血红蛋白基因在脱硫菌R-8中的表达及其在柴油脱硫中的应用

摘要：【目的】旨在构建一株优良的工程菌株,对血红蛋白基因在柴油的生物脱硫领域的应用做初步的探索。【方法】以德氏假单胞菌（Pseudomonas delafieldii） R-8为出发菌株,通过基因工程的手段,构建透明颤菌（Vitreoscilla）血红蛋白基因表达质粒并电击导入原始菌株,得到重组菌P. delafieldii R-8-2。【结果】R-8-2菌株的CO差光谱在419 nm处有特征峰出现,表明血红蛋白在脱硫菌中得到了有效表达。R-8-2菌株和R-8菌株相比,生长得到改善,相同培养条件下菌体密度比R-8提高了20%,最大脱硫活性能够达到R-8的2.4倍。在实际柴油脱硫实验中,R-8-2菌株能将柴油的硫含量降至96.6 mg/L,脱硫率达到69.9%,而R-8仅为57.2%。【结论】R-8-2是在较低溶氧条件下仍能保持较高的菌体密度和脱硫活性的基因工程菌株,具有良好的应用前景,该研究为血红蛋白基因在生物脱硫工业的应用提供参考。     

脱硫菌Fds-1的分离鉴定及其对柴油脱硫特性的研究

从含硫土壤中分离筛选出一株专一性脱硫菌Fds-1,经生理生化指标和16S rRNA序列分析鉴定其属于枯草芽孢杆菌(Bacillus subtilis)。用Gibb’s试剂显色和气相色谱-质谱联用分析表明,该菌株通过“4S”途径脱除有机硫。实验发现Fds-1的最佳脱硫活性在30℃,在此温度下72h内能脱除约0.5mmol/L DBT中的有机硫。Fds-1菌株对有机硫化合物的利用情况和柴油脱硫前后烃组分比较都进一步证明该菌株适合于柴油生物脱硫。利用休止细胞对不同组分柴油的脱硫研究表明,脱硫菌株Fds-1对精制柴油中的DBT类化合物的降解能力强。因此,该菌株对精制低硫柴油的深度脱硫具有应用意义。     

专一性脱硫菌脱硫活性比较与基因保守性研究

对几株能专一性脱除二苯并噻吩(DBT)中硫元素生成2-羟基联苯的细菌,即短芽孢杆菌(Bacillus brevis)R-6、德氏假单孢菌(Pseudomonas delafleldii)R-8、小球诺卡氏菌(Nocardia globerula)R-9、球形芽孢杆菌(Bacillus sphaericus)R-16、红平红球菌(Rhodococcus erythropolis)LSSE8-1和戈登氏菌(Gordonia nitida)LSSEJ-1展开研究。对照研究发现它们对DBT及其衍生物的代谢活性存在着一定的差异。为了从基因水平分析造成这些差别的原因,对这几株菌的脱硫基因展开了研究。根据Rhodococcus erythropolisIGTS8脱硫基因的保守区设计引物,PCR扩增了R-6、R-8的脱硫基因。测序结果表明脱硫基因高度保守,与IGTS8的相关脱硫基因相似性在99%以上。为了进一步验证不同专一性脱硫菌的脱硫基因的保守性,PCR扩增、克隆了LSSEJ-1和R-9的整个脱硫操纵子,结果表明脱硫基因在这两株菌中也是高度保守的。与IGTS8的相关脱硫基因相比较:R-9的dszA与IGTS8的dszA同源性为99.6%,LSSEJ-1的dszA与IGTS8的dszA的同源性为99.9%;R-9和LSSEJ-1的dszB的同源性与IGTS8的dszB都是99.9%;R-9的dszC与IGTS8的dszC同源性是99.9%,LSSEJ-1的dszC与IGTS8的dszC同源性为99.1%。对比研究认为专一性脱硫嗜温菌的脱硫基因的起源可能相同。     

一株高耐镉硫酸盐还原菌的分离及脱硫性能研究

本研究从镉污染稻田水稻根际土壤中分离、纯化出一株硫酸盐还原菌SRB1-1,并对该菌株的生理生态特征、镉和盐耐受性、16S rDNA、脱硫性能及影响因子进行了系列分析。结果表明,该菌为革兰氏阴性菌,菌体弧状,对镉离子的耐受浓度可达200 mg/L,在2%的氯化钠浓度下仍可生长。对其16S rDNA的序列分析表明该菌株属于脱硫弧菌属(Desulfovibrio)。单因子实验考察温度、pH及SO_4~(2-)浓度对该菌脱硫效率的影响,正交实验确定了该菌最佳脱硫工艺条件及影响因子顺序。结果表明最佳脱硫工艺条件为pH 7.5、温度40℃、SO_4~(2-)浓度为1 000 mg/L、培养时间56 h。     

一株异养脱硫反硝化菌株的筛选及其生物脱硫脱氮特性研究

【目的】从生物脱硫脱氮EGSB-DSR反应器的污泥中分离筛选出具有生物脱硫脱氮特性的细菌,并对其生物脱硫脱氮的性能进行研究。【方法】采用Hungate厌氧滚管技术筛选功能微生物,从稳定运行的生物脱硫脱氮EGSB-DSR反应器的污泥中分离筛选出一株高效的生物脱硫脱氮细菌A2。【结果】经过16S rRNA基因序列鉴定,菌株A2为固氮弧菌属(Azoarcus sp.)。其典型特征为能够以有机碳作为电子供体,将亚硝酸盐或者硝酸盐转化为氮气的同时还能将硫化物氧化为硫单质。因此具备了高效同步代谢有机碳、NO3–和S2–的特征。这是首次关于固氮弧菌属能够进行反硝化脱硫的相关报道。对菌株A2的生物脱硫脱氮能力的分析表明,在硫化物S2–浓度200 mg/L,NO3?浓度87.5 mg/L,乙酸根离子浓度200 mg/L的条件下,菌株A2在20 h内完成对碳、氮、硫的脱除。菌株对于碳、氮去除率均达到99%,对于硫的去除率达到95%。【结论】结果表明固氮弧菌属A2具有高效的生物脱硫脱氮功能,将有望成为强化生物脱硫脱氮工艺的潜在微生物资源。     

高效脱硫诱变菌株降硫性能的研究

目的:筛选具有脱硫功能的细菌,为采用生物法脱硫奠定理论基础.方法:从大庆石化废水曝气池中采集5个活性污泥样本,经过富集培养、分离、纯化获得具有典型特征的菌株,采用碘量法对这些菌株进行降硫能力测定,从中选择降硫效率较高的菌株进行诱变.结果:在30℃、转速160r/min的条件下,Z39ay1菌株的最佳生长pH值为7.0,对数生长期为12～32h,当硫离子为102.24mg/L时,该菌株对硫化物的降解率达42.60％,将其置于2000Gry的60Co射线下照射,从存活菌细胞中进行筛选获得1株诱变菌株Z39a,当硫离子浓度为60mg/L时,对硫化物的降解率达98.58％.结论:从大庆石化废水中分离纯化出1株代号为Z39ay1菌株,经鉴定为赖氨酸芽孢杆菌,诱变后获得菌株Z39a,其降硫效果比出发菌株有大幅度的提高.     

专一性脱硫菌的鉴定及高DBT降解力菌株的选育

从大庆油田土壤中分离得到1株可降解二苯并噻吩(DBT)的脱硫微生物HDBS-1,对该微生物的种属地位进行了鉴定并通过诱变手段提高了该菌株的脱硫能力。经过形态观察、生理生化特征分析及16S rDNA序列测定发现该微生物为坂崎肠杆菌(Enterobacter sakazakii),该菌种可以按特异性脱硫途径(简称4S途径)将DBT转化为2-羟基联苯(2-HBP)。利用紫外线(UV)、硫酸二乙酯(DES)和UV+DES对该菌株复合诱变后,得到菌株HDBS-4,其降解DBT生成2-HBP的能力得到了极大的提高,发酵液中2-HBP生成含量(2.574 mg/L)较原始菌株(0.434 mg/L)提高了5.93倍。     

生物脱硫菌根癌土壤杆菌UP-3的固定化研究

生物脱硫催化剂固定化研究对生物脱硫技术的推广应用具有重要的意义。该文以筛选出的具有脱硫能力的根癌土壤杆菌UP-3为固定化研究对象,二苯并噻吩(DBT)为生物催化脱硫的模型化合物,主要考察了菌株UP-3的培养条件、固定化方法和载体、固定化操作条件和固定化细胞的使用条件。结果表明：以桑特斯培养基在30℃下培养28h的根癌土壤杆菌UP-3具有最佳活性。采用3wt％海藻酸钠水溶液为包埋载体,液菌比为20：1,在4℃下1wt％CaCl2水溶液中固定化24h,得到的固定化细胞脱硫性能最好。在30℃下,反应6d可将浓度为625mg／L的DBT降解60％以上。     

一株CX-DBT脱硫菌的筛选及发酵条件优化

【目的】从大型工业油田石油污染土样中分离鉴定一株能专一性脱除CX-DBT的脱硫菌株,分析其对CX-DBT的脱硫途径,并确定菌体最优发酵条件。【方法】以二苯并噻吩(DBT)为唯一硫源底物,多次富集并分离可代谢CX-DBT菌株,通过形态学、生理生化实验及16S rRNA基因序列分析对筛选菌株JDZX13进行鉴定。采用GC-MS鉴定菌株对CX-DBT的代谢产物,确定其相应的脱硫途径。通过单因素发酵实验确定最佳碳源、氮源、微量元素、MgCl_2、温度及p H的水平范围,并采用正交实验进一步优化。【结果】该菌株鉴定为戈登氏菌属,命名为戈登氏菌JDZX13(KP993297),其CX-DBT代谢途径为"4S途径"。最佳发酵条件为:蔗糖15.0 g/L、NH_4Cl_2.0 g/L、MgCl_2 0.1 g/L、微量元素1.0 m L/L、pH 7.0、温度35°C。【结论】获得一株通过"4S途径"代谢CX-DBT的脱硫菌株JDZX13,经过进一步优化实验,强化了菌株的生长和脱硫能力,该研究结果对石油生物脱硫技术的开发具有重要参考意义。     

华癸中生根瘤菌共生质粒的定向标记、消除与结瘤功能的鉴定

采用Tn5-mob-sacB转座子对华癸中生根瘤菌(Mesorhizobium huakuii)菌株7653R的共生质粒进行定向标记,获得该质粒标记菌株7653RT14.利用sacB基因对蔗糖的敏感性,对标记质粒进行消除实验,获得7653R的共生质粒消除突变株7653R-1.测得Tn5-mob-sacB转座频率高于10-5.突变株的培养特征与出发菌株基本一致.采用琼脂管法对7653RT14和7653R-1进行回接实验,结果显示7653RT14能正常结瘤固氮,表明Tn5的插入并未影响其共生能力,但失去共生质粒的7653R-1则为不结瘤或只结个别小瘤.稳定性实验结果表明供试菌株的标记质粒在本实验条件下是稳定的,可以作为共生质粒转移的供体菌.     

Microbial desulfurization of alkylated dibenzothiophene and alkylated benzothiophene by recombinant Rhodococcus sp. strain T09

The dibenzothiophene (DBT) desulfurizing operon, dsz, was introduced into various benzothiophene (BT)-desulfurizing bacteria using a Rhodococcus-E. coli shuttle vector. Of the tested recombinant bacteria, only those from Rhodococcus sp. strain T09 grew with both DBT and BT as the sole sulfur source. These recombinant cells desulfurized not only alkylated BTs, but also various alkylated DBTs, producing alkylated hydroxybiphenyls as the desulfurized products. Recombinant strain T09 also desulfurized alkylated DBT in an oil-water, two-phase resting-cell reaction. The dsz operon had the same desulfurizing activity when inserted into the vector in either orientation, indicating that the promoter region of the operon was functional in strain T09.     

Isolation and characterization of a moderate thermophile,Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization

An organism, identified as Mycobacterium phlei GTIS10, was isolated based on its ability to use dibenzothiophene (DBT) as a sole source of sulfur for growth at 30-52 degrees C. Similar to other biodesulfurization-competent organisms, M. phlei GTIS10 converts DBT to 2-hydroxybiphenyl (2-HBP), as detected by HPLC. The specific desulfurization activity of the 50 degrees C M. phlei GTIS10 culture was determined to be 1.1+/-0.07 micromol 2-HBP min(-1) (g dry cell)(-1). M. phlei GTIS10 can also utilize benzothiophene and thiophene as sulfur sources for growth. The dszABC operon of M. phlei GTIS10 was cloned and sequenced and was found to be identical to that of Rhodococcus erythropolis IGTS8. The presence of the R. erythropolis IGTS8 120-kb plasmid pSOX, which encodes the dszABC operon, has been demonstrated in M. phlei GTIS10. Even though identical dsz genes are contained in both cultures, the temperature at which resting cells of R. erythropolisIGTS8 reach the highest rate of DBT metabolism is near 30 degrees C whereas the temperature that shows the highest activity in resting cell cultures of M. phlei GTIS10 is near 50 degrees C, and activity is detectable at temperatures as high as 57 degrees C. In M. phlei GTIS10, the rate-limiting step in vivo appears to be the conversion of DBT to dibenzothiophene sulfone catalyzed by the product of the dszC gene, DBT monooxygenase. The thermostability of individual desulfurization enzymes was determined and 2-hydroxybiphenyl-2-sulfinate sulfinolyase, encoded by dszB, was found to be the most thermolabile. These results demonstrate that the thermostability of individual enzymes determined in vitro is not necessarily a good predictor of the functional temperature range of enzymes in vivo.     

Sequence and molecular characterization of a DNA region encoding the dibenzothiophene desulfurization operon of Rhodococcus sp. strain IGTS8.

Dibenzothiophene (DBT), a model compound for sulfur-containing organic molecules found in fossil fuels, can be desulfurized to 2-hydroxybiphenyl (2-HBP) by Rhodococcus sp. strain IGTS8. Complementation of a desulfurization (dsz) mutant provided the genes from Rhodococcus sp. strain IGTS8 responsible for desulfurization. A 6.7-kb TaqI fragment cloned in Escherichia coli-Rhodococcus shuttle vector pRR-6 was found to both complement this mutation and confer desulfurization to Rhodococcus fascians, which normally is not able to desulfurize DBT. Expression of this fragment in E. coli also conferred the ability to desulfurize DBT. A molecular analysis of the cloned fragment revealed a single operon containing three open reading frames involved in the conversion of DBT to 2-HBP. The three genes were designated dszA, dszB, and dszC. Neither the nucleotide sequences nor the deduced amino acid sequences of the enzymes exhibited significant similarity to sequences obtained from the GenBank, EMBL, and Swiss-Prot databases, indicating that these enzymes are novel enzymes. Subclone analyses revealed that the gene product of dszC converts DBT directly to DBT-sulfone and that the gene products of dszA and dszB act in concert to convert DBT-sulfone to 2-HBP.     

Improvement of dibenzothiophene desulfurization activity by removing the gene overlap in the dsz operon

Dibenzothiophene (DBT) and its derivatives can be microbially desulfurized by Dsz enzymes. We investigated the expressional characteristics of the dsz operon. The result revealed that the ratio of mRNA quantity of dszA, dszB, and dszC was 11:3.3:1; however, western blot analysis indicated that the expression level of dszB is far lower than that of dszC. Gene analysis revealed that the termination codon of dszA and the initiation codon of dszB overlapped, whereas there was a 13-bp gap between dszB and dszC. In order to get a better, steady expression of DszB, we removed this structure by overlap polymerase chain reaction (PCR) and expressed the redesigned dsz operon in Rhodococcus erythropolis. The desulfurization activity of resting cells prepared from R. erythropolis DR-2, which held the redesigned dsz operon, was about five-fold higher than that of R. erythropolis DR-1, which held the original dsz operon.     

Desulfurization of light gas oil in immobilized-cell systems of Gordona sp. CYKS1 and Nocardia sp. CYKS2

Desulfurizations of a model oil (hexadecane containing dibenzothiophene (DBT)) and a diesel oil by immobilized DBT-desulfurizing bacterial strains, Gordona sp. CYKS1 and Nocardia sp. CYKS2, were carried out. Celite bead was used as a biosupport for cell immobilization. Seven-eight cycles of repeated-batch desulfurization were conducted for each strain. Each batch reaction was carried out for 24 h. In the case of model oil treatment with strain CYKS1, about 4.0 mM of DBT in hexadecane (0.13 g sulfur l(oil)(-1)) was desulfurized during the first batch, while 0.25 g sulfur l(oil)(-1) during the final eighth batch. The mean desulfurization rate increased from 0.24 for the first batch to 0.48 mg sulfur l(dispersion)(-1) h(-1) for the final batch. The sulfur content in the light gas oil was decreased from 3 to 2.1 g l(oil)(-1) by strain CYKS1 in the first batch. The mean desulfurization rate was 1.81 mg sulfur l(dispersion)(-1) h(-1), which decreased slightly when the batch reaction was repeated. No significant changes in desulfurization rate were observed with strain CYKS2 when the batch reaction was repeated. When the immobilized cells were stored at 4 degrees C in 0.1 M phosphate buffer (pH 7.0) for 10 days, the residual desulfurization activity was about 50 approximately 70% of the initial value.     

Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8

The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to remove sulfur from certain aromatic compounds without breaking carbon-carbon bonds. In particular, sulfur is removed from dibenzothiophene (DBT) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurization phenotype was imparted to Escherichia coli. Therefore, IGTS8 was mutagenized, and a new strain (UV1) was selected that had lost the ability to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype were isolated, though free plasmid could not be isolated. Instead, vector DNA had integrated into either the chromosome or a large resident plasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This procedure identified individual cosmid clones that, when electroporated into strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which these clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicated that the desulfurization genes reside on a 4.0-kb DNA fragment. Finally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV1, and R. fascians produced 2-hydroxybiphenyl from DBT when they contained appropriate clones, indicating that the genes for the entire pathway have been isolated.     

Microbial desulfurization of alkylated dibenzothiophenes from a hydrodesulfurized middle distillate by Rhodococcus erythropolis I-19.

Rhodococcus erythropolis I-19, containing multiple copies of key dsz genes, was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) found in a hydrodesulfurized middle-distillate petroleum (MD 1850). Initial desulfurization rates of dibenzothiophene (DBT) and MD 1850 by I-19 were 5.0 and 2.5 micromol g dry cell weight(-1) min(-1), more than 25-fold higher than that for wild-type bacteria. According to sulfur K-edge X-ray absorption near-edge structure (XANES) analysis, thiophenic compounds accounted for >95% of the total sulfur found in MD 1850, predominantly Cx-DBTs and alkylated benzothiophenes. Extensive biodesulfurization resulted in a 67% reduction of total sulfur from 1,850 to 615 ppm S. XANES analysis of the 615-ppm material gave a sulfur distribution of 75% thiophenes, 11% sulfides, 2% sulfoxides, and 12% sulfones. I-19 preferentially desulfurized DBT and C1-DBTs, followed by the more highly alkylated Cx-DBTs. Shifting zero- to first-order (first-order) desulfurization rate kinetics were observed when MD 1850 was diluted with hexadecane. Apparent saturation rate constant (K(0)) and half-saturation rate constant (K(1)) values were calculated to be 2.8 micromol g dry cell weight(-1) min(-1) and 130 ppm, respectively. However, partial biocatalytic reduction of MD 1850 sulfur concentration followed by determination of initial rates with fresh biocatalyst led to a sigmoidal kinetic behavior. A competitive-substrate model suggested that the apparent K(1) values for each group of Cx-DBTs increased with increasing alkylation. Overall desulfurization rate kinetics with I-19 were affected by the concentration and distribution of Cx-DBTs according to the number and/or lengths of alkyl groups attached to the basic ring structure.