蓝藻发酵生产微生物农药的影响因素研究

采用摇瓶发酵试验探讨了蓝藻为原料制备苏云金杆菌生物杀虫剂的可行性,并考察了不同培养条件（蓝藻含固率、种龄、接种量、初始pH、摇床转速、发酵温度）对苏云金杆菌生长增殖、产孢与产毒效果的影响。研究结果表明,无需任何预处理工序,Btk 130菌株能在蓝藻为唯一原料的培养基中正常生长发育,并且产孢产毒。发酵48 h后,芽孢产率达到86.7%,远高于常规培养基;生物毒效为282 IU.mL-1,与常规培养基相当。培养条件优化结果表明,在蓝藻含固率为2%、初始pH为7.0、接种物种龄为9 h、接种量为2%、培养温度为30℃、摇瓶转速为200 r.min-1的条件下培养48 h,Btk 130可达到较好的发酵效果,活菌数及抗热芽孢数可达7.32 CFU.mL-1和6.38 CFU.mL-1,生物毒效为528 IU.mL-1。该研究不仅为蓝藻提供了高附加值的处置新途径,而且可显著降低生物杀虫剂的生产成本,具有广阔的应用前景。     

一株产酸性α-淀粉酶菌株的筛选、纯化及酶学性质

从南宁酒厂附近土壤中筛选到一株产淀粉酶的野生菌株GXBA-4,经革兰氏染色、芽孢染色以及16SrDNA鉴定,初步确定为解淀粉芽孢杆菌(Bacillus amyloliquefacien).将该菌株发酵液经过硫酸铵沉淀和凝胶过滤纯化出一种α-淀粉酶,该酶相对分子质量约为57×103;反应最适温度为40~45℃,适应温度范围广,30℃时仍具有80%以上的相对活力;最适pH为5.0,为低温酸性淀粉酶.该酶水解可溶性淀粉的产物,经HPLC检测主要是以葡萄糖,麦芽糖以及麦芽三糖为主的低聚糖,分别以α-环糊精、β-环糊精、可溶性淀粉、玉米生淀粉为底物,还原糖产物比为0:0:99:3.4,表明该酶为典型的α-淀粉酶.     

响应面法优化锰氧化细菌发酵培养基

Bacillus sp.WH4是一株锰氧化能力强的锰氧化细菌,在锰污染的水源净化方面具有较好的应用前景.为提高发酵液中Bacillus sp.WH4的活菌数,应用响应面法优化其发酵培养基.Plackett-Burman设计获得对活菌数有显著影响的4个因素:豆粕粉、KH2PO4、MgSO4、FeSO4;最陡爬坡试验确定中心组合实验的最大响应区域;中心组合设计和响应面分析优化出4个显著因素的最佳浓度.优化后的最适培养基(w/%)组成为:蔗糖2、豆粕粉0.96、K2HPO4 0.8、KH2PO4 0.19、MgSO4.7H2O 0.11、CaCl2 0.02、NaCl 0.5、FeSO4.7H2O 0.02.发酵试验表明,最优培养基的活菌数可达4.2×109 CFU mL-1,比初始发酵培养基提高近3倍.     

抗真菌多肽捷安肽素发酵条件的研究

对筛选出的一株芽孢杆菌ZK发酵产物抗真菌多肽捷安肽素的发酵培养基组分(碳源、氮源及无机盐)和工艺条件(发酵温度、起始pH,摇床转速,装液量)进行了摸索,通过单因素实验和正交优化实验,确定了ZK菌株发酵培养基最佳组成:生物氮素3.5％、葡萄糖3％、酵母膏0.08％、MgSO4·7H2O 0.5％、KH2PO4·3H2O 0.1％;最适温度为32℃;最适初始pH为8.0.在250 mL三角瓶中装50 mL培养基,于150 r min-1的旋转摇床上32℃振荡培养72 h,ZK菌株产捷安肽素的量达到最大.图8表5参14     

苏云金芽孢杆菌BtZ01对小鼠胚胎和胚后发育的影响

为了探讨苏云金芽孢杆菌(Bacillus thuringiensis,Bt)Bt Z01对小鼠胚胎发育和胚后生长发育的影响,将48只孕鼠随机分组,试验组饲喂浓度分别为106CFU·m L-1、108CFU·m L-1、5×109CFU·m L-1的Bt Z01菌液,并设灭菌水为对照组。采用行为致畸学方法检查Bt Z01对小鼠胚胎和胚后发育的影响并制作脏器切片,测量脏器指数等指标。结果表明,与对照组相比,各试验组小鼠在胚胎和胚后发育各阶段体重和体长无显著差异(P0.05),胚胎畸形发生率无增加,骨骼染色观察发现高剂量组小鼠后囟门显著大于其他3组(P0.05)。18 d与48 d小鼠部分脏器指数存在显著差异,但病理切片观察发现仅高剂量组肾脏有病变发生。试验结果表明,苏云金芽孢杆菌Bt Z01在浓度为108CFU·m L-1及以下时对小鼠胚胎发育及胚后生长发育无不良影响。     

产耐温蛋白酶苏云金芽孢杆菌FS140液体发酵条件优化

应用快速有效的数学统计方法对苏云金芽孢菌FS140耐温蛋白酶的发酵条件进行优化.首先采用二水平Plackett-Burman设计对影响产酶的8因素进行筛选,获得培养基成分中3个重要影响的因子:黄豆饼粉、酵母粉、葡萄糖;再利用响应面分析法对该3个因素进行3水平的优化,获得它们的最佳组合:黄豆饼粉1.8%、酵母粉0.36%、葡萄糖0.14%.优化后产酶水平达到837.71U mL-1,与响应面数学模型的预测值只有1.34%的误差.同时进行了发酵温度、初始pH、摇瓶装量与接种量等发酵条件的优化,FS140最终发酵产酶水平达918.91U mL-1.图2表5参14     

微生物絮凝剂生产菌T1的鉴定及其对生活污水絮凝特性

从活性污泥中分离筛选絮凝剂产生菌,得到一株对高岭土悬浊液具有较高絮凝活性的微生物絮凝菌T1.根据菌株的生理生化鉴定以及16S r DNA基因序列分析,确定该菌株为芽孢杆菌(Bacillus sp),该菌的絮凝活性物质分布在发酵液上清液中.通过正交实验确定最佳生活污水絮凝条件为絮凝剂投加量5%,污水p H值7.0,助凝剂Ca Cl2投加量为2%,在此条件下,T1发酵上清液对生活污水的絮凝率为83.85%.     

生防枯草芽孢杆菌SQR9固体发酵生产生物有机肥的工艺优化

枯草芽孢杆菌SQR9是一株广谱性的拮抗菌,为促进农业废弃物的资源化利用以及植物土传病害的生物防治,分别以氨基酸有机肥、牛粪堆肥、猪粪堆肥、中药渣堆肥为原料,研究利用SQR9进行二次固体发酵生产微生物有机肥的最佳工艺条件及其原料最佳配比.结果表明,SQR9固体发酵的最佳参数为:发酵最佳原料为中药渣堆肥,接种量10%(φ/mL g-1),麸皮添加量15%,初始含水量65%,发酵温度37℃,翻抛次数1次/24 h,在此发酵条件下,SQR9菌株的活菌总量可达到1.20×1011CFU g-1.中药渣堆肥︰牛粪堆肥︰猪粪堆肥︰氨基酸有机肥的最佳配比为为40︰6︰0︰8,在此原料配比和最佳发酵条件下,SQR9活菌总量可达1.97×1010CFU g-1.因此,用农业废弃物采用合适的工艺条件可以生产出高质量的生物有机肥.     

苏云金芽孢杆菌生物杀虫剂发酵生产的影响因素及其工艺选择

化学杀虫剂的长期使用给生态环境造成了严重破坏,也使害虫种群的抗药性日益提高,生物杀虫剂以其＂绿色环保＂的特点引起人们的广泛关注。其中,苏云金芽孢杆菌（Bacillus thuringiensis）制剂是目前世界上产量最大、应用最广的生物杀虫剂。它对鳞翅目、双翅目、鞘翅目、螨类等许多有害昆虫有毒杀作用,而对人类、动物和农作物无害。长期以来,人们一直致力于苏云金芽孢杆菌发酵过程的研究,以期获得高毒效的生物杀虫剂产品。本文首先对苏云金芽孢杆菌发酵生产的各种影响因素进行了综合分析,将影响因素分为培养条件和培养基组分两类,得出最佳培养条件为温度：（30±1）℃,pH：7.0±0.1,搅拌速度：400～600r·min-1,通气量：1∶0.6～1.2（发酵培养基体积与每分钟通入空气的体积之比）,接种时间：对数期初;最佳培养基配比为碳氮比：8～10∶1,无机盐含量：KH2PO4或K2HPO4为0.075%～0.2%;MgSO4·7H2O为0.075%～0.3%;CaCO3为0.075%～0.15%;MnSO4·H2O、FeSO4·7H2O各为0.002%。其次,对当前研究与工业化生产中的各种发酵工艺进行了评述,总结了现有发酵工艺的优缺点。在现有研究基础上,降低培养基原料成本、改进发酵工艺和采用基因学手段构建高效工程菌株将成为未来研究热点。     

短小芽孢杆菌胞外蛋白质组双向电泳分析及碱性胁迫下胞外差异蛋白鉴定

短小芽孢杆菌Bacillus pumillus SCU11是本实验室从富含蛋白质的生活垃圾中分离并诱变得到的具有高碱性蛋白酶活性的菌株,其分泌的碱性蛋白酶具有很好的生皮脱毛效果,在生物制革工业具有良好的应用前景.短小芽孢杆菌胞外蛋白质多达数百种,为了解其组成情况,采用LB培养基培养短小芽孢杆菌SCU11,以三氯乙酸/丙酮法沉淀发酵上清液中的蛋白质,通过双向电泳分析,建立了具有较高分辨率的短小芽孢杆菌胞外蛋白的双向电泳图谱.在此基础上,分析了碱胁迫条件下短小芽孢杆菌胞外蛋白质的双向电泳图谱,利用液相色谱-串联质谱(LC-MS/MS)鉴定了10个差异蛋白点,其中有5种为胞外分泌蛋白,包含2种胞外蛋白酶.本研究结果表明与呼吸作用及运动相关的蛋白可能参与了短小芽孢杆菌碱胁迫的应答反应.     

Toxicity of cadmium nanoparticles to Bacillus subtilis

The study deals with the toxicological impact of cadmium nanoparticles (Cd NPs) on Bacillus subtilis as a model Gram-positive bacterium. Cadmium oxide (CdO) NPs (～22 nm) and cadmium sulfide (CdS) NPs (～3 nm) were used in this study. Both the NPs were found to inhibit the cell viability of B. subtilis when added to the culture at mid-log phase, the viable cell number declined with increasing concentration of Cd NPs. At mid-log phase, 15 mg L^?1 CdO NPs inhibited growth by ～50%, whereas at 30 mg L^?1 growth completely ceased. Under the same conditions, CdS NPs inhibited growth by ～50% at a concentration of 8 mg L^?1, and at 20 mg L^?1 growth was completely retarded. The cells changed their morphological features to a filamentous form with increasing Cd NPs exposure time, leading to associated with clumping. NPs treated cells when stained with 4′, 6-diamino-2-phenylindole, showed filamentous multinucleated bead structure, suggesting irregularities in cell division. Increasing intracellular oxidative stress due to Cd NPs exposure might be one of the reasons for the cell morphological changes and toxicity in B. subtilis.     

Aerobic granulation of pure bacterial strain Bacillus thuringiensis

The objective of this study is to cultivate aerobic granules by pure bacterial strain, Bacillus thuringiensis, in a sequencing batch reactor. Stable granules sized 2.0–2.2 mm were formed in the reactor after a five-week cultivation. These granules exhibited excellent settling attributes, and degraded phenol at rates of 1.49 and 1.19 g phenol/(g VSS?d) at 250 and 1500 mg/L of phenol concentration, respectively. Confocal laser scanning microscopic test results show that Bacillus thuringiensis was distributed over the initial small aggregates, and the outer edge of the granule was away from the core regime in the following stage.     

Bacillus thuringiensis pesticide exposures reported to Texas poison centers

One of the most common types of microbial pesticides used is Bacillus thuringiensis. Although generally considered safe, exposures to B. thuringiensis pesticide do occur where management by healthcare providers is sought. The objective of this investigation was to describe B. thuringiensis pesticide exposures reported to Texas poison centers. Cases were all B. thuringiensis pesticide exposures reported to Texas poison centers during 2000–2010. The distribution of cases was determined for various demographic and clinical factors. Of 115 total cases, 64% occurred during May–July. The rate per 1,000,000 population was 14.3 in central Texas and 4.4 in the rest of the state. Of the patients, 56% were of an age 20 years or older and 55% were male. The exposure routes were 57% by ingestion, 30% by dermal contact, and 11% by inhalation. The patient was managed on site in 93% of the exposures and only one exposure was judged to be potentially serious. B. thuringiensis pesticide exposures reported to Texas poison centers were more frequently reported from the central region of the state. The majority of exposures occurred during April–July, involved adults and males, occurred by ingestion or dermal route, were managed on site and did not result in serious outcomes.     

固定化枯草芽孢杆菌发酵生产捷安肽素

采用吸附固定化细胞技术发酵生产捷安肽素.首先对7种不同的吸附性固体载体进行筛选,发现木屑作为载体效果最好;然后通过正交优化实验和单因素实验确定载体木屑的最佳条件为:粒度0.5～0.6 mm,添加浓度10 g L-1发酵液.为避免吸附法固定的菌体细胞从载体上脱落,尝试在木屑固定化细胞体系中加入粘稠剂以增强吸附性能,运用单因素实验确定了粘稠剂的种类和浓度,即为15 g L-1海藻酸钠.最后应用响应面方法优化固定化细胞发酵培养基及培养条件,结果表明:接种量为10%、黄豆粉水解液总氮浓度为1.7 g L-1发酵液、葡萄糖浓度为33.7 g L-1发酵液时捷安肽素的生物效价最大,达到7 282.08 U,比非固定化细胞培养提高了53.9%.图3表7参17     

Decomposition of silicate minerals by Bacillus mucilaginosus in liquid culture

The extraction of K⁺ and SiO₂ from silicate minerals by Bacillus mucilaginosus in liquid culture was studied in incubation experiments. B. mucilaginosus was found to dissolve soil minerals and mica and simultaneously release K⁺ and SiO₂ from the crystal lattices. In contrast, the bacterium did not dissolve feldspar. B. mucilaginosus also produced organic acids and polysaccharides during growth. The polysaccharides strongly adsorbed the organic acids and attached to the surface of the mineral, resulting in an area of high concentration of organic acids near the mineral. The polysaccharides also adsorbed SiO₂ and this affected the equilibrium between the mineral and fluid phases and led to the reaction toward SiO₂ and K⁺ solubilization. These two processes led to the decomposition of silicate minerals by the bacterium.     

苏云金芽胞杆菌苏云金素缺失突变株的筛选及特性研究

利用高温和SDS对苏云金素高产菌株CT-43-1c进行了质粒消除,筛选得到一株不产苏云金素的无晶体突变株BMB0806;并通过SDS-PAGE、高效液相色谱(HPLC)和脉冲电泳(PFGE)分析了野生型菌株CT-43、出发菌株CT-43-1c和突变株BMB0806杀虫晶体蛋白的组成、苏云金素的产量和质粒图谱之间的差异.结果表明,突变株BMB0806与菌株CT-43和CT-43-1c相比,分别缺失了3个和2个大质粒,从而不再产生苏云金素和由cry1B基因编码的分子量为140×103的杀虫晶体蛋白.进一步分析三者质粒图谱后发现,突变株BMB0806中苏云金素的缺失和cry1B基因所在的大质粒A直接相关.图5参12     

地衣芽孢杆门菌（Bacillus licheniformis）对Cr^6＋的吸附动力学研究

从污染土壤中分离出地衣芽孢杆菌（Bacillus licheniformis），利用其死菌体对Cr^6＋溶液进行吸附动力学研究．在Ci=300mg／L、pH=2．5和日=50℃条件下，吸附120min获得最大吸附量60．5mg／g．应用Langmuir和Freundlich吸附等温线研究，结果表明，Langmuir吸附等温线更为适合．动力学研究显示，地衣芽孢杆菌对Cr^6＋的吸附动力学可以用拟二级速度方程进行描叙．图3表4参14     

地衣芽孢杆菌β-甘露聚糖酶的基因克隆和鉴定

通过PCR技术从Bacillus licheniformis CICIM-B2004染色体DNA中克隆出编码β-甘露聚糖酶成熟肽的基因manL,并对其进行了鉴定.manL由1110bp组成,编码由332个氨基酸残基组成的β-甘露聚糖酶成熟肽.将manL在大肠杆菌JM109中表达,在12h内可表达325u/mLβ-甘露聚糖酶.图4表1参23     

地衣芽孢杆菌(Bacillus licheniformis)对 Cr6+的吸附动力学研究

从污染土壤中分离出地衣芽孢杆菌(Bacilluslicheniformis),利用其死菌体对Cr6 溶液进行吸附动力学研究.在Ci=300mg/L、pH=2.5和θ=50℃条件下,吸附120min获得最大吸附量60.5mg/g.应用Langmuir和Freundlich吸附等温线研究,结果表明,Langmuir吸附等温线更为适合.动力学研究显示,地衣芽孢杆菌对Cr6 的吸附动力学可以用拟二级速度方程进行描叙.图3表4参14     

一株产生物絮凝剂的Bacillus sp.的分离与鉴定

从重庆城南污水处理厂的活性污泥中分离筛选到一株絮凝剂产生菌,革兰氏染色呈阳性,好氧,有芽孢,可运动,短杆;分离菌的基因组DNA(G +C)mol%含量测定为5 0 .6 % ,16SrRNA序列分析表明,分离筛选出的絮凝剂产生菌的16SrRNA基因序列与Bacillus菌有高度同源性,从而确定絮凝剂产生菌RL - 2归于Bacillusbrevis菌属。絮凝实验表明:在有CaCl2 存在时用Bacillussp .RL - 2生物絮凝剂处理土壤悬液时用量少(10 0mL样品只需2mL絮凝剂) ,絮体形成快,矾花大,泥少;体系温度对活性影响很小,且在酸性与碱性条件下,絮凝活性均很高