搅拌式反应器中生物催化降解含硫杂环化合物

【作者】 王妙冬；

【导师】 汪大翚；

【作者基本信息】 浙江大学 ， 环境工程， 2005， 博士

【摘要】 矿物燃料燃烧后产生的SO₂是形成酸雾、酸雨等环境污染的重要原因。随着燃料油（汽油、柴油）用量的不断增加,由此造成的环境污染也日趋严重。目前从石油及其产品中脱除有机硫普遍采用的方法为加氢脱硫（Hydrodesulfurization, HDS）。对于杂环类含硫化合物,如二苯并噻吩（DBT）等,HDS的催化剂很难脱除这部分硫。国际上近年来迅速发展中的生物脱硫（biodesulfurization, BDS）技术,可以选择性地脱除这类化合物中的硫而不损失燃料的热值,并以其温和的反应条件,较低的操作成本而将成为21世纪降低石油产品硫含量的有效途径。 以DBT作为模型脱硫化合物,从炼油厂污泥中分离出一株专一脱硫的棒杆菌Corynebacterium ZD-1,能降解DBT,终产物为2-HBP。DBT为硫源,甘油为碳源,摇瓶中30℃培养45小时后,棒杆菌ZD-1的质量浓度达到最大,并且指数生长后期收集的菌体具有最高的脱硫活性。对ZD-1生长条件如初始pH,温度等进行了优化。ZD-1适宜生长条件为:pH 6.0～7.5,30℃,10.0g/l的甘油,5.0g/l的氯化铵,0.20 mmol/l的DBT。同时首次在Tween-80存在下,研究了ZD-1对底物DBT的生长动力学,发现ZD-1对DBT的生长动力学符合Monod方程,其中μ_max为0.134 hr^-1,k_m为0.016 mmol/l。 ZD-1通过“4S”途径降解DBT,反应终产物2-HBP和Na₂SO₄对DBT的降解均有抑制作用,而且2-HBP对菌体的生长也有抑制作用。该菌株休止细胞降解DBT的最佳浓度为9.2gdry cell/l,其脱硫活力随着菌体浓度的降低而增大,同时休止细胞的脱硫活性随放置时间的延长变化不大,稳定性好。 考察了ZD-1在油-水双液相体系中降解DBT时油水比、水相介质、反应器中通气量以及搅拌速率等因素对该反应的影响。ZD-1脱硫时的最佳油水比为1:2,而水相介质以培养液为佳,通气量越高越好,但搅拌速度以1300 r/min比较适宜。在定时更换培养液的情况下,ZD-1生长细胞在间歇式搅拌反应器中能够持续高活性脱硫200小时以上。 由于在水相和有机相中,后来分离到的菌株ZD-M2的脱硫活性都高于ZD-1,并且ZD-M2具有更广的底物范围,后续的实验也对ZD-M2作了进一步的研究。 ZD-M2休止细胞脱硫时,最佳油/水两相配比为1:1,在生长介质和非生长介质中具有几乎相同的脱硫活性,休止细胞脱硫时,茵体浓度在5.6g dry cell/l到14.0g dry cell/l范围时,ZD-M2在油-水双液相体系中降解DBT的脱硫活性基本相同,可以达到0.200 mmol（DBT）/kg dry cell/min,即2.2ling（DBT）/g/h。搅拌式反应中ZD-M2休止细胞降解正十六烷中的DBT内,在实验所选的条件下,通气量也是越高越好,而最佳的搅拌速率为1140r/min。 正十六烷中DBT的浓度过高对ZD-M2脱硫有抑制作用,ZD-M2降解DBT的生化反应动力学符合底物抑制模型,其中最大降解速率r_max为0.806 mmol/kg dry cell/min,米氏常数K_M为4.395 mmol/l,底物抑制的解离常数K_s为1.984 mmol/l。 为了促进微生物在油-水双液相体系中对DBT的降解,并探讨表面活性剂的作用机理。选择了Brij35、Tween-80、Triton-100X和β-环糊精等四种表面活性剂,考察了不同表面活性剂对水溶液中DBT的增溶效应,同时研究了不同表面活性剂对ZD-1和ZD-M2两种不同更多还原

【Abstract】 Emission of sulfur-oxides to the atmosphere through combustion of fossil fuel is a main cause of serious environmental problem such as acid rain. Countries all over the word have respond to this problem by enacting more and more stringent regulations, demanding refiners to produce petroleum products such as diesel fuel and gasoline with lower sulfur levels. Hydrodesulfurization （HDS） is a traditional technology used to remove sulfur from petroleum, but this process is reaching the limits of its cost and technical effectiveness. Biocatalytic desulfurization （BDS） may offer an attractive alternative to conventional thermochemical treatment due to the mild operating conditions and greater reaction specificity afforded by the nature of biocatalysis.A strain, isolated from refinery sludge and identified as Corynebacterium sp. ZD-1, can metabolize dibenzothiophene （DBT） to 2-hydroxybiphenyl （2-HBP） through a sulfur-specific pathway. The ability of strain ZD-1 to metabolize DBT in the presence of glycerol was investigated. The experimental results showed that the strain has its maximal desulfurization activity in the late exponential growth phase; and that the specific production rate of 2-HBP is about 0.14 （mmol/kg dry cell/min）. The strain reached its maximum growth （1.80 g dry cell/1） in shaking flask culture after 45.5 h. Some effect factors including initial pH, temperature, carbon source, sulfur source, and nitrogen source on the growth of Corynebacterium sp. ZD-1 were also studied. The optimum conditions for the growth of ZD-1 were obtained at initial pH=6.0⁷.5, 10.0g/l of glycerol, 5.0 g/l of NH₄C1, 0.20mmol/l of DBT and 30℃. Because of the poor solubility of DBT in water, the growth kinetics of ZD-1 was studied with Tween-80 existence. The growth kinetics of ZD-1 met the Monod-equation, meanwhile, μ_max= 0.134 hr^<sub>-1 and k_m =0.016 mmol/l.The behavior of DBT degradation by ZD-1 in aqueous phase was investigated. 2-HBP inhibited the growth of strain ZD-1, the production of DBT degradation enzymes, and the activity of enzymes. Sulfate inhibited the production of dibenzothiophene （DBT） degradation enzymes but had no effect on the enzymes’ activity. The production rates of 2-HBP at lower cell densities were higher and the maximum amount conversion of DBT to 2-HBP （0.067mmol/l） after 8 h was gained at 9.2 （g dry cell/1） rather higher cell density.The effects of oil/water volume ratio, composition of aqueous phase, gas flow rate, and stirrer speed on the DBT degradation were studied. It was found that the optimum oil-water ratio and rotation rate were 1: 2. Growing cells could degrade DBT for 48 h, which was much longer than resting cells in potassium phosphate buffer （pH 7.0） or physiological saline （both only 7h）. Meanwhile, Growing cells had higher desufurization activity than the others. As the culture media were refreshed regularly, ZD-1 could keep high desulfurization activity up to 200 h. The optimum conditions in stirred reactor were high gas flow rate and 1300 r/min.Microbacterium sp. ZD-M2, a strain isolated lately, had higher desulfurization activity thanZD-1 weather in aqueous phase or in oil-water system. Furthermore, ZD-M2 could degrade 4,6-dimethyl-DBT, thiophene, benzothiophene and 70% diphenylsulfid. Thus, more research work was done for ZD-M2.The optimum conditions for ZD-M2 were 1:1 oil/water ratio, 1140r/min stirrer speed. The cells almost had same desulfurization activity in culture media, potassium phosphate buffer （pH 7.0） or physiological saline. When the cell concentration of ZD-M2 was from 5.6 g dry cell/1 to 14.0 g dry cell/1, the specific production rate of 2-HBP were almost same, 0.200 mmol （DBT） /kg dry cell/min, or 2.21mg （DBT）/ g/ h. It was found that high concentration of DBT inhibited biodesulfurization of ZD-M2. Biochemical reaction kinetics of desulfurization by ZD-M2 met substrate inhibition model, r_max =0.806 mmol/kg dry cell/min, K_m =4.395 mmol/1, K_s=1.984 mmol/l.The effects of surfactants enhanced on the DBT degradation were researched. Among Brij, Tween-80, Triton-100X更多还原