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。     

固定化苏云金杆菌以色列变种控制水中红虫的研究

采用聚乙烯醇(PVA)加少量海藻酸钠及活性炭的方法固定一株对红虫具有高效毒力的苏云金杆菌以色列变种(B.t.i).结果表明,控制质量分数分别为10%PVA、1%海藻酸钠和20%活性炭,采用4%CaCl₂的饱和硼酸溶液(pH为6.7)作为交联剂且交联时间为24h时,制得的凝胶颗粒机械强度好,操作简单,制作成本低.B.t.i固定化微球缓慢释放,且对红虫具有很高的毒力作用.     

胶质芽孢杆菌对印度芥菜富集土壤Cd及土壤pH的影响

采用根袋法盆栽试验,研究接种活菌量浓度分别为1×1010 cfu·kg-1 (T1)和2×1010 cfu·kg-1(T2)的胶质芽孢杆菌,对印度芥菜富集土壤Cd、土壤有效态Cd含量及土壤pH的影响.结果表明,以不接种菌液作为对照,处理T1、T2地上部分生物富集系数分别为13.58、16.83,与对照相比分别提高42.95％、77.16％;地下部分生物富集系数分别为4.35、4.83,分别比对照提高24.64％、38.40％;土壤的净化率分别是对照的1.73、2.20倍.对照组土壤有效态Cd含量随着时间的延长而降低,根际和非根际土壤分别降低32.93％和45.54％;但T1和T2处理中,根际、非根际土壤有效态Cd含量分别提高15.15％、31.73％和36.54％、45.28％.收获时,处理T1、T2根际土壤pH降低率分别是对照的1.08、1.10倍.不同浓度胶质芽孢杆菌处理下土壤pH与有效态Cd含量均呈显著负线性关系.综上,胶质芽孢杆菌主要是通过改变土壤pH来影响土壤Cd生物有效性,从而促进超富集植物的修复效果.本研究可为微生物辅助植物修复重金属污染土壤提供一定理论依据和实践指导.     

Stability of green fluorescent protein using luminescence spectroscopy: is GFP applicable to field analysis of contaminants?

Green fluorescent protein (GFP) was first isolated in the early 1970s for experimental use from coelenterates or the Pacific jellyfish. Aequorea victoria (Morin and Hastings, 1971). GFP has since become a favored biomarker in the photophysical analysis of molecular and cell biology because of its strong intrinsic visible fluorescence and the feasibility of fusing it to other proteins without affecting their normal functions (Creemers et al., 2000). Here we report using Bacillus subtilis expressing GFP to evaluate the influence of different environmental pH conditions on GFP fluorescence. Emission acquisitions were configured to excite at 471 nm and detect at an emission from 490 to 650 nm at 1-nm increments. Fluorescence intensity was significantly better at pH 7 (4.2 x 105 cps; P-value < 0.01) than at acid or alkaline conditions. GFP is a good biomarker for environments near netural conditions: however, GFP may be unsuitable where soils or waters are below or above pH 7 because of loss in fluorescence intensity. Alternative fluorescent markers and delivery systems must be examined in different environments to optimize responses from bioreporter molecules.     

一株芽孢杆菌在低氮源浓度培养基中的生长与氨氮去除特性

对菌株YB3进行了16S rRNA基因序列进化分析,并分别以NH_4Cl、NaNO_2、NaNO_3、尿素和蛋白胨为单一氮源,配制了5种低氮源浓度培养基,研究YB3在与养殖水体相近营养水平条件下的生长与氨氮去除特性.结果显示,菌株YB3属于蜡样芽孢杆菌(Bacullis cereus),在5种培养基中均能够生长,菌悬液(吸光度OD600为1.0)接种量为1.0%(v/v)时,OD600由0.010增长到0.100~0.117.在NH_4Cl培养基中,YB3的氨氮去除速率为1.23 mg·L~(-1)·d~(-1),去除率为93.5%.在尿素、蛋白胨等有机氮源培养基中,YB3将首先导致氨氮的积累,累积倍数分别为51.69和3.38,之后开始去除,去除速率为1.56和0.29 mg·L~(-1)·d~(-1),去除率为93.7%和26.8%.结果也表明,提高YB3接种量至8.0%(v/v),可以使蛋白胨培养基氨氮累积倍数下降至2.02,去除速率提高至1.07 mg·L~(-1)·d~(-1),去除率最终达到98.4%.NaNO_2和NaNO_3培养基中均未检测到氨氮,而NH_4Cl、尿素和蛋白胨培养基中也未检测到NO_2~--N和NO_3~--N,表明YB3的硝化、亚硝化和反硝化作用均不强烈,去除氨氮的同时将不会造成NO_2~--N和NO_3~--N等的大量积累.本文为菌株YB3在养殖水体调控与净化中的应用研究提供了实验基础和理论支持.     

蜡样芽孢杆菌中甲酸脱氢酶基因产氢研究

通过基因筛选,成功分离并克隆到蜡样芽胞杆菌XN12（Bacillus cereus XN12）的甲酸脱氢酶基因fdhF（formate dehydrogenase）,该基因全长2937bp,GC含量39.3%,编码978个氨基酸,与已报道的蜡样芽孢杆菌Q1的fdhF基因（GenBank No.CP000227.1）同源性达到100%.将其连接在表达载体pET32a上并融合His标签,构建了重组质粒pET32a-FDHF-His,转入大肠杆菌BL21（Escherichia coli BL21）后获得了高效表达.重组菌株经IPTG诱导后经WesternBlot分析表明,重组蛋白分子量约为108kDa.通过对重组菌株产氢性能试验表明,重组菌对提高产氢率具有一定促进作用,产氢量为每消耗1mol的葡萄糖和甲酸盐分别能产生0.73mol和0.20mol的氢气.     

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     

原生质体融合构建高产碱性蛋白酶工程菌

用原生质体融合的方法，将碱性蛋白酶生产菌2709与含有碱性蛋白酶基因克隆载体pDW2的工程菌枯草杆菌BD105进行细胞融合，得到1株高产碱性蛋白酶的工程菌A16，菌落的原位杂交表明，该菌株携有双亲的遗传物质，A16的表型与生长特征与2709相似，但同样条件下发酵酶活比2709高50%-100%，摇瓶发酵的产酶水平最高可达30000u/mL,A16发酵所产酶的最适pH与耐温性及发酵条件等特征均与2709相同。适用于工业生产。     

溶胞菌的筛选、特性及其污泥减量效果

采用抑菌圈法从活性污泥中筛选溶胞菌,研究溶胞菌用于污泥减量的效果。在5种溶胞菌中RJM-2对金黄色葡萄球菌的溶胞能力最好。根据形态、生理生化及16SrRNA测序,鉴定RJM-2为短小芽孢杆菌属。为了分析RJM-2的活性物质成分,分别对发酵上清液进行121℃高压蒸汽处理及蛋白酶K处理。结果表明,10 min内RJM-2发酵上清液与对照组相比能使金黄色葡萄球菌(S.aureus)的OD580(580 nm条件下检测溶液的吸光度)下降0.12,而经过处理的发酵上清液OD580在2 h内无下降,这表明溶胞能力主要来自蛋白酶的作用。RJM-2对受试的5株革兰氏阴性菌及多株革兰氏阳性菌具有溶胞能力。RJM-2接种到污泥中24 h后能够使污泥SCOD(溶解性化学需氧量)达到111.96 mg/L,与不接种RJM-2的对照组相比SCOD增加了90.50 mg/L;在36 h内TSS(悬浮物固体)去除率达到56.68%,使污泥比阻下降了47.73%,污泥滤饼的含水率从83.6%下降到78.0%。研究表明,RJM-2能有效减少污泥TSS含量,降低污泥的比阻,改善污泥脱水性能。     

Identification and comprehensive evaluation of a novel biocontrol agent Bacillus atrophaeus JZB120050

Abstract

Bacillus spp. have long been used as biocontrol agents because of their efficient broad-spectrum antimicrobial activity. We identified a novel strain of Bacillus atrophaeus, named JZB120050, from soil. B. atrophaeus JZB120050 had a strong inhibitory effect against Botrytis cinerea and many other phytopathogens. Gas chromatography-mass spectrometry showed that B. atrophaeus JZB120050 produced many secondary metabolites, such as alkanes, alkenes and acids; some of which were related to pathogen inhibition. Enzyme activity analysis showed that B. atrophaeus JZB120050 secreted cell-wall-degrading enzymes, including chitinase, glucanase and protease, which degraded fungal cell walls. Both the novel glucanase gene bglu and chitinase gene chit1 were cloned and heterologously expressed in Escherichia coli and the products showed strong enzyme activity. In addition, B. atrophaeus JZB120050 secreted siderophores and formed a significant biofilm. Future studies should focus on these antimicrobial factors to facilitate widespread application in the field of agricultural biocontrol.