Coupled magnetic nanoparticle-mediated isolation and single-cell image recognition to detect Bacillus' cell size in soil

Microbial morphology fundamentally constrains how species interact with their environment, and hence ultimately affects their niche. However, the methodology of functional microbes in the soil ecosystem is still poorly studied since it is difficult to capture and identify the active monospecific community from the complicated environment and enormous number of microbial species in soils. To comprehensively reveal the morphology of active microbes in soil ecosystem, magnetic nanoparticle-mediated isolation (MMI) and single-cell image recognition (SCIR) were employed to study soil active Bacillus community, which functionally boosted the soil fertility in organic fertilisation compared to mineral fertilisation and unfertilised control treatments in our previous study. The results showed that MMI and SCIR can efficiently isolate active Bacillus from soil particles and other microorganisms. High throughput sequencing showed that the captured Bacillus showed similar community structure in different long-term fertilisation soils, while SCIR revealed that the active Bacillus was greater in number and larger in size in organic fertilisation treatment compared to mineral fertilisation and unfertilised control treatments. Our study demonstrates that the combination of MMI and SCIR is a potentially powerful tool to capture and identify the morphology of active and functional microbes in the soil ecosystem.     

Source of soil protease based on the splitting sites of a polypeptide

Abstract

Soil protease is an important enzyme in the nitrogen cycle which plays an essential role in the growth of various crops. We have attempted to identify a microbial source of soil protease. Selective soil incubation using antibiotics was suitable for a rough estimation of the groups of microorganisms responsible for the production of soil protease (Hayano and Watanabe 1990; Hayano 1993). Enumeration of proteolytic microorganisms in the field and analysis of their protease-producing ability enabled to evaluate the potential of various soil microorganisms for soil protease production (Watanabe and Hayano 1993a; Watanabe et al. 1994). For the accurate estimation of the soil protease source, the characteristics of the soil protease and microbial protease must be compared directly based on enzymatic properties as indices.     

添加巨大芽孢杆菌与胶质芽孢杆菌对土壤DTPA提取态Cd的影响

采用短期和长期恒温培养的方法,研究巨大芽孢杆菌、胶质芽孢杆菌及两菌混合液对土壤DTPA提取态Cd的影响.结果表明,短期试验中巨大芽孢杆菌对土壤Cd有一定的活化作用,不同Cd污染水平条件下,巨大芽孢杆菌使土壤DTPA提取态Cd增加比例在3.8％～24.4％之间,其中50 mg/kg CdCO3污染水平条件下,巨大芽孢杆菌加菌量为2 ml时活化效果最好,而胶质芽孢杆菌和混合菌则对土壤Cd无显著影响或有一定的钝化作用.长期培养试验中,巨大芽孢杆菌也表现出了对土壤Cd的活化作用,未添加Cd条件下,巨大芽孢杆菌加菌量为10,20 ml时,土壤DTPA提取态Cd较对照分别增加了44.0％,28.9％,而胶质芽孢杆菌和混合菌对土壤Cd无显著影响或有一定的钝化作用.     

普施特降解菌Bacillus sp.zx2和zx7生长及降解特性

芽孢杆菌zx2和zx7是普施特高效降解菌,研究其生长和降解特性旨在为普施特污染土壤的生物修复提供科学依据。采用瓶培养法,对芽孢杆菌zx2和zx7的生长特性及单菌和复合菌对普施特的降解特性进行了研究。结果表明,zx2和zx7均可在普施特初始浓度≤200mg·L-1的无机盐培养液中生长良好,zx2在温度25～35℃和pH4.0～7.0时生长良好,而zx7适宜在温度30～35℃和pH5.0～8.0时生长,可见在适应性上二者互补。在最佳条件（温度32℃、pH6.0和普施特初始浓度为200mg·L-1）下,zx2和zx7在无机盐培养液中对普施特降解动态均符合阻滞动力学,半衰期分别为3.8d和2.8d,培养6d时普施特降解率分别为85.81%和90.27%。在培养过程中,zx2的pH是降低的,而zx7的pH基本不变,可初步表明二者降解机理不同;zx2和zx7复合菌（1∶1）对普施特降解率比单菌低,为82.70%,这可能是因为zx2或zx7降解普施特的过程中利用了对方产生的降解产物。     

钾细菌制剂在花生上的应用研究

在种植花生时采用7．5kg／hm2作拌种肥处理，与增施KCl效果相似，可促进花生生长，增加花生有效根瘤、单株结荚数、百果重和出仁率，最终比对照增产l7．93％，每公顷投入产出比达1：40．8，具有明显的经济和生态效益。     

乙酸盐对几种地带性土壤吸附Bt毒素的影响

苏云金芽胞杆菌(Bacillus thuringiensis,简称B t)近缘于蜡状芽胞杆菌,革兰氏染色阳性,在其芽孢形成过程中产生B t杀虫晶体蛋白。B t是目前应用最广的生物杀虫剂之一,其杀虫原理也被广泛应用于转基因抗虫育种实践中。转B t基因作物的外源基因表达产物B t杀虫晶体蛋白可通过根系分泌物或作物残茬等方式进入土壤[1,2],其残留会影响土壤生态系统平衡[3]。纯化的B t毒蛋白可被黏土矿物、腐殖酸和有机矿质复合体等活性颗粒快速吸附,且不易解吸[3~5]。吸附后毒素蛋白仍具有杀虫活性,毒性甚至较游离态的还强[6];红外光谱和X射线衍射表明,吸附后毒素蛋白结构仅有微小变化[4]。毒素     

枯草芽孢杆菌sacB基因的功能验证及应用

本研究将枯草芽孢杆菌(Bacillus subtilis)基因组DNA中克隆的1.4 kbsacB基因片段连接到表达载体pET-28a(+),构建了sacB基因表达载体pSacB。通过测定蔗糖水解产生的葡萄糖具有的还原性,确定sacB基因产物具有蔗糖果聚糖酶活性。该基因表达后,导致大肠杆菌(Escherichia coli)细胞不能在含5%蔗糖的培养基中生存。同时发现sacB基因也能赋予耐辐射菌株Deinococcussp.BR501蔗糖敏感性。利用条件致死sacB基因的这一特性,我们分析了D.sp.BR501的突变率,结果表明DNA错配修复缺失突变株(ΔmutS1)的突变频率明显高于野生型。因此枯草芽孢杆菌sacB基因可作为验证DNA损伤修复能力的一种筛选标记。     

一株新的短芽孢杆菌低温弹性蛋白酶的发酵优化简

[目的]优化短芽孢杆菌XZE116菌株的培养条件使之产生高活性的胞外弹性蛋白酶.[方法]通过单因子和正交试验对XZE116菌株产胞外弹性蛋白酶的培养基及发酵条件进行优化.[结果]该菌株产酶最佳培养基组成为：葡萄糖0.5％、蔗糖0.1％、干酪素0.5％、酵母膏0.5％,K2HPO40.1％,KH2PO40.5％,MgCl20.01％;最佳发酵条件为：250 ml三角烧瓶中装液量40 ml、接种量5％、摇瓶转速200 r/min、pH 8.4、发酵温度为27℃、发酵时间为72 h.XZE116菌株在最佳培养条件下,该菌株的酶活力提高了1.6倍.[结论]研究可为这株新的短芽孢杆菌进行发酵工业生产提供依据,同时为进一步利用该菌株产生的弹性蛋白酶奠定基础.     

柑橘砂皮病菌生防菌的筛选及其发酵条件优化

以柑橘砂皮病菌为靶标菌株,采用平板对峙法从土壤中分离到的227株细菌中获得4株对柑橘砂皮病菌有明显抑制活性的菌株,其中Y2菌株发酵液稀释5倍和10倍后,对柑橘砂皮病菌菌丝生长的抑制率分别为69.85%、61.76%。生理生化测定与16S r RNA基因序列分析结合,确定Y2菌株为短小芽孢杆菌(Bacillus pumilus)。Y2菌株培养的最佳氮源和最佳碳源分别为蛋白胨和可溶性淀粉,最佳发酵温度28℃,转速180 r/min。Y2菌株产生的抑菌活性物质经80℃高温处理和强酸、强碱处理,活性明显下降。     

Differentiation Between Bacillus thuringiensis and Bacillus cereus by 16S rDNA-PCR and ERIC-PCR

16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus＂ strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn＇t have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.