Screening Of Biodesulfurization Bacteria And Inspection Of Biodesulfurization Conditions

Fossil fuels, including petroleum,coal,natural gas are the most extensive applied energy materials,especially petroleum,it connect intimate with commercial industry and the lifeline of world industrial economy,so its called the"blood of industry". In all the vocations of agriculture and various fields of our daily life we can not live without fossil fuel. Owning to the special causes of formation and structure,all kinds of fossil fuels contain sulfur ubiquitously, inorganic sulfur mostly exist in the form of FeS2,organic sulfur exist in the form of thiol,thioether,thiophene and other more complicated heteroatomic ring structure,the amount of the sulfur element differ from the type of the fossil fuels. During the usage of fossil fuels,sulfur element discharged into the environment in various types,that bring about severe pollution and demolish.High-level sulfur fuels only can be used after the desulfurization process, so removal of the sulfur in the fuels to some extent come to be a big issue that the fossil fuel exploiter must be confronted with, research and develop economic effective desulfurization method is coming to be one of the most urgent tasks for the fossil fuel industries,it also benefit in the aspects of improving applying efficiency,cutting down processing costs and ameliorating zoology environment,etc..From the summer of 2000,our country began carrying out the new criteria of fuel. According to the new criteria,sulfur concentration in gasoline can not excess 800 mg/L. This new criteria firstly put into practice in Beijing,Shanghai and Guangzhou on July 1,2000,then other parts of china follow out before January 1,2003. The new criteria is more strict contrast to the past criteria which limit sulfur concentration to 1200 mg/L.Fossil fuels contain sulfur organic compounds and their combustion release sulfur oxides into the atmosphere. These emissions imply serious environmental problems such as acid rain. Government have recognized the worldwide problems associated with sulfur oxide emissions and have taken messures to reduce sulfur content in fuels through technique improvement and strict environmental legislation. Sulfur levels in typical crude oil range from 1000 to 30 000 mg/L, while typical concentration of sulfur in diesel oil is 5000 mg/L. In the last decade, the sulfur concentration has reduced from 5000 mg/L to less than 500 mg/L. Sulfur level in diesel oil is to be less than 10–15 mg/L according to the angency `s prediction in the years of 2005–2007. To decrease the amount of sulfur oxides leaving out into the environment, the reduction of the sulfur content in oil fractions is usually carried out traditionally by hydrodesulfurization (HDS). HDS is a catalytic hydrogenation technique at high temperature (>300℃) and high pressure (>100 atm), transforming sulfur element to H2S or into organic molecules. To remove sulfur from heterocycle compounds such as benzothiphene (BT) or dibenzothiphene (DBT) and/or their derivatives, a more stricter treatment is required,while this treatment is cost unaccessible and the caloric value of oil is reduced at the same time.Biocatalytic desulfurization(BDS) is another possible way to remove sulfur in diesil oil which is to be used after the HDS process,which has developed rapidly in recent years, due to the environmental benefits,high efficiency and high specificity of the desulfur enzymes to excise the bond between sulfur and carbon atom in the heterocycle compounds. For the high specificity of the enzyme and low cost of the transformating reaction, BDS is going to be a deeplevel desulfurization technique,and maybe used to produce low sulfur content commercial gasoline and diesel oil fractions. The specific enzyme in the microorganism seletive catalysis heterocycle sulfur atom, break down the"C-S"bonds, let out water soluble sulfate,thus the calorific value of the fuel oil is maintained. BDS don`t need hydrogen,which also cut down enery consume and reduce carbon dioxide discharge,so avail very much to environmental protection. BDS is a prospective method in desulfurization field.In this paper, DBT has been used as a model compound to study biodesulfurization. These Gram-positive bacteria YZ-1～YZ-13 are able to metabolize sulfur atom from DBT by the"4S"pathway, in which DBT is ultimately transformed to 2-hydroxybiphenyl (2-HBP) and sulfate. 2-HBP react with Gibb`s agent and produce a blue derivative.The strain YZ-9 was purified as a represent for its stable ability to metabolite DBT as sole sulfur source,we observed its ability to ultilize DBT and optimized its growing conditions when cultivated in basic salt medium when DBT was added as the sole sulfur source.At last, we applied YZ-9 into diesel oil gained from Guangzhou petrochemical company in order to observe its desulfurize effect in oil.This paper is mainly composed of four parts as follow:(1) Screening of desulfurization bacteria.We adopted two ways to screen out the specific sulfur degrading bacteria from long term contaminated soil by oil and its cut fractions.One way is operated by adding the inorganosulfur (sulfate),organosulfur(DMSO),heterocycle sulfur(thiophene) and at last the model compound DBT into the media by sequence when the YZ-9 was cultivated after 36 hours respectively,that means the desulfurization ability maybe inhanced and also the range of bacteria selectivity to sulfur compounds would be broaden.The result of this experiment shows eight strains YZ-1～YZ-8, they all follow the specific sulfur pathway of"4S",but after a serious of cultivation they lost the ability rapidly to degrade DBT to form 2-HBP,which react with Gibb`s agent form a blue compound that shows a significant absorption at 610nm. In another screening way, we gained YZ-9～YZ-13,by adding 1.0mmol/L DBT into the basic salt media(BSM), especially YZ-9 showed the long lasting desulfrization ability. So YZ-9 was selected as aim strain, and was identified as Rhodococcus sp. by physiological and biochemical methods.(2) Observation of the ferment conditions of YZ-9 in DBT mediumThe enzyme produced by the desulfurization bacteria shows high selectivity and high specificity, the concentration of the enzymes and their catalytic activity were affected both by cell concentration and other influence factors,thus the most suitable growth conditions of YZ-9 was inspected. When 5% volume culture at last phase of logarithm was inoculated into new BSMD media,detect the cell concentration at 620 nm and DBT desulfurization rate.The experiment result ensured glycerine as suitable carbonic source as contrasted to glucose,cane sugar,alcohol,citric acid at 5 g/L, and NH4Cl was a better nitrogen source when compared with NH4NO3.While MgSO4,DMSO,thiophene,DBT were respetively used as sulfur source in seed bacteria cultivation, the seed cultivated with DBT shows better desulfurization effect. The suitable pH and temperature were inspected under the above-mentioned suitable conditions, the result discovered that pH range was 7.0～8.0 and the temperature variation scope was 30～35℃. To further inspect the better suitable fermentation conditions of YZ-9, orthogonality experiment was carried out, the experiment discoverd the most suitable fermentation conditions: KH2PO4 2.44 g/L,Na2HPO4?12H2O 14.04 g/L,MnCl2 0.20 g/L,NH4Cl 2.00 g/L,CaCl2 0.02 g/L,FeCl3?6H2O 0.001 g/L,NaCl 0.01 g/L,Glycerine 6.5 g/L,microelement mother liquor 3.0 ml/L,vitamin solution 1.0 ml/L,pH 7.0.Under these conditions the desulfurization effect of DBT at different concentrations and diesel oil at different water/oil ratios were inspected with YZ-9.(3)Inspection of desulfurization effect to DBT.YZ-9 could transform sulfur atom in DBT into water soluble sulfate, DBT was transformed to two intermediate products DBTO and DBTO2 under this mechanism,and finally DBT was metabolited as 2-HBP.The concentration of DBT and 2-HBP in the culture both affect the growth of YZ-9 and desulfurization activity.2-HBP react with Gibb`s agent to form a kind of blue derivative which absorb light significantly at 610nm, so by detecting the absorbance at 610nm,we can optimize the media where the bacteria grow and metabolite.In our experiment, YZ-9 can degrade DBT to the extent of 60% at 73h when 1.0 mmol/L DBT was added as initial sulfur concentration and can endure 1.5～2.0 mmol/L DBT in culture medium.(4)Desulfurization experiment on diesel oil.The ultimate purpose of all the experiments carried out above is to apply the specificity strain YZ-9 into desulfurization of diesel oil ,thus we select distillated diesel oil from Guangzhou Petrochemical Company, in which sulfur concentration vary between 700～800 mg/L according to our test.When the ratio of oil to water vary from 1:3～1:6,the effect of desulfurization differ from 8.08%,13.11%,15.88% to 19.25%, while industrial alcohol was selected as deemulsifier.YZ-9 maybe studied further for the puopose of industry usage.(5)Inspection of YZ-9 resting cell desulfurization rate with DBT. Harvest YZ-9 at its logarithm growth phase and centrifugate the culture so to get solid cell to make resting cell at different concentrations by dilute it with phosphate salt buffer. Inspecting resting cell desulfurization effect in 2 hours,the result shows that when initial DBT concentration is 2.0 mmol/L in BSMD,incubating volume rate is 10%,resting cell has the biggest ratio desulfurization activity 0.48μmmolg-1min-1 while the concentration of the resting cell is 3.0 g/L。When 3.0 g/L resting cell was added into BSMD with different initial DBT concentrations,the result shows that when the initial DBT concentrations are more higher, the sulfur reduced rate is more lower. While initial DBT concentration is 0.5,1.0,1.5 and 2.5 mmol/L the DBT reduced rate is 83.2%,78.4%,64.2% and 53.1% respectively.(6)Inspection of YZ-9 resting cell desulfurization rate with diesel oil. Mixing resting cell with diesel oil for 12 hours in rotation bed,when oil/water rate is 1:6,incubating volume is 10% of the culture,the result of desulfurization test discovered that , resting cell behaved the best desulfurization effect 25.7% and 24.6% when resting cell concentrations are 5.0 and 6.0 g/L respectively. Resting cell shows the better desulfurization effect both to DBT and diesel oil than live cell for the reason that the specific enzymes are highly concentrated.From the experiments above we can safely draw the conclution that YZ-9 is a valuable desulfurization bacterium with great developing potential for its enzyme could remove sulfur atom in commercial diesel oil,so it has the potential to be used to develop a reinforced desulfurization process after HDS process to produce low sulfur or super low sulfur diesel oil ,so YZ-9 has the potential for further study.