苏云金芽孢杆菌培养基优化及间歇发酵

对苏云金芽孢杆菌的培养基配方进行室内摇瓶优化筛选,首先用摇瓶培养筛选到Ⅱ号培养基,在此配方的基础上,将培养基组分划分为氮源、碳源及无机盐三因素,采用三因素二水平正交旋转组合设计的方法进行培养基优化组合研究,建立其芽孢产量依氮源、碳源、无机盐的响应面方程。借助此方程获得响应面最佳点即培养基各组分的最佳配比。实验结果表明,该方法是苏云金芽孢杆菌培养基优化中十分简便、实用、快速的途径。此外,对其间歇发酵过程也进行了初步考察。     

苏云金杆菌Bt0601菌株发酵培养基的优化

运用正交试验L18(37)设计对携带杀虫基因cry1Ac和虎纹捕鸟蛛毒素基因hwtx-Ⅰ的苏云金杆菌工程菌Bt0601的发酵培养基进行了优化研究.试验获得的最佳优化复合培养基配方为(%)玉米粉1.0,黄豆饼粉0.50,酵母膏0.15,蛋白胨0.05,磷酸二氢钾0.75,碳酸钙0.05,硫酸镁0.035.Bt0601在该优化培养基下发酵,48 h每mL发酵液产芽孢11.4×109个,伴孢晶体23.0×107个,对3龄小菜蛾(Plutella xylostella)48 h的致死率达96.7%.普通发酵培养基下相应的芽孢产量为9.33×109个,伴孢晶体9.57×107个,毒力为68.5%.     

苏云金杆菌33菌株最佳培养基和发酵条件研究

应用正交试验L27(313)对鳞翅目昆虫高毒效的苏云金杆菌33菌株的发酵培养基进行研究,得到了适合其发酵培养的优化复合培养基配方—0.5％玉米粉 2．00％黄豆粉 0．15％酵母粉 0．25％鱼粉 0．075％蛋白胨 0．25％磷酸二氢钾 0．05％碳酸钙 0．035％硫酸镁。另外实验还证实适当增加通气量、发酵初始酸碱度控制在pH7．5、发酵温度30℃、转速180r／min、培养40h更适合该菌株液体深层发酵。     

蜡质芽孢杆菌DLSL-2发酵条件探讨及培养基优化

对蜡质芽孢杆菌(Bacilluscereus)DLSL2深层液体发酵的主要影响因子温度、转速、初始pH值等进行了单因素实验探讨,确定了最佳培养条件:温度为30℃、转速为250r/min、初始pH值为7.0。并用均匀设计法对其发酵培养基进行了优化,优化验证实验结果为7.1×109cfu/mL明显高于原发酵培养基结果3.2×109cfu/mL。     

苏云金杆菌SD—5菌株发酵生理学研究及生产

通过苏云金杆菌SD－5菌株对混合氨基酸的抗性及在3吨发酵罐中发酵生理学的研究,显示SD－5菌株具有优良的发酵性能。用正交试验设计方法筛选出SD－5菌株发酵培养基配方,菌数可达110－130亿／ml,芽孢率达95％以上。     

苏云金杆菌“79007”菌株发酵工艺的研究

对苏云金杆菌79007菌进行了生长条件的研究,获得了该菌最佳发酵培养基配方和最优发酵工艺。最佳培养基配方(g/100 mL):豆饼粉2.5,淀粉2.0,酵母粉2.0,玉米浆2.0,添加剂Ⅰ号0.1,葡萄糖1.0,KH2PO40.3,CaCO30.1,CaCl20.05,MgCl20.01,CuCl20.005,ZnSO40.002,CoCO30.005;15 m3发酵罐最优工艺:通气量1∶0.5~1.1,培养温度28~33℃,搅拌转速0~200 r/min。40 m3发酵罐连续4批试验表明,平均发酵效价6 053.5 IU/μL、平均晶体蛋白含量达0.5%,产品质量达到原粉优质品。     

蜡质芽孢杆菌AR156发酵培养基及发酵条件的优化

对前期筛选得到的在田间试验中防治根结线虫效果较好的蜡质芽孢杆菌(Bacillus cereus)AR156,通过单因素筛选及正交试验的方法进行了发酵培养基优化,得到的最佳配比为:麦芽糖0.25%,玉米粉0.5%,黄豆粉0.5%,胰蛋白胨0.5%,CaCl2·2H2O0.05%,MnSO4·H2O0.05%,K2HPO40.1%。同时对实验室摇瓶条件下液体发酵的主要影响因子温度、转速、初始pH值等进行实验探讨,确定了最佳培养条件:初始pH值7.0,装液量200mL/L,接种量5%,发酵温度28℃,转速200r/min,发酵时间48h。优化后芽孢产量为1.03×109CFU/mL,芽孢生成率在97%以上,明显高于初始发酵培养基发酵结果。     

苏云金芽孢杆菌发酵实验参数的分析

采用最小二乘法对苏云金芽孢杆菌发酵实验测试数据进行回归分析,得到了还原糖含量、PH值及溶氧值随时间的变化规律,并对其进行误差分析。为苏云金芽孢杆菌发酵实验过程的参数设计和控制提供了可靠的理论依据。     

短小芽孢杆菌HR10产孢培养基及发酵条件优化

短小芽孢杆菌(Bacillus pumilus)HR10是一株具有促生抗逆作用的优良菌株.探究菌株HR10产孢的最佳发酵培养条件,对于在更大规模上进行生产发酵具有重要的指导意义.以稀释涂布平板法计数活菌数和芽孢数并计算芽孢率;对菌株HR10产孢培养基的碳源、氮源和无机盐进行单因素分析及正交试验,并采用摇瓶发酵法对影响菌...     

高效杀蚊苏云金芽孢杆菌BRC-LLP29的发酵优化

苏云金芽孢杆菌BRC-LLP29为新型高效杀蚊菌株,应用快速有效的数学统计方法对其杀蚊毒力的发酵培养进行优化.通过单因素筛选确定最佳碳源为葡萄糖、麦芽糖、可溶性淀粉,氮源为typetone、大豆蛋白胨、干酪素;最佳金属离子为Mg²⁺、Al³⁺.采用二水平Placlkett-Burman设计对影响毒力的8因素进行显著性筛选,获得培养基成分中3个重要影响因子:葡萄糖、干酪素和Al₂（SO₄）₃;运用爬坡路径法对这3种因子进行试验,获得3种重要因子的最适浓度范围;通过响应面分析法得到3个重要因子的交互作用和最佳条件,确定BRC-LLP29菌株最佳毒力水平的发酵培养基为:葡萄糖19.8g/L、干酪素28.4g/L、Al₂（SO₄）₃1.2g/L、MgSO₄ 2g/L、K₂HPO₄ 3g/L、CaCO₃ 0.5g/L,优化后毒力水平达到致死率61.11%,与响应面数学模型的预测值只有5.91%的误差.发酵条件优化结果表明:发酵温度为31°,发酵初始pH为7.0,摇瓶装量为40mL/250mL三角瓶,每瓶的接种量为3.5%,发酵72h,对致倦库蚊最终致死率达到最高为83.33%.     

苏云金芽胞杆菌Bt 0601发酵条件的优化研究

应用正交实验和单因子实验,结合杀虫毒力测定,对携带杀虫基因cry1Ac和虎纹捕鸟蛛毒素基因Hwtx-I的苏云金芽胞杆菌工程菌Bt 0601的发酵工艺条件进行研究。实验获得的苏云金芽胞杆菌Bt 0601工程菌的最佳发酵条件为:发酵初始酸碱度控制在pH 7.5、发酵温度(30±0.5)℃、转速200 r/min、通气量为30 mL/300 mL,接种量为4%,培养44 h。此外,本文还对Bt 0601菌株在30 L发酵罐中的发酵特性进行了研究,摸索了通气量对其大罐发酵的影响,确定大罐发酵的最佳通气量为1.75 L/min。     

土壤放线菌FX05发酵培养基及发酵条件的优化

以高氏1号培养基为基础,通过单因素分析和正交试验,对土壤放线菌FX05最佳培养基扣最优培养条件进行了研究。结果表明：适宜FX05产抑绿脓杆菌活性物质的发酵培养基配方为玉米粉1％、NaNO30．3％、K2HPO40．075％、MgS040．075％,发酵条件为初始pH为7．0,培养温度为28℃,培养96h产抑菌物质迭最大值。     

1株纳豆菌抑菌活性及其培养基优化

对1株纳豆菌的体外抑制金黄色葡萄球菌作用进行动力学分析,考察了其培养基组成对纳豆菌增长倍数及抑菌效果的影响,通过正交试验确定纳豆菌对金黄色葡萄球菌起抑菌作用的优化培养基组合为乳糖2%,蛋白胨4%,KH2PO40.2%,甘氨酸0.2%。     

苏云金杆菌WG-001工程菌发酵培养基的筛选

探讨了BtWG001工程菌以农副产品为主要碳、氮源的利用情况,为该菌株发酵培养基的筛选提供了广谱的原料资源。利用高速上升浓度加倍法进行了发酵培养基的优选,获得一个优良配方。发酵水平达到4024IU/μL,0.39%,与对照配方(2800IU/μL,0.26%)相比,分别提高44%,50%。证明该方法为一种筛选Bt发酵培养基配方的新模型。     

A New Medium for Bacillus thuringiensis Berliner

S ummary : A new solid medium for culturing Bacillus thuringiensis consists of groundnut cake, 10%; tamarind kernel powder, 1·5% and agar, 0·5%. The quantity of agar in the medium could be decreased from 1·5 to 0·5% by adding tamarind kernel powder. A spore yield (85% sporulation) of 3·2 g/l was obtained. Bacterial spores produced on the new solid medium were pathogenic to the larvae of the almond moth, Cadra cautella.     

Characterization of a Novel Strain of Bacillus thuringiensis

Bacillus thuringiensis is a well-known species of entomopathogenic bacteria that is widely used as a biopesticide against many insect pests. Insecticidal proteins, coded for by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. In this report, an unusual strain of B. thuringiensis subserovar oyamensis (LBIT-113), isolated from living larvae of Anopheles pseudopunctipennis in Mexico, was characterized by its ultrastructure, the protein composition of its parasporal crystal, plasmid pattern, and toxicological properties against several insect and noninsect targets. The parasporal crystal is enclosed within the spore's outermost envelope (exosporium), as determined by transmission electron microscopy, and exhibits a square, flat shape. Its main components are two proteins with sizes of 88 and 54 kDa. Despite some crystal morphology resemblance, both proteins are immunologically unrelated to the Cry IIIA protein, as shown by immunoblot analysis, when probed with antisera raised against the 88-kDa protein and the Cry IIIA protein. Partial N-terminal sequence of the 88-kDa protein revealed a unique amino acid arrangement among the Cry proteins. Solubilization of the crystal proteins was achieved at 3.3 M NaBr, and its digestion with trypsin showed only one ca. 60-kDa peptide, as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The patterns of three plasmids of strain LBIT-113 were considerably different from those of B. thuringiensis subspp. kurstaki, tenebrionis, and israelensis. Parasporal crystals showed no toxicity to larvae of four species of caterpillar, three species of mosquito, two species of beetle, one species of cricket, one species of ant, one species of aphid, one species of nematode, one species of ostracod, one species of ameba, and one species of rotifer.     

Complete Genome Sequence of Bacillus thuringiensis Mutant Strain BMB171

Bacillus thuringiensis has been widely used as a biopesticide for a long time. Here we report the finished and annotated genome sequence of B. thuringiensis mutant strain BMB171, an acrystalliferous mutant strain with a high transformation frequency obtained and stocked in our laboratory.Bacillus thuringiensis is an insect pathogen which is widely used as a biopesticide due to its various endogenous crystal proteins and spores (12). To improve the virulence and practical effectiveness of B. thuringiensis, genetic transformation of different genes with beneficial traits is a fundamental procedure. Simultaneously, genetic transformation can facilitate functional genomic research. However, wild-type strains are not suitable to be used as recipient strains because of low transformation efficiency. This obstacle is mainly caused by the thick cell wall layer of B. thuringiensis together with multiple plasmids inside the cell, which harbor genes encoding insecticidal crystal proteins. We used the method of elevating the growth temperature and adding 0.05% sodium dodecyl sulfate to treat several parental strains and finally obtained mutant strain BMB171, with no resident plasmid, from wild-type crystalliferous strain YBT-1463 (9). The electrotransformation frequency of mutant BMB171 could reach up to 10⁷ transformants/μg DNA after optimization of the electrotransformation parameters (7), which was 4.8 × 10⁴-fold higher than that of the parental strain (8). Moreover, mutant strain BMB171 exhibited the same characteristics as YBT-1463, such as metabolic abilities and growth properties, as well as sensitivity to 10 antibiotics (8). Of course, BMB171 could produce parasporal crystals with characteristic geometric shapes through the expression of relevant cry genes carried by plasmids (7). Thus, B. thuringiensis mutant strain BMB171 has become a major recipient strain and is widely used for insecticidal crystal protein-encoding gene expression (14, 15), cell surface display (10, 13), gene function and regulation researches (2, 5), etc.The B. thuringiensis mutant strain BMB171 genome was sequenced by using a massive parallel pyrosequencing technology (454 GS-FLX). A total of 448,963 high-quality reads with an average read length of 391 bp were produced, providing about 32-fold coverage of the genome. Assembly was performed using the Newbler software of the 454 suite package (454 Life Sciences), which resulted in 193 large (defined as >500 bp) contigs. The relationship of contigs was determined by multiplex PCR, and gaps were filled through sequencing of PCR products by primer walking or shotgun sequencing with an ABI 3730 sequencer. The Phred/Phrap/Consed software package (3) was used for final sequence assembly and quality assessment. Protein-coding genes were predicted by combining the results of Glimmer 3.02 (1) and ZCURVE (4), followed by manual inspection. Both tRNA and rRNA genes were identified by tRNAscan-SE (11) and RNAmmer (6), respectively. Functional annotation was performed by searching against a protein database of the microbial genome developed in house.The 5.64-Mb genome of B. thuringiensis mutant strain BMB171 contains two replicons: a circular chromosome (5.33 Mb) encoding 5,088 open reading frames (ORFs) and a circular plasmid (0.31 Mb), which is named pBMB171, encoding 276 predicted ORFs. The G+C content of the chromosome is 35.3%, while that of the plasmid is 33.3%. The mutant strain BMB171 genome encodes 104 tRNAs and 14 rRNA operons. A previous study indicated that BMB171 is a plasmid-free mutant (9); however, our sequencing results demonstrated that a large plasmid still remains. The reason why the plasmid was not detected previously might be its large size and low copy number. We did not find any crystal protein genes in either chromosome or plasmid sequences, which was consistent with previous observations (9).In summary, the complete B. thuringiensis mutant strain BMB171 genome provides a better-defined genetic background for gene expression and regulation studies, especially crystal protein production and metabolic network construction.     

苏云金芽胞杆菌6618杀虫菌株的分离和鉴定

从我国不同地区采集118份土样,利用温度筛选法分离获得一株苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)6618,镜检观察发现该菌株能产生典型的菱形晶体,PCR分析表明其含有cry1类杀虫基因。采用SDS-PAGE和质谱分析发现该菌株主要产生130 k D原毒素,其组分由Cry1Ae和Cry1Ac原毒素组成。基因序列分析表明该菌株的cry1Ac基因为已知的cry1Ac1,而cry1Ae为新型杀虫基因。毒力生测表明该菌株对棉铃虫(Helicoverpa armigera)幼虫具有显著的杀虫效果。筛选获得的高毒力Bt菌株6618为丰富我国苏云金芽胞杆菌储备和研发新型高效生物杀虫剂提供了菌株资源。     

苏云金芽胞杆菌CTC菌株的S-层蛋白可以形成伴胞晶体

苏云金芽胞杆菌(Bacillus thuringiensis)CTC菌株产生卵圆形伴胞晶体,晶体蛋白分子量为100kD;透射电子显微镜观察结果表明该菌株有S层结构,而且在母细胞内可以形成伴胞晶体和S层的初体结构;其蛋白基因导入苏云金芽胞杆菌无晶体突变株BMB171后,扫描电子显微镜观察结果表明转化子能形成晶体,而其形状与CTC菌株的相同;转化子晶体蛋白的分子量大小也与CTC菌株的相同,为100kD。以上实验结果结合以前晶体蛋白N末端测序和基因核苷酸序列,表明苏云金芽胞杆菌CTC菌株的S层蛋白可以形成伴胞晶体。