一株氯嘧磺隆降解菌分离鉴定及降解条件优化

为解决氯嘧磺隆残留对土壤、水体污染及后茬敏感作物药害问题,为污染土壤微生物修复提供降解菌种资源,文中采用富集培养、逐级驯化等方法,从氯嘧磺隆污染土壤中分离到1株高效氯嘧磺隆降解菌T9DB-01,经形态特征、生理生化及16S rDNA序列分析,鉴定为假单胞菌Pseudomonas sp.。采用单因素实验探究温度、pH值、底物浓度、装液量和接种量对菌株T9DB-01降解氯嘧磺隆的影响,采用正交试验及验证,优化菌株T9DB-01对氯嘧磺隆降解条件。结果表明,在30℃,pH 8.0,底物浓度200 mg/L,装液量100 mL/250 mL,接种量4%的条件下,5 d后降解率达到93.7%。该降解菌株对氯嘧磺隆污染土壤原位生物修复具有一定的应用潜力。     

氯嘧磺隆降解菌的分离鉴定及其降解特性

利用富集培养技术从长期施用氯嘧磺隆的土壤中分离得到1株能够降解氯嘧磺隆的细菌L-7。通过生理生化特性和16SrRNA序列分析,初步鉴定L-7为寡养单胞菌属(Stenotrophomonas sp.)。并分析了氯嘧磺隆的初始浓度、接种量、温度和pH值对L-7菌株降解氯嘧磺隆效果的影响,确定了最佳降解条件。结果表明,该菌在氯嘧磺隆浓度为100mg/L、接种量为5%、pH4.0、温度30°C条件下,接种5d后对氯嘧磺隆的降解效率达到80%以上。     

一株吡嘧磺隆降解菌S8-1的分离鉴定及生物学特性

以多年连续施用吡嘧磺隆除草剂的水稻试验田泥土为材料,采用富集培养、平板反复划线分离方法,分离到一株能以吡嘧磺隆为唯一碳源和能源生长的光合细菌(Photosynthetic Bacterium,简称PSB),命名为S8-1。通过菌落形态特征观察、菌体形态学观察、活细胞吸收光谱特征、培养特性、生理生化特性及16SrRNA同源性序列分析(GenBank登录号:GQ180069)等试验,初步鉴定该菌为红假单胞菌属(Rhodopseudomonas sp.)。利用高效液相色谱(HPLC)测定了菌株S8-1的降解性能,得出该菌降解吡嘧磺隆的最佳条件为:pH值为7.0-7.5,温度为30°C-35°C;在30°C与pH7.0的光合细菌培养基中培养7d,对100mg/L的吡嘧磺隆降解率为52.07%;并且该菌对浓度高达800mg/L的吡嘧磺隆仍保持降解活性,外加发酵提取物能够明显提高菌体的生长及其降解效率,显示了该菌在高浓度农药废水处理及土壤农药残留生物修复方面潜在的开发价值。     

几种除草剂对薇甘菊的防控效果

【背景】薇甘菊是一种外来入侵的恶性杂草,对当地农、林业造成了严重的危害。【方法】研究了草甘膦、氨氯吡啶酸、甲嘧磺隆、噻吩磺隆、苯磺隆、百草枯、五氟磺草胺、单嘧磺隆和单嘧磺脂9种除草剂对薇甘菊的化学防治,调查各药剂对薇甘菊的植株鲜重抑制率、叶片覆盖度和抑花率。【结果】氨氯吡啶酸25～100gai.hm-2、草甘膦1538～2306gai.hm-2、甲嘧磺隆100～400gai.hm-2、百草枯750、1125gai.hm-2、五氟磺草胺125gai.hm-2、噻吩磺隆11.25～33.75gai.hm-2和单嘧磺脂15～60gai.hm-2对薇甘菊营养生长控制效果较好。其中,百草枯短时间有效,草甘膦、五氟磺草胺、单嘧磺脂、单嘧磺隆、氨氯吡啶酸和甲嘧磺隆对薇甘菊的抑花率较高,苯磺隆和单嘧磺隆基本无效,且9种药剂对橡胶树均未表现出药害。【结论与意义】适当降低氨氯吡啶酸、甲嘧磺隆、草甘膦、五氟磺草胺、噻吩磺隆和单嘧磺脂的浓度可以保持薇甘菊营养生长而扼制种子蔓延。本研究首次发现五氟磺草胺、噻吩磺隆和单嘧磺脂对薇甘菊有良好的抑制效果。     

不同土壤细菌种群结构对氯嘧磺隆胁迫的响应及降解菌系的获得

应用PCR-DGGE、DNA测序等方法,在室内驯化条件下研究了8种来源于中国不同地区土壤样品细菌种群结构对氯嘧磺隆胁迫的响应。结果表明:在氯嘧磺隆100～500mg·L-1浓度梯度下,土壤细菌群落组成有明显的更迭现象,多样性发生明显变化,驯化至10周,绝大部分细菌种群消失,样品的细菌种群结构趋于简单并呈现趋同效应;DNA测序结果表明,在驯化第10周可培养Methylophilus sp.、Beta proteobacterium、uncultured bacterium成为优势菌属,所获细菌种群出现的16个优势种群中有10个与已知的具有有机污染物降解功能和有机污染环境修复功能细菌的相似性大于97%;其中5个与嗜甲基菌16S rDNA部分序列相似性达98%以上。获得了一组对氯嘧磺隆具有降解作用的细菌菌系,可在5d内将100mg·L-1氯嘧磺隆降解67%;其主要组成为嗜甲基菌属(Methylophilus sp.)、丛毛单胞菌属(Comamonas sp.)、鞘酯杆菌属(Sphingobacterium sp.)和嗜氢菌属(Hydrogenophi-lus sp.)。     

氯嘧磺隆对土壤微生物群落结构的影响

采用磷脂脂肪酸(PLFA)法研究了黑龙江省苇河地区不同氯嘧磺隆施药历史下土壤微生物群落结构的差异, 并测定了土壤中氯嘧磺隆的残留量.结果表明：不同施药历史下氯嘧磺隆在土壤中的残留量均很低;随着施用氯嘧磺隆年限的增加,土壤微生物的PLFA总量减少, 革兰氏阴性菌/革兰氏阳性菌（GN/GP）和真菌/细菌比降低,土壤微生物的压力指数增加.主成分分析表明,氯嘧磺隆显著改变了大豆田土壤微生物群落结构.     

噻吩磺隆降解菌Bacillus subtilis LXL-7的分离与应用

从土壤中分离到一株能高效降解噻吩磺隆的细菌LXL-7,经鉴定LXL-7为枯草芽孢杆菌(Bacillus subtilis)。在含100 mg/L噻吩磺隆的培养基中,菌株LXL-7降解噻吩磺隆的72 h降解率可达99.6%以上。菌株LXL-7的最适p H为7.5,适宜温度为30-35℃,该菌还对噻吩磺隆和盐有很好的耐受性,至少能耐受400 mg/L的噻吩磺隆和4.0%的Na Cl。另外,还通过在商品化的土壤微生物改良剂中添加菌株LXL-7开发了多功能微生物制剂,新制剂除原有功能外还增加了降解噻吩磺隆的新功能,故认为菌株LXL-7能够被用于噻吩磺隆污染的处理以及相关微生物制剂的开发。     

橡胶树胶孢炭疽菌T-DNA插入突变体库构建及其致病缺陷转化子筛选

【目的】进一步研究橡胶树胶孢炭疽菌致病分子机理。【方法】通过含ILV1基因(具氯嘧磺隆抗性)的pSULF.gfp双元载体农杆菌AGL-1介导进行橡胶树胶孢炭疽菌遗传转化,利用氯嘧磺隆抗性标记筛选转化子,对转化子PCR验证及荧光显微观察;采用离体古铜期橡胶树叶无伤接种法进行致病性缺陷转化子筛选,并对转化子进行遗传稳定性检测。【结果】获得含3 721个转化子的T-DNA插入突变体库,转化效率为150 400个转化子/106孢子,从3 721个转化子中筛选得到致病性缺陷转化子25个;随机选取20个转化子进行遗传稳定性测定,在不含氯嘧磺隆PDA平板上继代培养10次后仍保持氯嘧磺隆抗性,且表型稳定,表明插入外源基因能够稳定遗传。【结论】可以利用根癌农杆菌介导橡胶孢炭疽菌转化,构建橡胶树胶孢炭疽菌T-DNA插入突变体库,筛选致病缺陷突变菌,为进一步研究该菌致病相关基因提供材料。     

氯嘧磺隆与尿素对土壤微生物生物量碳、氮及无机氮的影响

通过室内培养试验,研究了不同浓度氯嘧磺隆(20、200、2000 μg·kg-1土)单一施用及与尿素(120 mg· kg-1土)配合施用情况下,土壤微生物生物量碳、氮和土壤铵态氮、硝态氮随时间的动态变化规律.结果表明:各浓度氯嘧磺隆单独处理在整个培养期(60 d)中对微生物生物量碳、氮均有抑制作用,且浓度越高,后期抑制作用越强;各浓度氯嘧磺隆处理在培养前期对硝态氮、铵态氮没有明显影响,中期(15 d)能显著提高土壤中铵态氮的含量,后期(30 d后)显著提高了土壤中硝态氮的含量.尿素单独施用及与氯嘧磺隆配施均能在短时间内增加微生物生物量碳、氮,但随后配施处理的促进作用减弱;尿素单独和配施均能持久增加土壤中铵态氮、硝态氮含量.     

节杆菌PJ3降解咔唑和联苯

菌株PJ3经16SrDNA鉴定为节杆菌(Arthrobacter sp.),同源树表明,该菌株与已报道咔唑降解菌株IC177同源关系最近,其次是联苯降解菌株K37和R04。为了明确PJ3菌株对咔唑和联苯的降解能力,在PJ3生长的最适pH值进行优化的基础上,利用分光光度法测定了该菌株在不同浓度咔唑和联苯的矿物培养基中的生长速率以及对咔唑和联苯的降解性能。结果表明,在pH为7、8、9时,PJ3菌株的生长速率一致,而且高于其他pH值的生长速率。在pH为7、咔唑浓度为0.1mg.ml-1的CNFMM培养基中,PJ3菌株生长速度较快,而且15d咔唑的降解率可达到73%。在pH为7、联苯浓度为0.5mg.ml-1的MSB培养基中PJ3菌株生长最好。在0.1和0.5mg.ml-1联苯浓度下,15d对联苯的降解率可达80%～85%。综合评价,PJ3菌株降解咔唑比较适宜的浓度范围应该为0.1～0.2mg.ml-1,降解联苯较适宜的浓度范围为0.1～0.5mg.ml-1。     

Thermophilic protease-producing Geobacillus from Buranga hot springs in Western Uganda

Two proteolytic thermophilic aerobic bacterial strains, PA-9 and PA-5, were isolated from Buranga hot springs in western Uganda. The cells were rods, approximately 10–12 μm in length and 3 μm in width. Isolate PA-9 grew at between 38°C and 68°C (optimum, 62°C), and PA-5 grew at between 37°C and 72°C (optimum, 60°C). Both isolates grew optimally at pH 7.5–8.5. Their 16S rRNA gene sequences indicated that they belong to the newly described genus Geobacillus. Zymogram analysis of the crude enzyme extracts revealed the presence of two extracellular proteases for isolate PA-5, and at least eight for isolate PA-9. The optimum temperature and pH for casein-degrading activity were 70°C, pH 6.5 for isolate PA-9, but caseinolytic activity could also be observed at 2°C. In the case of isolate PA-5, optimal activity was observed over a temperature and pH range of 50–70°C and pH 5–10, respectively. Received: 26 November 2001 / Accepted: 12 December 2001     

枯草芽胞杆菌L-天冬氨酸a脱羧酶的酶学性质

b-丙氨酸是一种重要的医药化工原料,目前主要依靠化学法进行生产。探寻更为环保和高效的生物生产法是未来研究的一个方向。L-天冬氨酸a脱羧酶 (PanD) 能特异地脱去L-天冬氨酸的a羧基,生成b-丙氨酸。本文比较了3种分别来源于大肠杆菌、谷氨酸棒状杆菌及枯草芽胞杆菌的PanD比酶活 (分别为0.98、7.52和8.4 U/mg)。后两者的最适pH均为6.5,最适反应温度分别为65 ℃及60 ℃。与目前研究最多的来源于大肠杆菌和谷氨酸棒状杆菌的PanD相比,来源于枯草芽胞杆菌的PanD具有更好的活性和热稳定性,具有更强的工业应用潜力。同时,本文对该酶特有的翻译后自剪切及机理性失活现象进行了分析和讨论。     

Box-Behnken design in the development of optimized complex medium for phenol degradation using Pseudomonas putida (NICM 2174)

The biodegradation of phenol by Pseudomonas putida (NICM 2174), a potential biodegradent of phenol has been investigated for its degrading potential under different operating conditions. Box-Behnken design has been employed to study the effect of different experimental variables. Four variables of maltose (0.25, 0.5, 0.75?g/l), phosphate (3, 12.5, 22?g/l), pH (7, 8, 9) and temperature (30?°C, 32?°C, 34?°C) were used to identify the significant effects and interactions in the batch studies. A second order polynomial regression model, has been developed using the experimental data. It was found that the degrading potential of Pseudomonas putida (NICM 2174) was strongly affected by the variations in maltose, phosphate, pH and temperature. The experimental values were in good agreement with the predicted values, the correlation coefficient was found to be 0.9980. Optimum conditions of the variables for the growth of Pseudomonas putida (NICM2174) and for maximum biodegradation of phenol are maltose (0.052?g/l), phosphate (8.97?g/l), pH (7.9) and temperature (31.5?°C).     

Effects of cooling and freezing on pH of semen extender

The pH change of 10 different buffering systems with temperature ranging from room to 5 °C was examined; three were conventional buffers which included phosphate yolk, citrate yolk, and skim milk. Seven were Good's buffers with egg yolk which included TES, TRIS, BES, MOPS, PIPES, MES, and TEST. The pH of the three conventional buffers did not change with decreasing temperature, but Good's buffers showed an increase in pH with decreasing temperature from room to 5 °C. The pH change due to temperature was measured for TEST buffer solution with and without 20% egg yolk containing 2 or 6% of five different cryoprotective compounds. The pH at 5 °C was significantly higher than at room temperature. The addition of egg yolk and/or cryoprotective compound did not alter the pH significantly during cooling, even though a slight drop in pH was noted with the addition of egg yolk indicating that the change in pH is primarily due to the buffer. The pH of TEST yolk buffer (pH 7.2 at room temperature) was measured continuously from 37 °C to below freezing (?18 °C). The pH increased with decreasing temperature to 8.0 ± 0.2 from 37 to ?14 °C at which point it dropped abruptly to pH 6.5 ± 0.2.     

一株可乳化降解原油的Compostibacillus的分离及特性

【背景】通过实施多轮次微生物采油,华北油藏产出液菌浓达到了106个/mL以上,油藏内部已经形成了较稳定的微生物发酵场,从其中筛选出能够乳化降解原油的微生物,并在地面对其进行扩大培养,然后再应用到微驱油藏,以进一步提高微生物采油实施效果。【目的】筛选乳化降解原油性能良好的菌株,对其进行多相分类学鉴定和性能评价。【方法】利用原油为底物筛选乳化降解性能良好的菌株,通过形态特征观察、生理生化测定、16S rRNA基因序列分析等确定菌株的分类地位。通过乳化能力、降解率等方法确定菌株的原油乳化降解特性。【结果】从华北油田采集的地层水样品中分离得到一株乳化原油的菌株BLG74,经多相分类鉴定表明其是土壤堆肥芽孢杆菌(Compostibacillus humi)的新菌株,亲源性99.6%。该菌株的生长温度为30-60℃ (最适温度45℃),pH6.5-9.5(最适pH7.0),NaCl浓度0%-7%(质量体积比)。菌株BLG74在玉米浆培养基中培养,其发酵液的表面张力为56.3 mN/m,乳化力约95%,在初始原油质量浓度0.5%、温度45℃的条件下培养20d,对原油的降解率可达40.8%。【结论】菌株BLG74是可乳化降解原油的新成员,其在热盐条件下乳化降解原油的特性在石油开采中有一定的潜力。     

Estimates of Frankia growth under various pH and temperature regimes

Summary The growth of Frankia isolates was monitored by dry weight, total protein and total ATP measurements under different temperature and pH regimes. Significant correlations (P<0.01) were found among all growth measures which meant that similar general conclusions were reached irrespective of the study method involved. The assessment of protein was the method of choice for regular assessments of Frankia growth due to its facility and relatively high sensitivity. The optimum temperature for growth of isolate LDAgp1 and AvcI1 was about 30°C while for CpI1 it lay between 30° and 35°C. No growth was observed at 40°C but some growth was observed at 10°C with isolate CpI1 and LDAgp1 over an extended growth period of 39 days. The range of pH favouring growth lay between 6 and 8. The optimum for LDAgp1 lay between 6.5 and 7, that for AvcI1 and CpI1 is close to 6.5. The pH response was medium dependent. Increases in biomass were observed for some isolates at 4.6 and above 8.0 on some media.     

Isolation and characterization of β-galactosidase fromLactobacillus crispatus

β-Galactosidase was isolated from the cell-free extracts ofLactobacillus crispatus strain ATCC 33820 and the effects of temperature, pH, sugars and monovalent and divalent cations on the activity of the enzyme were examined.L. crispatus produced the maximum amount of enzyme when grown in MRS medium containing galactose (as carbon source) at 37°C and pH 6.5 for 2 d, addition of glucose repressing enzyme production. Addition of lactose to the growth medium containing galactose inhibited the enzyme synthesis. The enzyme was active between 20 and 60°C and in the pH range of 4–9. However, the optimum enzyme activity was at 45°C and pH 6.5. The enzyme was stable up to 45°C when incubated at various temperatures for 15 min at pH 6.5. When the enzyme was exposed to various pH values at 45°C for 1 h, it retained the original activity over the pH range of 6.0–7.0. Presence of divalent cations, such as Fe²⁺ and Mn²⁺, in the reaction mixture increased enzyme activity, whereas Zn²⁺ was inhibitory. TheK _m was 1.16 mmol/L for 2-nitrophenyl-β-d-galactopyranose and 14.2 mmol/L for lactose.     

Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> Si3

During the industrial stabilization process, lactic acid bacteria are subjected to several stressful conditions. Tolerance to dehydration differs among lactic acid bacteria and the determining factors remain largely unknown. Lactobacillus coryniformis Si3 prevents spoilage by mold due to production of acids and specific antifungal compounds. This strain could be added as a biopreservative in feed systems, e.g. silage. We studied the survival of Lb. coryniformis Si3 after freeze-drying in a 10% skim milk and 5% sucrose formulation following different fermentation pH values and temperatures. Initially, a response surface methodology was employed to optimize final cell density and growth rate. At optimal pH and temperature (pH 5.5 and 34 °C), the freeze-drying survival of Lb. coryniformis Si3 was 67% (±6%). The influence of temperature or pH stress in late logarithmic phase was dependent upon the nature of the stress applied. Heat stress (42 °C) did not influence freeze-drying survival, whereas mild cold- (26 °C), base- (pH 6.5), and acid- (pH 4.5) stress significantly reduced survival. Freeze-drying survival rates varied fourfold, with the lowest survival following mild cold stress (26 °C) prior to freeze-drying and the highest survival after optimal growth or after mild heat (42 °C) stress. Levels of different membrane fatty acids were analyzed to determine the adaptive response in this strain. Fatty acids changed with altered fermentation conditions and the degree of membrane lipid saturation decreased when the cells were subjected to stress. This study shows the importance of selecting appropriate fermentation conditions to maximize freeze-drying viability of Lb. coryniformis as well as the effects of various unfavorable conditions during growth on freeze-drying survival.     

Partial purification and properties of an endonuclease from germinating pea seeds specific for single-stranded DNA.

An enzyme that rapidly catalyzes the hydrolysis of denatured DNA has been partially purified from germinated pea (Pisum sativum) seeds. The nuclease has been characterised as having endonucleolytic activity degrading single stranded DNA at a 15- to 20-fold higher rate than native DNA. From exclusion chromatography on Sephadex G-200 the molecular weight of the enzyme was calculated to be 42,000. The small extent of hydrolysis of native DNA is suggested to be due to the degradation of partially denatured areas in the native molecule. The enzyme shows activity over a broad range of pH but was most active between pH 6.5 and 8.0. The maximum hydrolysis of denatured DNA was observed at 45 °C while with native DNA the temperature optima was 60 °C. The nuclease does not show an absolute requirement for added divalent cations. However, the addition of Mg²⁺ and Ca²⁺ results in 40 and 60% stimulation, respectively. EDTA has no effect on enzymatic activity, whereas 8-hydroxyquinoline was inhibitory.