纤维素降解菌CMC-4的分离鉴定、诱变和酶学特性研究

从秸秆还田土壤中初筛获得一株高效纤维素降解菌CMC-4,根据菌株形态、理化性质及16S rDNA序列分析,初步鉴定为地衣芽孢杆菌(Bacillus licheniformis)。以此为出发菌株,经亚硝酸钠诱变获得一株稳定高产纤维素酶突变株CMC-4-3。对CMC-4和CMC-4-3的产酶条件和酶学性质进行比较分析,结果表明:CMC-4-3纤维素酶活力较CMC-4提高67.5%;其最适产酶条件是:37℃、pH 6.0、葡萄糖为碳源、蛋白胨为氮源、接种量2.0%、装液量60 ml/250 ml。该菌株所产纤维素酶的最适反应pH为6.0,且在4.0~7.0酶活力较稳定;最适反应温度为50℃,在20~80℃范围内均保持稳定;金属离子Fe~(2+)、Mg~(2+)、Co~(2+)、K~+对酶有激活作用,其余离子均有不同程度抑制作用,而Cu~(2+)和Ca~(2+)抑制作用最强,酶活力减弱近50%,是该酶的强效抑制因子。诱变前后菌株产酶条件和酶学性质等部分表型发生了变化,而突变菌株显示出了更宽泛的环境适应范围。据此,获得一株高效产纤维素酶、耐受范围广的具纤维素降解能力的地衣芽孢杆菌,而CMC-4-3和CMC-4的表型可作为深入探讨基因型变化的线索。     

一株高活性纤维素降解细菌的筛选鉴定及酶学特性

利用小麦/玉米秸秆还田土壤样品,通过富集培养和刚果红平板染色法筛选分离出纤维素降解细菌XWS-12;对分离的菌株进行16S rRNA基因序列系统发育分析,初步鉴定为伯克氏菌属(Burkholderia),定名为Burkholderia sp.XWS-12。以玉米秸秆和麸皮为碳源,研究了氮源、发酵时间、初始发酵温度、培养基初始pH等条件对该伯克氏菌产纤维素酶的影响。结果显示,该菌株产纤维素酶最适氮源为硝酸钠,培养时间为60 h,培养温度为37℃,培养基初始pH为4,该菌株的CMC酶活力最高,可达25 U/ml。其粗酶液的最适反应温度为50℃,最适反应pH为5,在pH 4~8的范围内酶活力较稳定。粗酶液的热稳定较差,当温度超过50℃时,该酶活力显著下降;当温度为50℃时,保温1 h,该酶活力损失53%。     

纤维素降解菌的分离、鉴定与产酶条件探究

从林下腐熟土壤中筛选可降解纤维素的天然微生物,经过初筛、复筛得到降解能力较强的菌株DT13,革兰氏染色和分子生物学鉴定结果显示该菌株为枯草芽孢杆菌(Bacillus subtillis DT13)。以羧甲基纤维素酶活性(Carboxymethyl Cellulase Activity,CMCA)为指标,设计单因素试验与正交试验对菌株DT13产纤维素酶条件进行优化,确定其最佳产酶条件为初始pH值7.0、发酵温度40 ℃、发酵时间96 h。     

一株产常温葡甘聚糖酶的芽孢杆菌的分离、鉴定及产酶发酵条件的研究

以魔芋生粉为唯一碳源的筛选培养基,从魔芋废渣中分离筛选出一株可以降解魔芋生粉的细菌菌株,编号为LS-6。根据16SrDNA序列和生理生化特性,将该菌株鉴定为枯草芽孢杆菌Bacillus subtilis。胞内外葡甘露聚糖酶酶活分析结果表明,LS-6产生的葡甘聚糖酶为胞外酶。LS-6产生葡甘聚糖酶的最佳培养基组分和条件是：1%魔芋生粉,0.5%酵母粉,0.5%NaCl,培养基初始pH为6.0,最佳发酵条件是25℃,摇床转速200r/min,培养48h。酶解产物的高效液相色谱分析结果表明,LS-6粗酶液的酶解产物主要为葡萄糖和甘露糖。LS-6的分离为进一步克隆常温葡甘聚糖酶基因和产酶工程菌的构建奠定了基础。     

一株芽孢杆菌属的苄嘧磺隆降解菌的分离鉴定和降解研究

The objectives of this study were to isolate a bensulfuron-methyl (BSM)-degrading strain of Bacillus spp. and to evaluate its effectiveness in remediation of a BSM-contaminated soil. A BSM-degrading bacterium, strain L1, was successfully isolated in this study. Strain L1 was identified as Bacillus megaterium based on its morphological, physiological, and biochemical properties, G+C content, phylogenetic similarity of 16S rDNA, and fatty acid compositions. Two experiments were used to examine BSM degradation by strain L1. When BSM was used as a sole carbon source in a mineral salt medium, the average degradation rate of BSM by strain L1 was 12.8%, which suggested that the strain was able to utilize BSM as a sole carbon and energy source. In addition, supplement of yeast extract (200 mg L-1) significantly (P ≤ 0.01) accelerated the degradation of BSM by strain L1. Almost complete degradation (97.7%) of BSM could be achieved in 84 h with addition of yeast extract. In addition, when a sterile soil was supplemented with BSM (50 mg L-l), BSM degradation rate was 94.3% in 42 d, indicating the potential of using microbes for the remediation of BSM-contaminated soils in fields.     

一株产高温蛋白酶耐热菌BY25的产酶条件与酶学性质研究

对菌株BY25的生长条件、产酶条件及其产生的蛋白酶的酶学性质进行了研究。结果发现,BY25的最高生长温度为55℃,最适生长温度为50℃,最佳产酶温度为30℃,最佳培养起始pH为8.0,最佳C源为葡萄糖,高通气量明显提高菌株产酶能力。在以上条件下培养52 h,上清液蛋白酶活力达4 170 U/ml。酶学性质研究表明：该蛋白酶为高温中性金属蛋白酶,最适反应pH为7.0,最适反应温度为55℃,具有良好的pH耐受性和较好的热稳定性;EDTA能强烈抑制酶活力,而Fe2+、Cu2+对酶活力也具有一定抑制作用。     

热稳定性β-葡聚糖酶菌种选育及产酶特性

从土壤中筛选到一株热稳定性β－1，3－1，4－葡聚糖产生菌ZJF－1，发酵60h酶活性为64U／mL，经鉴定该菌株为枯草芽孢杆菌（Bacillus subtilis)。紫外线和硫酸二己酯复合诱变，获得的突变株ZJF－1A5发酵60h酶活性达154．7U／mL，是出发菌株酶活性的2．42倍，对B.subtilis ZJF-1A5产酶特性的研究发现：大麦粉，糊精，可溶性淀粉等糖有利于β－1，3－1，4－葡聚糖酶的产生，葡萄糖，麦芽糖等单糖和双糖不利于菌体生长和产酶。B.subtilis ZJF-1A5 β-1,3-1,4-葡聚糖酶的产生与菌体生长部分相关，在细胞进入对数生长后期至稳定期，酶活性显著增加，且β－葡聚糖酶活性与菌体生物量密切相关，B.subtilis ZJF-1A5 α－淀粉酶的产生也与生长部分相关，细胞进入对数生长期，α－淀粉酶开始大量产生，而中性蛋白酶的产生与菌体生长同步。     

白酒糟纤维素降解菌的优选及酒糟降解工艺

白酒酿造行业产生的酒糟含有大量纤维素,不仅原料利用率低,而且丢弃的酒糟会对环境产生污染。为了获得酒糟纤维素降解能力强的微生物并进行应用,从酱香型酒醅、清香型酒醅、浓香型大曲、竹林里的土壤腐殖质中分离筛选酒糟纤维素降解菌,并对菌种分类、理化特征和降解酒糟的特性进行研究。pH值、温度和酒精胁迫性试验进一步确定了最佳酒糟纤维素降解菌为B2菌株,该菌株在pH值3.0、温度44 ℃、酒精含量为体积分数4%环境中生长良好。基于形态学、生理生化和分子生物学分析,鉴定B2菌株为枯草芽孢杆菌（Bacillus subtilis）。在单因素试验结果的基础上,采用Box-Benhnken响应面法进行优化,确定了B2菌株降解酒糟的最佳工艺条件为酒糟添加量71 g/L、温度37 ℃、pH值6.4、接种量8%,此时酒糟降解率为15.23%。该研究丰富了酒糟纤维素降解菌的微生物资源库,同时为酒糟的资源化利用提供技术参考。     

尼古丁降解菌L1的分离鉴定与降解特性分析

通过对分离于烟草叶片的154株具有降解尼古丁细菌菌株筛选，获得能高效降解尼古丁的L1菌株。通过形态观察，16S rDNA序列分析和生理生化测定将其鉴定为Bacillus simplex。菌落生长密度测定和高压液相色谱分析表明， L1菌株最适生长尼古丁浓度为1g/L，随着菌落密度增加，尼古丁降解效率逐渐升高，36 h最高降解率达到75.0%。而低尼古丁含量不利于L1菌株的生长，过高尼古丁含量对L1菌株的生长和降解效率具有反馈抑制作用。菌株L1降解尼古丁过程中无色素产生，说明其尼古丁代谢途径有别于节杆菌。     

一株黄曲霉拮抗菌的筛选、鉴定及特性研究

为获得拮抗黄曲霉和降解黄曲霉毒素的生物防治菌株,以烟台黄曲霉污染区域土壤为材料,采用稀释分离法和琼脂扩散法,筛选到一株对黄曲霉有抑制作用且可降解黄曲霉毒素的菌株Y-17-3。根据菌株的形态学特征、生理生化试验结合16S rDNA基因序列分析和系统发育树构建,确定菌株 Y-17-3 为空气芽孢杆菌(Bacillus aerius)。该菌株发酵液经硫酸铵沉淀的抗菌活性及稳定性试验,发现70%饱和硫酸铵沉淀的蛋白粗提物抑制黄曲霉活性最强,可降低黄曲霉孢子的萌发率,抑制菌丝的生长和菌丝球的形成,且该蛋白粗提物对温度、pH值和蛋白酶敏感。菌株Y-17-3能够降解黄曲霉毒素B₁,培养6 d时的降解率达90%。综上,所分离的空气芽孢杆菌Y-17-3菌株能够通过产生拮抗蛋白来抑制黄曲霉生长,且具有较强的黄曲霉毒素降解能力,在农业生物防治领域具有一定的应用前景。     

2种菌联合修复农田土壤镉污染的研究

采用盆栽试验,运用BCR四步法、火焰原子吸收法和稀释涂布平板法等方法,研究了沼泽红假单胞菌(Rhodopseudanonas palustris)和枯草芽孢杆菌(Bacillus subtilis)联合作用对修复农田土壤重金属镉污染盆栽种植小白菜(Brassica campestris L. ssp.chinensis Makino)的效果及安全性,旨在探讨降低土壤镉生物有效性的有效途径,为新型生物菌肥研究提供理论依据。结果表明,沼泽红假单胞菌、枯草芽孢杆菌单独作用以及联合作用均能使农田土壤中镉的固定态增加,降低农田土壤中镉的生物有效性,联合菌处理组镉的生物有效性降低最显著,为32.70%,均对农田土壤中微生物各生理类群数量有显著性影响(P0.05),其中,细菌的数量达到10~5 CFU/g;均显著降低了小白菜根、茎中镉含量,联合菌处理组根、茎中镉的去除率分别达到58.06%～71.37%和73.66%～76.70%;均增加了小白菜株高、茎鲜重、叶绿素a、b的含量和叶绿素a/b的比值,联合菌处理组小白菜的生物量增加18.13%～69.55%。研究结果说明,沼泽红假单胞菌和枯草芽孢杆菌联合作用,对农田土壤镉污染的修复效果显著,且促进了小白菜的生长,具有修复农田土壤重金属污染的潜力。     

PATS结合山梨酸钾对枯草杆菌芽孢的灭活机理

枯草杆菌芽孢是极难被杀灭的细菌之一,为探究山梨酸钾与压力辅助热杀菌（pressure assisted thermal sterilization,PATS）对枯草杆菌芽孢的联合作用效果,该研究以OD₆₀₀值（optical density at 600 nm）、OD₂₆₀值（optical density at 260 nm）、OD₂₈₀值（optical density at 280 nm）、2, 6-吡啶二羧酸释放率表征芽孢内容物的释放情况,扫描电镜、流式细胞术、傅里叶红外光谱仪、激光粒度仪检测芽孢结构变化情况。结果表明,山梨酸钾强化了PATS的杀菌效果,200 MPa-75 ℃处理杀灭了2.63 log芽孢,添加2 g/L山梨酸钾后,杀灭了3.24 log芽孢。较单独PATS处理,添加山梨酸钾后,促进了芽孢内容物的释放,内膜通透性增加,芽孢粒径显著减小,内膜、细胞壁等结构受损程度加剧。同时,Na⁺ /K⁺-ATPase活力显著降低,膜电位平衡被破坏,基本生理代谢功能紊乱。总之,PATS结合山梨酸钾对枯草芽孢杆菌芽孢有协同杀菌作用,且随着山梨酸钾添加量或温度的升高而增强,有利于促进该技术在食品杀菌中的应用。     

溶剂萃取与生物降解法联合修复老化石油污染土壤

This study aimed to evaluate the efficacy, practicality and sustainability of a combined approach based on solvent extraction and biodegradation to remediate the soils contaminated with high levels of weathered petroleum hydrocarbons. The soils used in this study were obtained from the Shengli Oilfield in China, which had a long history of contamination with high concentrations of petroleum hydrocarbons. The contaminated soils were washed using a composite organic solvent consisting of hexane and pentane (4:1, v/v) and then bioremediated in microcosms which were bioaugmentated with Bacillus subtilis FQ06 strains and/or rhamnolipid. The optimal solvent extraction conditions were determined as extraction for 20 min at 25 °C with solvent-soil ratio of 6:1 (v/w). On this basis, total petroleum hydrocarbon was decreased from 140 000 to 14 000 mg kg-1, which was further reduced to < 4 000 mg kg-1 by subsequent bioremediation for 132 d. Sustainability assessment of this integrated technology showed its good performance for both short- and long-term effectiveness. Overall the results encouraged its application for remediating contaminated sites especially with high concentration weathered hydrocarbons.     

Poultry manure and banana waste are effective biofertilizer carriers for promoting plant growth and soil sustainability in banana crops

The aims of our study were to compare the effectiveness of poultry manure (PM) and banana waste (BW), with regard to their use as inoculant carriers of a bacterial consortium constituted by strains of Azospirillum, Azotobacter and P-solubiliser bacteria and to establish the most efficient dose of biofertilizer for a soil cultivated with banana (Musa paradisiaca AAA Simmonds), with respect to improving plant performance and soil physical and microbiological properties. Six months after planting, plant growth had increased with increase in dose of the biofertilizers applied. The biofertilizer prepared on BW enhanced the density of P-solubiliser bacteria, the concentrations of available P and foliar P to a greater extent than with the biofertilizer prepared on PM. The increases produced by the biofertilizer prepared on PM for the soil aggregate stability, enzymatic activities and the labile carbon fractions were highly correlated to the dose applied. Both biofertilizers can be considered potentially useful as inoculant carriers of PGPR but the usefulness of BW appears to be restricted to moderate doses of application (≤3%).     

Control of Fusarium wilt disease of cucumber plants with the application of a bioorganic fertilizer

Two field experiments were conducted to evaluate the effect of organic fertilizer application either with or without antagonistic bacteria (Bacillus subtilis SQR-5 and Paenibacillus polymyxa SQR-21) on the control of Fusarium oxysporum f. sp. Cucumerinum J. H. Owen wilt disease in cucumber. The incidence of Fusarium wilt disease was 5.3–13.5% for cucumber plants treated with bioorganic fertilizer, while it was 30.3–51% in controls (only with organic fertilizer). Higher yields and lower disease incidences were observed in the dry season when compared with the wet season for both types of organic fertilizer treatments. Biolog analysis showed a significant change in soil bacterial composition and activity after bioorganic fertilizer application. The numbers of colony-forming units of F. oxysporum f. sp. Cucumerinum J. H. Owen for bioorganic-fertilizer-treated soils were significantly decreased compared with control. Scanning electron micrographs of cucumber basal stems showed a presence of mycelia-like mini strands accompanied by an amorphous substance within the xylem vessels. This amorphous substance and mini strands were richer in calcium and phosphorus but had low carbon and oxygen than the living mycelia. Reverse-phase high-pressure liquid chromatography and mass spectroscopic analysis showed that the antagonistic bacteria produced the antifungal compounds fusaricidin A, B, C, and D with molecular weights of 883.5, 897.5, 947.5, and 961.5 Da, respectively. The application of bioorganic fertilizer has a great potential for the control of F. oxysporum wilt disease in cucumber plants.     

Colonization of wheat seedling (Triticum aestivum) roots by Pseudomonas fluorescens and Bacillus subtilis

Summary Colonization patterns of Pseudomonas fluorescens and Bacillus subtilis on roots of wheat seedlings growing on water agar were studied qualitatively by replica printing and quantitatively by the plate count method. The results indicated a stronger colonization potential for P. fluorescens (up to 10⁷ cfu/cm root) than for B. subtilis (up to 10⁵ cfu/cm root). Although the numbers of both species were lower when inoculated together, the observed colonization patterns on the roots were comparable to those found with single inoculations. For none of these bacteria was active migration along the root surface in any direction observed, indicating that distal positions are reached mainly by a passive displacement on the root tip and elongating cells. Ecological implications of the observed phenomena are discussed.     

Mechanisms of production of soil protease by proteolytic Bacillus subtilis in wetland rice soil

In sterile soil inoculated with proteolytic Bacillus subtilis, the correlation between variations in protease activity and cell numbers was investigated during incubation, and the results were compared with those obtained in different growth media. In sterile soil inoculated with B. subtilis, the extracted soil z-FLase activity (hydrolytic activity towards benzyloxycarbonyl-L-phenylalanyl-L-leucine extracted with 0.1 M phosphate buffer) was correlated with the extracted soil protein content (r=0.95; P<0.01) and the number of vegetative cells (r=0.88, P<0.05), while the extracted soil caseinase activity was well correlated with the number of spores (r=0.82, P<0.05) and not with the number of vegetative cells. Extracellular z-FLase activity in different growth media, inoculated with the proteolytic B. subtilis, was correlated with the extracellular protein concentration (r=0.73, P<0.01) and the number of cells (r=0.73, P<0.01), but extracellular caseinase activity was not correlated with either of these measurements. From these results, we concluded that soil z-FLase production might relate to vegetative growth of the proteolytic B. subtilis and soil caseinase might relate to B. subtilis sporulation.