【作者】 张英；

【导师】 施耀； 李伟；

【作者基本信息】 浙江大学 ， 环境工程， 2005， 硕士

【摘要】 燃料油燃烧引起的硫氧化物的排放是形成酸雨的主要原因。低含硫矿物燃料的稀缺、高品质清洁燃料油的需求和日益严格的控制标准迫切需要开发燃烧前深度脱硫技术。生物脱硫（BDS）具有选择性高、反应条件温和、设备投资和操作费用低等优点,是可能取代或补充传统的加氢催化脱硫法、实现燃料油深度脱硫最有效的技术之一。论文工作围绕着新分离的一株微杆菌（Microbacterium sp.）ZD-M2展开,主要研究了该菌的脱硫特性以及在气升式反应器内的脱硫性能。 从不同污泥与堆煤土样中分离出七株菌株,均能专一地切断二苯并噻吩（DBT）中的C-S键,沿4S途径代谢,生成2-羟基联苯（2-HBP）。通过对七株菌株46h休止细胞和繁殖期生长细胞脱硫活性的比较,筛选出具有较高脱硫活力的2^#和7^#菌株,并在生长温度30℃,pH 7.0,0.5 mmol·L^-1DBT作为唯一硫源,以甘油为碳源,用摇瓶培养法测定了它们的生长和脱硫曲线。两菌还能以燃料油中的其他含硫有机化合物:噻吩、苯并噻吩、二苯硫醚和4,6-二甲基二苯并噻吩为硫源生长,表现出较广的底物范围,具有较好的燃料油生物脱硫工业应用前景。系列实验结果显示2^#的各项脱硫特性均优于7^#,因此进一步的研究采用2^#菌株。通过生理生化和16S rDNA的鉴定,2^#菌株属于微杆菌属,并命名为微杆菌（Microbacterium sp.）ZD-M2,国内外未见该类属性的脱硫菌株的报道。 研究培养条件对该菌生长和脱硫的影响,发现该菌生长适宜的温度为30℃;对环境pH变化有良好的耐受性,适宜的初始pH为6.5～9.5;有机氮源比无机氮源更有利于细胞的生长,然而有机氮源对ZD-M2脱硫活性的表达有抑制作用,以氯化铵为最佳氮源来生长的最佳浓度为1.0 g·L^-1;该菌能利用多种碳源和硫源进行生长,但以甘油和DBT为最佳,最适甘油浓度为2.0 g·L^-1、DBT投加量为0.2 mmol·L^-1。在水溶液和存在有机相的溶液中,ZD-M2的脱硫活性都较高,以正十六烷为有机相时能提高菌株对DBT的脱硫活性,但活性持续时间只有9h,较水溶液中的24 h短。而有机相和水相的不同配比也会影响ZD-M2降解DBT的效率,通过实验得出两相的最佳配比为1:1,此时微生物的脱硫活性最高。 微杆菌ZD-M2的脱硫代谢途径基本上遵循“4S途径”,但同时发现另外两种新产物2-甲氧基联苯和联苯,这两种产物能部分消除2-HBP对酶的抑制和对细胞的毒害,从而更有利于生物脱硫。产物2-HBP不仅对ZD-M2的生长有抑制作用,对细胞脱硫酶的产生和脱硫酶的活性也有较强的抑制作用。在水溶液中当2-HBP初始投加浓度达到0.1 mM时DBT就不再降解,此时降解率为25%左右,而以正十六烷为有机相的加入能有效降低2-HBP的抑制作用,当2-HBP初始投加浓度达到0.2mM时DBT降解率也有50%左右。初次尝试研究了微杆菌ZD-M2更多还原

【Abstract】 Vast amounts of sulfur oxides released into the atmosphere by combustion of fossil fuels are the principal sources of acid rain. High sulfur contents of the crude oil reserves, the need for fuels ultra-low in sulfur and continued stringent regulations call for deep desulfurization technologies. Biodesulfurization （BDS） offers an attractive alternative or may be used complementary to hydrodesulfurization （HDS） due to the mild operating conditions, low costs and great reaction specificity afforded by the nature of biocatalysis. In the present work, BDS was studied with a newly isolated bacterial strain Microbacterium sp. ZD-M2.Seven bacterial strains capable of converting dibenzothiophene into 2-hydroxybiphenyl by cleaving the carbon-sulfur bonds were isolated from sludge and soil samples. Among them two strains, numbered 2^# and 7^# , were chosen for further characterization because of their relatively high desulfurization activities. Their growth curves were investigated under the condition of 30 ℃, PH 7.0, 0.5 mmol·L^-1DBT as the sole sulfur-source, glycerol as carbon nutrient in shaking flask. Other organic sulfur compounds such as thiophene, benzothiophene, diphenylsulfid and 4,6-dimenthylDBT, can also be used as sulfur sources to growth by the two strains, showing a broad substrate range. Strain 2^# is chosen for further study since the desulfurization activity of strain 2 was better than that of strain 7^# . This organism, designated ZD-M2, clustered most closely with members of the genus Microbacterium, as determined by 16S rRNA gene sequence analysis, and was firstly found to our knowledge.In aqueous the strain grew well in the pH range of 6.5⁹.5, the optimal temperature for growth was 30 ℃. Though organic nitrogen was better than inorganic nitrogen for strain growth, it inhibited the desulfurization activity, and the appropriate concentration of ammonium chloride as the source of nitrogen was 1.0 g·L^-1. Glycerol and DBT were selected as the best carbon and sulfur sources for its growth, and the optimal concentration and amount were 2.0 g·L^-1 and 0.5 mmol·L^-1, respectively. Desulfurization ability was studied in the presence of hexadecane as model oil with an optimal volume ratio of oil-to-aqueous of 1:1. In the aqueous and model oil-aqueous system, the desulfurization activities of strain ZD-M2 and 7^# were compared. It showed that the presence of hexadecane enhanced the activity of desulfurization, however the stability of the desulfurizing activity was worse than that in aqueous.Metabolites produced by DBT desulfurization were identified by GC-MS, and two sulfur-free products, 2-methoxybiphenyl （2-MBP） and biphenyl, were detected in addition to 2-hydroxybiphenyl （2-HBP）, the end product of the previously reported sulfur-specific pathway （also called 4S pathway）. The results suggest that the production of 2-MBP and biphenyl has the advantage of partially eliminating the inhibitory effect of 2-HBP and the pollution from diesel oil combustion. Not only the growth of ZD-M2, but the production and the activity of desulfurization enzyme were inhibited by 2-HBP. In aqueous phase, the degradation of DBT was stopped with a degradated amount of 25% once the concentration of 2-HBP was above 0.1 mM,however, when the concentration of 2-HBP was above 0.2 mM, the amount of degradated DBT was 50%. It showed the presence of hexadecane effectively eliminated the inhibitory of 2-HBP. Kinetic model of product （2-HBP） formation have firstly been attempted to develop. The model was found to meet Garden’s growth-associated scheme, i.e. 2-HBP is reffered to as growth-associated product, its rate of production parallels the growth of the cell population.Biodesulfurization of DBT by strain ZD-M2 in an oil-aqueous system was carried out in a self-designed airlift bioreactor. The optimal gas volume was 180 L·h^-1. The desulfurization of resting cells of ZD-M2 could be proceeded in the non-growth media （saline and phosphate buffer） as efficiently as in the growth medium （BSM）. In aqueous system, the concentration of更多还原