and does not require costly energy savings, while reducing carbon dioxide emissions, is extremely beneficial to environmental protection. Therefore, BDS desulfurization technology is a useful development prospects. The study of the subject content and select DBT diesel model compounds of sulfur-containing heterocyclic compounds, from the long-term oil-contaminated soil in 13 specific desulfurization bacteria isolated YZ-1 - YZ-13. The DBT desulfurization strains \matter, blue derivative absorbance value of quantitative basis, examine strains of DBT desulfurization capacity. YZ-9 desulfurization activity test, therefore, the YZ-9 further isolated and identified as the only sulfur source culture DBT investigated the ability of the strains of different concentrations of DBT desulfurization, and YZ-9 fermentation conditions optimized. Then YZ-9 inoculated into the Guangzhou Petrochemical straight run diesel, to inspect finished diesel desulfurization effect of the YZ-9 living cells. Finally, the YZ-9 made quiescent cells, to examine the desulfurization effect of the quiescent cells to different concentrations of DBT and diesel. Experimental specific points following sections expand: (1) Screening of Strains to be the petroleum fractions of contaminated soil samples for bacteria source, sieve bacteria used in two ways. One way is to inorganic sulfur (Na2SO4), the organic sulfur (DMSO), a heterocyclic thio (thiophene), model compounds DBT as sole sulfur source, sequentially forwarding mixed bacteria culture soil samples in every 24 h, gradually domesticated strains and enhanced desulfurization strains desulfurization capacity, dilution the coated tablet purified YZ-to YZ-8 eight specific desulfurization bacteria. YZ-1 ~ YZ-8 performance in basic salt medium containing DBT desulfurization capacity (basic salt medium, referred BSMD) unstable when adapter culture found. Another filtering method is, as a selective medium, the culture was centrifuged supernatant Gibb `s reagent under alkaline conditions becomes bluish as the basis, filtering separation purposes containing 1.0 mmol / L of DBT BSMD strains. Laboratory screened five specificity desulfurization bacteria were named as YZ-9 to YZ-13, adapter culture found YZ-9 on the model compound DBT desulfurization capacity of more stable performance, thereby determining the YZ-9 experimental strains, its physiological and biochemical identification, identification results show that the YZ-9 Rhodococcus. (2) YZ-9 fermentation conditions investigated, microbial body produces specific enzymes, with the specificity of the height of the substrate selectivity and reaction type, the concentration of enzyme and the concentration of the enzyme catalytic activity and bacterium, but also with strains of other influencing factors in the growth environment. Therefore, the YZ-9 optimal growth conditions were investigated. Will the late logarithmic phase the seed liquid volume ratio of 5%, were inoculated to the initial concentration of DBT, by the level of cell concentration and DBT removal rate of 0.5 mmol / L of fresh BSMD medium investigated carbon source, a nitrogen impact of source, sulfur source, pH value, temperature and other factors on the the strains growth conditions and desulfurization effect. In respectively contrast cultured with 5.0 g / L glucose, glycerin, sucrose, ethanol, citric acid, etc. as the carbon source, it is determined that the glycerin as the optimum carbon. Determine ammonium chloride for optimum carbon, ammonium nitrate and ammonium chloride as the nitrogen source compared cultivate visits. Incubated over magnesium sulfate, dimethyl sulfoxide, thiophene and DBT as sulfur source, investigated the growth and the desulfurization effect of the YZ-9, the results show that the seed culture DBT as sulfur source in cells that have a high desulfurization activity, other sulfur source seed cells cultured in growth in transit BSMD situation better desulfurization activity is relatively low. Investigated the initial pH value of the liquid fermentation with more optimization of the medium, and the culture temperature, and determine the optimal pH is between 7.0 to 8.0 and a temperature of 30 to 35 ° C. Further identified by orthogonal experiment, the medium formulation and optimal liquid fermentation conditions. Test medium formulation (g / L): KH2PO42.44 Na2HPO4? 12H2O 14.04, MnCl2 0.2, NH4Cl 2.0, CaCl2 0.02, FeCl3? 6H2O 0.001, NaCl 0.01, glycerol 6.5, trace elements solution mother liquor of 3.0 ml / L, the vitamin solution mother liquor 1.0 ml / L, fermentation pH value of 7.0. YZ-9, cultured under the optimized culture conditions the broth with the end of the logarithmic growth phase, the effects of different DBT concentration gradient of the desulfurization efficiency and diesel desulfurization effect. (3) YZ-9 living cells to DBT desulfurization capacity investigated specificity desulfurization bacteria the YZ-9 will be generated by the model compound DBT metabolism 2-HBP, the catalytic reaction occurs in the key of the \maintaining the \DBT catalytic after microbes specific enzymes, sequentially generates three metabolites of DBTO DBTO2 and 2-HBP (\Test using gas chromatography mass spectrometry determination of fermentation products, and found the existence of DBTO2 and 2-HBP, indicating that the metabolic pathways of this strain for the \Fermentation process, DBT metabolism, the final concentration of the product of 2-HBP and DBT initial concentration, have a certain degree of the desulfurization efficiency of the strain. Gibb `s reagent and 2-HBP to generate blue complex under alkaline conditions, there is a linear relationship between the concentration by mass of 2-HBP in the absorbance value at 610 nm and, for quantitatively determining the fermentation broth generated The concentration of 2-HBP, thereby calculating the degradation rate of the DBT. The experimentally determined strain effective desulfurization concentration of 0.5 ~ 1.0 mmol / L, the desulfurization rate at this time is more than 60%, the strain tolerance of DBT can range from 1.5 to 2.0 mmol / L. (4) YZ-9 living cells diesel desulfurization test, screening the ultimate purpose of the cultured bacteria strains used for biocatalytic desulfurization of diesel, select the sulfur content of 600 ~ 700 mg / L of Guangzhou Petrochemical straight-run diesel as processing objects, examine strains desulfurization capacity. Experiment, by YZ-9 on the oil-water volume ratio 1:3,1:4,1:5,1:6 proportion cultured 120 h, micro coulombs measured sulfur content after the culture, the use of industrial ethanol as demulsifiers The test showed that the ratio of oil volume 1:6 the best effect of desulfurization, desulfurization rate of 19.25%. (5) YZ-9 inspection of DBT desulfurization rate resting cells, YZ-9 centrifugal separation logarithmic growth of the late harvest bacteria, dubbed as the resting cells with different concentrations of bacterial suspension investigated resting cells 2 h of DBT off In addition to the effect, results show that DBT initial concentration of 2.0 mmol / L BSMD medium in a volume ratio of 10% inoculation, quiescent cell concentration was 3.0 g / L ratio desulfurization activity maximum 0.48μmmolg-1min-1. DBT removal rate of resting cells to different concentrations of 3.0 g / L of resting cells concentrations investigated, the results show that DBT initial concentration is higher, the lower the removal rate of 0.5 mmol / L the removal rate of up to 83.2 %, 1.0,1.5,2.5 mmol / L DBT removal rate were 78.4%, 64.2% and 53.1%, respectively. (6) YZ-9 resting cells investigated diesel desulfurization capacity. Resting cells mixed with diesel oscillating reactions visits diesel desulfurization rate 12 h, and the oil-water volume ratio of 1:6 repose cell volume fraction of 10%, the the diesel desulfurization test results show that resting cells concentrations of 5.0 and 6.0 g / L of diesel desulfurization best results, the desulfurization rate reached 25.7% and 24.6%, respectively. As can be seen from the experimental results, YZ-9 is an excellent strains, the enzyme produced by this strain can effectively removing the sulfur of the DBT and diesel laboratory diesel selected is straight run diesel, this strain of hope for hydrodesulfurization supplement desulfurization process research and development, with the value of further research.
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