生物催化脱除柴油中有机硫的研究

国际上对燃料油的硫含量要求越来越严格,生物脱硫法(BDS)相比于传统的加氢脱硫(HDS)具有更广阔的应用前景.我们从自然界筛选到一组能专一性脱除二苯并噻吩(DBT)中硫元素的菌株YS-S-4,高效液相色谱(HPLC)对代谢产物的分析表明该菌能选择性地脱除DBT中的硫,生成2-羟基联苯(2-HBP).以DBT为硫源考察了pH值,碳源,氮源,硫源等对菌株YS-S-4生长及脱硫效果的影响.并在最佳条件下,能脱除正十六烷模拟柴油相中83·14%的DBT硫.     

燃油生物催化脱硫过程中的产物分析方法

燃油生物催化脱硫代谢产物结构复杂，种类多，分析测定有一定困难，现存在的测定方法之间有一定的差异，文章综述了现在常用的测定生物脱硫代谢产物的分析方法：如高效液相色谱法，气相色谱法，气相色谱-质谱联用法，液相色谱-质谱联用法，分光光度法，傅立叶红外光谱法，薄层色谱法等，以及这些方法的原理，样品前处理的程序，各种方法所能检测到的代谢产物种类，讨论并比较了方法的优缺点．     

氧化亚铁硫杆菌生长特性的研究

采用单因素实验研究实验室分离的氧化亚铁硫杆菌的生长特性.通过测定培养过程中吸光度、Fe2 浓度和总Fe浓度的变化,计算Fe2 氧化速率,研究初始pH值、初始接种量和温度对细菌生长的影响.结果发现,该氧化亚铁硫杆菌在接种初期,生长缓慢,对Fe2 的氧化速度较慢,12h后繁殖速度加快,氧化速率开始增加,27h后对Fe2 的氧化速率达最大值0.52g/(L.h),30h后Fe2 转化率接近100%;适宜于该氧化亚铁硫杆菌生长和代谢的初始pH值为2.30,接种量为10%,培养温度为28℃.     

Desulfurization metabolite of Rhodococcus erythropolis LSSE8-1 and its related desulfurizational gene fragments

Rhodococcus erythropolis LSSE8-1 is a newly isolated biodesulfurizaion strain from the soil of Chishui gas field, Guizhou Province, China. The analysis of its metabolism product shows that the strain is a kind of biocatalyst able to oxidize dibenzothiophene (DBT) to 2-hydroxydi-phenyl (HBP), and therefore the sulfur in DBT is selectively removed. By using DBTO2 (dibenzothiophene 5,5-dioxide) as substrate, both DBT and HBP are found in the culture,which shows that the reaction from DBT to DBTO2 is reversible in the cell. While using 0.5 mmol/L DBT as control,0.01-0.4 mmol/L DBTO2 shows poisonous effect to the cell,which will explain why there is no DBTO2 accumulation in the process of biodesulfnrization. After treatment by lysozme,the plasmid DNA of the strain is isolated by alkaline method to be used as the template of PCR reaction. Three dsz gene fragments of 1.3, 1.0 and 1.2 kb respectively were amplified.Each fragment is ligate with PGEM-T vector, and cloned into E. coil DH5a. The clone DNA is sequenced and the result shows that dsz related genes are highly conservative. The identities of dszA and dszB with respect to IGTS8 are 100%,and the identity of dszC with that of IGTS8 is 99%.     

二苯并噻吩单加氧酶基因(dszC)的克隆及其在大肠杆菌中的表达

利用PCR法从红球菌DS-3中克隆了dszC基因,序列测定结果与报道的Rhod ococcus ery throp olisIGTS8中d szC基因有99%的同源性.构建了含dszC基因的表达载体paC 5并在E.coli中实现高效表达,重组蛋白(D szC)在降温条件下重组菌表达量可达20%.可溶性D szC经N i2 亲和层析纯化达到电泳纯(95%以上).用纯化的D szC制备一抗血清进行免疫印记检测,证明工程菌表达产物中存在目的蛋白.纯化的D szC酶活可达0.36 U.D szC能作用于DBT及其甲基取代物,但不能利用无硫的DBT类似物及无机硫源.     

红串红球菌USTB-03对4,6-二甲基二苯并噻吩的脱硫研究

分离出1株能够对4,6-二甲基二苯并噻吩(4,6-DMDBT)进行高效脱硫的微生物菌种,经中国科学院微生物研究所鉴定为红串红球菌USTB-03。研究结果表明,与甘油和乙醇相比,葡萄糖是促进红串红球菌USTB-03生长和提高其脱硫比活性的最好碳源,最高脱硫比活性可达298.1mmol4,6-DMDBT/(kg·h)。在初始pH从4.0到9.0范围内,pH7.0可以使红串红球菌USTB-03保持最大的脱硫比活性。进一步研究显示,红串红球菌USTB-03对4,6-DMDBT脱硫后的最终代谢产物是3,3-二甲基-2-羟基联苯(2-HDMBP),只有C-S键被切断,而C-C键未受到破坏。该脱硫途径类似于二苯并噻吩(DBT)特异性脱硫的途径。     

Preparation of microbial desulfurization catalysts

A Rhodococcus sp. lawq, a bacterium isolated from the soil cleaving the C—S bond of dibenzothiophene (DBT) via specific pathway, was investigated for cell growth and for its role in desulfurization. Clearly, the end product, 2-hydroxybiphenyl, inhibited the growth of the strain, the synthesis of the desulfurization enzymes, and the activity of the enzymes. The effects of sulfate on the DBT degradation enzymes were examined in the Rhodococcus sp. lawq growth system with DBT; the sulfate served, concurrently, as the sulfur source. The condition of the resting cells that were used in desulfurization, was also studied. The optimal concentration of the resting cells and the reaction conditions were determined. It was documented that there is no difference between desulfurization activity for resting cells cultured with sulfate as the sole sulfur source and that with the mixture of DBT and sulfate as the sulfur source.     

石油生物脱硫菌UP-1培养及反应条件的优化

利用从胜利油田的土壤和污水中筛选到的能降解二苯并噻吩(DBT)的施氏假单胞菌UP 1,考察了培养温度、时间、pH值对菌种生长的影响,以及底物浓度、反应温度和反应时间对菌株UP 1脱硫性能的影响,并考察了UP 1静息细胞的重复使用性能。结果表明,UP 1最佳培养温度为30℃,培养时间为24h,培养基的pH为7.5;脱硫反应适宜的DBT质量浓度为500mg/L,反应温度为30℃,反应时间为20h;UP 1静息细胞具有可循环使用的脱硫活性。     

二苯并噻吩生物降解菌培养和反应条件的优化

考察了培养温度、时间、pH值对根癌土壤杆菌UP-3生长的影响,选择了最合适的培养条件.通过考察底物浓度、反应温度和反应时间对菌株UP-3降解二苯并噻吩(DBT)性能的影响,优化了生物降解的反应条件.结果表明:UP-3最佳培养条件为培养温度31℃,培养时间30 h,培养基pH值7.3;UP-3催化降解DBT反应的适宜条件为DBT质量浓度500 mg/L,反应温度31℃,反应时间35 h;适宜条件下UP-3生长细胞对DBT的降解率达到85%,说明其具有较好的应用潜力.