瘤胃中纤维素分解菌的分离、鉴定及其产酶条件的优化

[目的]从新疆细毛羊、牛和骆驼瘤胃液中分离出具有分解纤维素能力的好氧细菌,用于绿色粗饲料微生物添加剂的研发.[方法]采取新鲜瘤胃液,接种于羧甲基纤维素钠平板,通过刚果红染色和液体复筛培养基,筛选出分解纤维素能力强的好氧细菌;形态学、生理生化试验与16S rDNA序列分析方法相结合对细菌进行鉴定;同时对纤维素分解能力强的4株细菌进行不同条件下酶活力测定.[结果]共分离到84株具有分解纤维素能力的细菌,其中筛选出较强分解纤维素能力的40株.该菌包括37株G-菌和3株G+菌;经鉴定40株纤维素分解菌分别属于6个属10个种,其中16株为寡养单胞菌属,10株为苍白杆菌属,5株为鞘氨醇杆菌属,3株为微杆菌属,3株为副球菌属,2株为假单胞菌属.其中产酶能力强的4株菌在不同条件下的酶活力表明,它们在最佳碳源为秸秆粉、pH5.5-6.0的偏中性条件和37℃培养条件下的酶活力较高.不同菌株对不同纤维素类物质的分解能力不一样,同一菌株对不同纤维素碳源的利用能力也不相同.[结论]分离获得的瘤胃纤维素分解菌是从新疆不同地区、干旱半干旱环境下饲养的动物胃液中分离、筛选出来的,有较强的纤维素分解能力,将为高品质、高消化率的青贮饲料生产提供优质菌种资源及一定的科学依据.     

一株培菌白蚁后肠木聚糖降解细菌的分离与鉴定

【目的】从培菌白蚁——黄翅大白蚁肠道微生物菌群中分离能降解木聚糖的细菌。【方法】以木聚糖为唯一碳源,利用刚果红染色,根据透明圈大小进行筛选。通过显微形态,革兰氏染色及16S r RNA基因序列分析进行菌株鉴定。二硝基水杨酸(DNS)法测定细菌生长过程中木聚糖酶酶活变化,比较酶活与菌株生长状况的关系。【结果】从黄翅大白蚁肠道中筛选到一株具有较高木聚糖降解活性的革兰氏阳性菌Mb1,16S r RNA基因序列分析表明为类芽孢杆菌属细菌,命名为Paenibacillus sp.Mb1。该菌培养72 h后菌体浓度达到最高,木聚糖酶酶活主要存在于培养液上清中,酶活在对数期增长快,在培养96 h时达到最高值,之后趋于稳定。【结论】从黄翅大白蚁肠道中分离出一株具有较高木聚糖酶活的类芽孢杆菌,可作为产细菌木聚糖酶的潜在优良菌株。     

纤维堆囊菌生理生化及药敏特征

对纤维堆囊菌模式菌DSM14627的生理生化和药敏性进行了系统的考察,研究显示该茵能利用葡萄糖及纤维素,不能分解利用麦芽糖、蔗糖等12种碳源;磷酸酶、β-葡萄糖苷酶及明胶液化实验为阳性,过氧化氢酶、β-木糖苷酶等13项实验均阴性;对丙氟哌酸、万古霉素、多粘霉素B等15种抗生素敏感,对氨苄西林及苯唑青霉素等10种抗生素有极强抗性.     

桉树EST-PCR产物测序方案的优化

EST-PCR产物测序是EST标记开发的重要步骤.本研究利用乙醇-醋酸钠法、清洁试剂盒、虾碱磷酸酶(SAP)-核酸外切酶Ⅰ(Exo Ⅰ)法和凝胶回收试剂盒4种方法,对6个桉树EST-PCR产物的纯化效果和测序结果进行了比较,推荐乙醇-醋酸钠法为首选的纯化方法;利用测序试剂盒Bigdye TerminatorV 3.1,对1/2、1/4、1/8、1/16和1/32标准用量(8.0μL)的测序结果的分析表明,1/16和1/32用量仍可有效测序,均可作为优先的测序试剂用量.同时,也发现EST-PCR扩增谱带单一、明亮的序列与设计的目标EST具有较好的同源性,开发SNP和Indel标记的潜力极大,但较弱的谱带则可能是非特异性扩增的产物.乙醇-醋酸钠法纯化和BigdyeTerminator 1/16或1/32用量的测序方案,具有成本低和操作简便的优点,适合96孔板或384孔板大规模EST-PCR产物的测序.     

一种产邻苯二酚菌株高通量筛选方法

目的：对产邻苯二酚菌株进行筛选。方法：采用前期筛得的产邻苯二酚菌为出发菌株,通过硫酸二乙酯诱变的方法使该菌株突变,同时建立96孔板培养和酶标仪检测方法对产邻苯二酚菌株进行高通量筛选。结果：硫酸二乙酯的终浓度为0.1%,诱变时间为8 min的条件下,突变菌致死率为84.5%,突变效果最好。筛选培养基中吸光值（495nm）和富集培养基中菌液浊度值（OD630）的加和值较大的突变菌株产邻苯二酚能力高。通过诱变和筛选得到的菌株,产邻苯二酚浓度可达0.87mg/ml,比出发菌株的提高了262.5%。经过形态学和生理生化反应,初步鉴定该菌株属于假单胞菌属（Pseudomonas sp.）。结论：硫酸二乙酯诱变和96孔板筛选的方法能以高通量方式快速筛选出产邻苯二酚菌株。     

绿脓杆菌的快速鉴定和耐药谱分型的探讨

本文用绿脓杆菌12群“O”多价血清辅以乙酰胺酶试验对临床分离的118株绿脓杆菌作了快速鉴定,2小时即可获得满意结果,与生物学鉴定的符合率达100%。对10种抗生素的药敏试验结果表明,本菌对丁胺卡那霉素和多粘菌素 B 较敏感,敏感率分别为98.31%和100%。118株菌可用8种抗生素分成20种不同的耐药谱型(A—T),其中属耐药谱 D 型(对多粘菌素、丁胺卡那霉素、新霉素敏感)的菌株在分布上最多,占28.8%(34/118)。     

微孔板改良法评价多种国内外单花蜜的体外抑菌活性

目的：开展国内外多种单花蜜体外抑菌活性差异的系统评价。方法：以无菌Mueller Hinton Broth培养基稀释得到3.75%～90%（w/v）不同浓度的单花蜜,采用96微孔板改良法,用空白无菌培养基和标准菌株作对照孔,测定18种单花蜜对金黄色葡萄球菌、沙门氏菌和大肠埃希氏菌的最低抑菌浓度、最低杀菌浓度、迟缓期持续时间、对数期持续时间和最大细菌密度5个抑菌活性指标。结果：18种单花蜜均具有不同程度的抑菌活性,且抑菌生长曲线均优于标准菌株对照孔,延长了3种致病菌的迟缓期持续时间、缩短了对数期持续时间和降低了最大细菌密度,其中小茴香蜂蜜最低抑菌浓度为7.5%,最低杀菌浓度为15%,均优于麦卢卡蜂蜜。结论：96微孔板改良法灵敏、快速、客观、可直观显示不同的单花蜜具有抑菌活性差异,其中小茴香蜂蜜、葵花蜂蜜、椴树蜂蜜活性最为显著,本文为蜂蜜的功能评价与市场开发提供了科学依据。     

斑马鱼饲养循环装置中的水生环境微生物群落代谢

目的在斑马鱼标准化养殖中,建立饲养环境中微生物群落代谢指纹数据库,用于分析和判断饲养环境是否出现病原菌感染。方法在不当的饲养过程中,斑马鱼会出现死亡或疾病,其中一个重要原因是病原菌感染。采用MicroStation细菌鉴定系统,微生物群落将鉴定板上的95种碳源作为唯一可利用碳源,当各孔中微生物利用碳源底物时,呼吸作用引起了氧化还原反应指示剂颜色变化,板上出现的颜色反应作为鉴定的指纹。结果多变量分析代谢的类型与程度,得出健康的斑马鱼饲养环境的微生物群落对GN和GP板上的22种有共同利用的模式。结论本文通过微生物群落代谢单碳源方法分析微生物群落的结构和功能变化,多变量分析后定位健康的斑马鱼饲养环境中的微生物群落代谢特征。     

两株土壤耐镉细菌的筛选分离及与CdS-TGA纳米颗粒相互作用的研究

由受重金属镉污染严重的化工厂土壤中,筛选分离获得两株镉耐受性加强的菌株,菌体长1.0~2.0μm,宽0.5~0.8μm,单个细胞呈杆状,单菌落分别呈淡黄色与乳白色,经鉴定为Enterobacter sp.与Serratia sp.。对所分离细菌的最适生长条件、镉离子最小抑制浓度及抗生素抗性进行了检测和优化,并进一步研究了硫化镉-巯基乙酸纳米颗粒与细菌的相互作用,发现纳米颗粒浓度在150μg/m L时细菌增殖基本停滞;过氧化物酶和超氧化物歧化酶酶活测试显示纳米颗粒对酶抑制性较Cd2+更为强烈,细胞形态受到严重破坏。     

1株大熊猫肠道纤维素降解菌的分离鉴定及其酶学性质

以羧甲基纤维素钠为唯一碳源,利用刚果红染色法,从大熊猫粪便内筛选具有降解纤维素能力的菌株,并研究其酶学特性。分离获得1株酶活力较高的菌株A1,通过形态学和BD PhoenixTM-100全自动细菌鉴定仪鉴定为蜡样芽胞杆菌(Bacillus cereus)。菌株A1最适生长条件和酶活力测定表明,其最适生长温度为37℃,Na Cl浓度为0.5%,p H值为7.0,内切葡聚糖苷酶、外切葡聚糖苷酶、β-葡萄糖苷酶和总酶活的最大值分别为0.139、0.074、0.126、0.108 5 IU/m L。丰富了大熊猫肠道纤维素降解菌的种类,为后续研究大熊猫如何消化利用竹纤维提供了菌源。     

Kinetics of the utilization of different C sources and the cellulose formation by Acetobacter xylinum

Different methylated glucose derivatives and cellobiose were examined as the carbon sources for growth and cellulose formation by Acetobacter xylinum. HPLC studies were carried out to gain information about the kinetics of the utilization of the C sources used. The type and yields of the synthesized cellulose were described. Besides glucose, cellobiose was a substrate for the synthesis of this polysaccharide by the bacteria. Other methylated derivatives of glucose were not accepted for a comparable synthesis of this polymer. An estimation of citrate in an unmodified culture liquid (SH medium) showed utilization in a late phase of cultivation. The influence of this organic acid on the pH value, cellulose synthesis and growth is described. By the application of citric acid as a sole carbon source “gel-like” forms of cellulose were formed generally.     

产紫杉醇内生真菌EFY-21的鉴定与生物学特性研究

利用PCR技术克隆了产紫杉醇内生真菌EFY-21的18S rDNA序列,通过同源性分析,初步确定该菌与拟盘多毛孢属(Pestalotiopsis)有较高的同源性,相似性为99%。为了进一步了解EFY-21的有关生物学特性,分别选用PDA、PSA、查氏、玉米粉琼胶、牛肉膏蛋白胨5种培养基,按照常规方法培养,用十字法测量菌落直径;同时选用查氏培养基为基本培养基,分别观察不同碳源葡萄糖、甘露醇、麦芽糖、果糖、可溶性淀粉,不同氮源KNO3、Ca(N03)2、(NH4)2SO4、NH4N03、(NH4)2HPO4、蛋白胨、尿素,不同培养温度10,15,20,25,28,30,37℃,不同pH值4,5,6,7,8,9对内生真菌菌丝的影响。试验结果表明:EFY-21在PDA培养基上生长最快,生长状况最好;供试的碳氮源中,对EFY-21菌丝生长影响的大小顺序为葡萄糖甘露醇果糖麦芽糖可溶性淀粉;蛋白胨KNO3Ca(N03)2NH4N03(NH4)2HPO4(NH4)2SO4尿素;最适培养温度为25～30℃;最适pH为5～7。     

一株毛栓菌静置液体发酵产纤维素酶条件研究

考察一株新分离的毛栓菌静置产纤维素酶的条件,通过单因素试验和正交设计试验分别对培养基初始pH、培养基中的碳源、氮源及诱导物进行研究。利用DNS（3,5-二硝基水杨酸）法测定CMC-Na（羧甲基纤维素钠）纤维素酶活。结果表明：该毛栓菌静置发酵产纤维素酶的最适pH为5.0,最适碳源、氮源和诱导物分别是淀粉、酵母粉和干麸皮,且质量浓度分别为淀粉1%、酵母粉0.4%、干麸皮1.5%,在此条件下,进行静置发酵3d,可获得CMC-Na（羧甲基纤维素钠）纤维素酶活力最高为56.62U/mL。此研究开创了纤维素酶生产菌的新发现,与以往所报道的好氧产纤维素酶菌种黑曲霉、木霉等相比,具有发酵时间短、耗能小等优点,具有一定的潜在应用价值。     

Assessment of the importance of similarity in carbon source utilization profiles between the biological control agent and the pathogen in biological control of bacterial speck of tomato

Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, was used to determine whether similarity in carbon source utilization between a preemptive biological control agent and the pathogen was significant in determining the ability of the bacterium to suppress disease. Similarity in carbon source utilization was quantified as the ratio of the number of tomato carbon sources utilized in vitro by the biological control agent to the number of tomato carbon sources utilized in vitro by the target pathogen (the niche overlap index [NOI]). Suppression of the disease was quantified as the percent reduction in disease severity compared to the pathogen-only control when nonpathogenic bacteria were applied to foliage 48 h prior to the pathogen. In the collection of 36 nonpathogenic bacterial strains, there was a significant (P < 0.01), but weak (r(2) = 0.25), correlation between reduction in disease severity and similarity in carbon source utilization, suggesting that similarity in carbon source use was significant in determining ability to suppress disease. The relationship was investigated further using catabolic mutants of P. syringae strain TLP2, an effective biological control agent of speck. Catabolic mutants exhibited lower levels of similarity (NOI = 0.07 to 0.90) than did wild-type TLP2 (NOI = 0.93). With these catabolic mutants there was a significant (P < 0.01), and stronger (r(2) = 0.42), correlation between reduction in disease severity and similarity in carbon source utilization. This suggests that similarity in carbon source utilization was a more important component of biological control ability for the catabolic mutants than for the nonpathogenic bacteria. Together, these studies indicate that suppression of bacterial speck of tomato was correlated with nutritional similarity between the pathogenic and nonpathogenic bacteria and suggest that preemptive utilization of carbon sources was probably involved in the biological control of the disease by both the naturally occurring nonpathogenic bacteria and the catabolic mutants.     

Characterization of three lactic acid bacteria and their isogenic ldh deletion mutants shows optimization for YATP (cell mass produced per mole of ATP) at their physiological pHs

Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y(ATP) (cell mass produced per mole of ATP). The pH that showed the highest Y(ATP) corresponded to the pH of the natural habitat of the organisms.     

培养基的组成及培养条件对茶树菇多糖产量的影响

本文研究了不同碳源,氮源以及发酵条件对茶树菇多糖产量的影响,并通过正交试验优化了深层发酵培养基。结果表明,茶树菇深层发酵最适的碳源为玉米粉,最适的氮源为酵母粉,最适的发酵条件为温度25℃,摇瓶装液量100mL/250mL,转速150r/min,接种量15%,起始pH自然。     

红树林溶磷菌的初步鉴定、溶磷能力测定及其优化培养

对分离来自华南红树林的溶磷菌进行16S rDNA或ITS等基因水平上的初步鉴定, 测定其溶解无机磷的能力, 并对溶磷菌的生长培养基条件进行优化。结果表明, 溶磷真菌的溶磷效果明显强于溶磷细菌, 且溶磷真菌的溶解无机磷能力与培养液的pH呈极显著负相关, 而溶磷细菌的溶磷能力与pH没显著相关关系。单因素实验表明, 对供试菌株生长的合适碳源为麦芽糖, 氮源为尿素。通过正交实验得到的优化培养基为麦芽糖5 g/L、尿素0.05 g/L、NaCl 5 g/L、pH 5, 在30°C下培养48 h菌落总数可达6.06×     

Gas chromatographic analysis of in situ cyclitol utilization by Klebsielleae growing in redwood extracts.

Gas chromatographic analysis was employed to demonstrate in situ cyclitol utilization in aqueous extracts of redwood by isolates of Klebsiella, Enterobacter, and several other genera of gram-negative bacteria. In aqueous redwood extracts, all but one of the Klebsiella and Enterobacter isolates tested reached densities exceeding 5.0 x 10(6) cells/ml within 4 days, and all utilized pinitol and sequoyitol. Other enteric bacteria did not utilize cyclitols in this extract. A defined minimal medium, containing the carbohydrates and cyclitols (including myo-inositol) in redwood, was used to determine which carbon sources are preferentially utilized by Klebsielleae and other bacteria. It was found that D-glucose and L-arabinose were consumed by Klebsiella before the three cyclitols were utilized. Pinitol utilization proceeded in more slowly than that of sequoyitol and myo-inositol. Cyclitol utilization in the defined medium was also observed for Yersinia, Erwinia, and Salmonella. Escherichia coli isolates did not utilize cyclitol compounds. The ability to use cyclitols as a sole source of carbon can explain the high cell densities of Klebsielleae in redwood water reservoirs and in redwood lumber.     

Gas chromatographic analysis of in situ cyclitol utilization by Klebsielleae growing in redwood extracts

Gas chromatographic analysis was employed to demonstrate in situ cyclitol utilization in aqueous extracts of redwood by isolates of Klebsiella, Enterobacter, and several other genera of gram-negative bacteria. In aqueous redwood extracts, all but one of the Klebsiella and Enterobacter isolates tested reached densities exceeding 5.0 x 10(6) cells/ml within 4 days, and all utilized pinitol and sequoyitol. Other enteric bacteria did not utilize cyclitols in this extract. A defined minimal medium, containing the carbohydrates and cyclitols (including myo-inositol) in redwood, was used to determine which carbon sources are preferentially utilized by Klebsielleae and other bacteria. It was found that D-glucose and L-arabinose were consumed by Klebsiella before the three cyclitols were utilized. Pinitol utilization proceeded in more slowly than that of sequoyitol and myo-inositol. Cyclitol utilization in the defined medium was also observed for Yersinia, Erwinia, and Salmonella. Escherichia coli isolates did not utilize cyclitol compounds. The ability to use cyclitols as a sole source of carbon can explain the high cell densities of Klebsielleae in redwood water reservoirs and in redwood lumber.     

Microbiological basis of phosphate removal in the activated sludge process for the treatment of wastewater

Several strains resembling members of theAcinetobacter-Moraxella-Mima group of bacteria were isolated from activated sludge-type sewage treatment plants designed for phosphate removal. The bacteria are obligate aerobes but utilize as carbon and energy sources low-molecular intermediates generated anaerobically, particularly acetate and ethanol. These bacteria can be shown to be responsible for the phosphate luxury uptake occurring in these treatment plants. The bacteria are physiologically unusual in that they perform luxury uptake of phosphates in a complete growth medium. Phosphate release occurs on addition of a carbon source to the carbon-starved bacteria, lowering pH or both. The bacteria persist in the system by virtue of their ability to form floc.