16S rDNA测序快速鉴定废水生物处理系统目标细菌

在废水生物处理系统中建立分子生物学技术快速鉴定有关微生物,具有十分重要的意义．以发酵法生物制氢系统的活性污泥分离培养的厌氧发酵细菌为研究对象,采用16S rDNA碱基测序分子生物学技术,通过生物信息学数据库NCBI将DNA序列输入、比对,可以直接鉴定从废水生物处理系统中分离培养的细菌进行种属科的系统发育学地位的判定,从而简化了细菌鉴定程序．通过16S rDNA测序技术,进行了分离产氢细菌的分子生物学鉴定,发现生物制氢厌氧活性污泥中所分离的细菌可能存在的菌属有：Lactobacillus,Clostridium,Klebsiella,Pmpionibacterium和可能新发现的1个新属Biohydrogenbacterium等5个菌属的菌种,其中新属Biohydrogenbacterium的菌种都以利用碳水化合物生产氢气为其主要特征．16S rDNA测序技术为废水生物处理系统的细菌学研究提供了方便、准确和经济的技术基础．     

海洋潮差区混凝土表面微生物16S rDNA分子鉴定

针对目前海洋混凝土工程的腐蚀现状,进行了海洋微生物对潮差区混凝土的作用机理及完善海工混凝土的腐蚀机理探索.通过海工混凝土潮差区部位取样,对暴露于潮差区中部22 y的混凝土试样表面进行FE-SEM、EDAX分析,结果表明:潮差区混凝土表面腐蚀严重,结构疏松,表面覆盖有有机物质和微生物.并将暴露于潮差区中部22 y的混凝土试样和该取样位置挂置7 d的混凝土试样表面的微生物群落分别分离,克隆了其中30株和12株细菌的16S rDNA基因,测定其序列且进行比对,其分别属于15个和7个种属中.优势种属均为假交替单胞菌,但随着暴露时间的增加,总细菌株数和总菌属数分别增大到2.5和2.14倍,且种属的变化也很大.同时,建立了海洋混凝土工程表面细菌鉴定方法,以及分离、鉴定的菌种为后续细菌对混凝土作用的试验提供菌种.     

几种金属离子对高效产氢细菌产氢能力的促进作用

金属离子Fe2 + 、Ni2 + 、Mg2 + 在生物产氢机制中有着重要作用 .在适当浓度下 ,研究金属离子对高效产氢细菌产氢能力的促进作用 ,是解决细菌产氢效率不高这一关键问题的有效手段之一 ,也加快了发酵法生物制氢技术产业化进程 .间歇实验结果表明 ,铁、镍和镁等金属离子对高效产氢细菌———B4 9产氢能力有明显的促进作用 ,使B4 9的最大产氢速率和平均产氢速率分别由 8 6 5和 0 36mmol·(g·h) -1提高到 2 8 0 1和1 95mmol·(g·h) -1.3种离子对B4 9产氢能力促进作用的顺序为Fe2 + >Ni2 + >Mg2 + ,Fe2 + 和Ni2 + 是促进发酵产氢的直接因素     

混合菌种非固定化技术制氢反应器产氢效能

为了研究混合菌种非固定化技术生物制氢反应器的产氢效能，实验采用厌氧Hungate技术和MPN法，从采用混合菌种非固定化技术的生物制氢反应器厌氧活性污染中分离到210株优势发酵菌株，其中18株为产氢细菌（HPB）。实验结果表明，主要决定反应器产氢效能的因素是反应器内HPB的数量和活性。采用混合菌种非固定化技术可以充分发挥HPB的产氢活性，但是由于反应器内HPB的数量和比例不高，大大制约了混合菌种非固定技术生物制氢反应器效能的充分发挥。针对这一结论，提出采用有自絮凝能力的高效产氢细菌进行快速启动和投加高效产氢菌株的方法提高生物制氢反应器的产氢效能。     

发酵产氢细菌分离培养的厌氧实验操作技术

厌氧产氢细菌的分离纯化与培养是发酵法生物制氢技术的基础课题之一,现有厌氧细菌的分离制备技术和培养基分别存在着操作繁琐和成份复杂问题．通过厌氧培养技术比较和细菌生长试验,确定了“煮沸吹氮去氧,固液交替分离”的厌氧产氢细菌分离纯化培养程序,改进了Hungate技术和建立了新的宽体窄颈培养瓶平板技术,通过培养基组成成份的增减和细菌增长、种类和数量的研究确立了厌氧发酵产氢细菌的分离和富集培养基．利用这些厌氧培养技术,已成功分离培养了550余厌氧菌株系．     

高效石油烃降解菌的分离鉴定及降解特性

为获得更为丰富的石油降解微生物资源,从沈抚污灌区石油污染土壤和实验室高浓度柴油胁迫土壤中筛选出了4株高效石油烃降解菌SF-422、SF-428、SF-433和SYS-1.这4株菌总石油烃(Total petroleum hydrocarbon/TPH)生物降解率为67.4%~73.6%.经过16项生理生化特性实验和16S rDNA序列分析鉴定,SF-433,SF-428,SF-422和SYS-1分别为蜡状芽孢杆菌(Bacillus cereus),木糖氧化无色杆菌(Achromobacter xylosoxidans),施氏假单胞菌(Pseudomonas stutzeri)和洋葱伯克霍尔德氏菌(Burkholderia cepacia).纯烃降解定性实验表明所筛选出的4株高效降解菌均能够利用正十六烷、苯、菲和环己烷为唯一碳源生长,其中菌株SF-428和SYS-1显示了对芳烃及环烷烃较强的利用能力.     

一株微生物采油菌的16S rDNA鉴定和绿色荧光蛋白分子标记

筛选到一株高效生产多糖的YX菌,油田先导试验证明该菌可以用于微生物调剖提高原油采收率（Microbial Enhanced Oil Recovery,MEOR）.经16SrDNA分析鉴定,YX菌属于肠内杆菌属（Enterobacter.）,为跟踪监测其动态信息,构建表达绿色荧光蛋白（green fluorescent protein,GFP）的重组质粒pET-30a（＋）-EGFP,利用CaCl2法将其转化至YX菌,利用荧光显微镜可以观察到重组YX（GFP）工程菌的绿色荧光.结果表明YX菌可以被绿色荧光蛋白基因标记,用于微生物采油研究.     

产絮凝剂微生物的分离与鉴定及其絮凝剂特性

从污水处理厂活性污泥中经富集、分离得到一株产絮凝剂的细菌Z-052，其发酵液对高岭土的絮凝效果达90％以上。采用16S rDNA序列分析法对该株菌进行鉴定．其核苷酸序列与Dia—phorobacter nitroreducens的同源性为98％，登录号AB076856．1．在细菌系统发育分类学上属于Diaphorobacter属。Zn^2＋离子对絮凝的促进作用效果最好。絮凝剂在100℃沸水中加热35min絮凝活性下降小于5％。Z-052絮凝剂对活性炭、土壤、酵母等固体颗粒悬液的絮凝活性分别达到95．1％、80．4％和72．4％。     

产氢发酵细菌培养基的选择和改进

高效产氢菌株的筛选与分离是生物制氢技术应用的重要基础之一，现有培养基都存在一定缺陷．为分离到高效产氢菌株，提出LM系列培养基配方，并通过产氢发酵细菌的分离和生长实验，确定了培养基的强还原剂为半胱氨酸，PH值为6．根据培养基对厌氧菌的分离数量、种类和培养基成分的分析，选择LM—1培养基为产氢发酵细菌的分离培养基．LM—1培养基分离出的5株高效产氢发酵细菌属于4个菌属，其中拟杆菌属、克雷伯氏菌属是国际上尚未分离到的高效产氢发酵细菌．利用LM—1培养基进行高效产氢细菌分离操作，得到较好的效果。     

养殖河鱼屯肠道细菌16S rDNA多样性分析

从养殖河鱼屯肠道内容物中提取细菌基因组DNA,通过PCR和TA克隆构建了细菌的16SrDNA基因文库研究肠道内容物细菌的多样性。结果表明,大部分序列通过NCBI数据库的BLAST搜索发现相似率为88%~99%,共有8个OTUs的序列相似率小于98%;鉴定出4类系统发育菌群,分别是变形菌门γ亚群(Gamma-Proteobacteria,44.8%)、CFB菌群细菌(Cytophaga-Flexibacter-Bacteroides,6.9%),厚壁菌门(Firmicutes,13.8%)、Unclassified group(34.5%),其中变形菌门γ亚群为肠道内容物中的优势细菌类群。文库多样性分析结果显示养殖河鱼屯肠道内容物中有着丰富的细菌多样性群落。     

Characterization and phylogenetics of a new species of genus LactobaciUus from the activated sludge in biohydrogen production

Anaerobic process of biohydrogen production was developed. There is a great deal of Lactobacillus bacteria in the activated sludge of biohydrogen reactor. The isolation and identification of different anaerobic bacteria in the reactor is important for fermented biohydrogen production process by anaerobic digesting organic wastewater. Considering with the physiological and biochemical traits, morphological characteristics and 16SrDNA sequence, the isolated Rennanqilyf13 is a new species in Lactobacillus genus. And the temporary no- menclature of the species is Lactobacillus Strain Rennanqilyf13 sp. nov.     

Characterization and phylogenetics of a new species of genus Lactobacillus from the activated sludge in biohydrogen production

Anaerobic process of biohydrogen production was developed. There is a great deal of Lactobacillus bacteria in the activated sludge of biohydrogen reactor. The isolation and identification of different anaerobic bacteria in the reactor is important for fermented biohydrogen production process by anaerobic digesting organic wastewater. Considering with the physiological and biochemical traits, morphological characteristics and 16SrDNA sequence, the isolated Rennanqilyfl3 is a new species in Lactobacillus genus. And the temporary nomenclature of the species is Lactobacillus Strain Rennanqilyfl3 sp. nov.     

The molecular biological characterization of a strain of biohydrogen-producing anaerobe in Clostridium Genus

The anaerobic process of biohydrogen production was developed recently. The isolation and identification of biohydrogen producing anaerobic bacteria with high evolution rate and yield is an important foundation of the fermented biohydrogen production process through which anaerobic bacteria digest organic wastewater. By considering physiological and biochemical traits, morphological characteristics and a 16S rDNA sequence, the isolated Rennanqilyf33 is shown to be a new species.     

Length polymorphisms for intergenic spacer regions of 16S-23S rDNA in members of the new hydrogen-producing bacteria

A method based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic spacer regions (ISR) was developed for the identification of species within the novel group hydrogen-producing anaerobes. The sizes of the PCR products varied from 1264 to 398 bp. Strain of isolate Rennanqilyf 3 was characterized as having products of 1262,398,638,437 and 436 bp. The isolate Rennanqilyf 1 had product of 1264 bp. The isolate Rennanqilyf 13 had products of 1261,579 and 485 bp. Of the 3 species of the novel group hydrogen-producing anaerobes examined, no one was indistinguishable. Two environmental isolates were identified as hydrogen-producing bacteria, which were new species in present taxon. Rennanqilyf 3 could not be associated with any Clostridium sp. studied. Rennanqilyf 1 could be classified into Clostridium genus. The combination between 16S rDNA equencing and length polymorphisms of IRS in 16S-23S rDNA is a better method for determining species of the hydrogen-producing bacteria.     

Isolation and identification of a sulfate reducing bacteria and sequence analysis of its dissimilatory sulfite reductase gene

A sulfate reducing bacteria was isolated from mining sewage of Daqing Oilfield by Hungate anaerobic technology. Physiological-biochemical analysis showed that the strain could utilize polyacrylamide as sole carbon and nitrogen source. The sequence analysis of 16S rDNA illustrated that the similarity of F8 and Desulfovibrio desulfuricans (AF192153) was 99%, and the similarity sequence of dissimilatory sulfite reductase gene (DSR) cloned from the strain and Desulfovibrio desulfuricans (AF273034) was 98%. Their phylogenitic analysis was basically anastomosed, and thus temporarily named as Desulfovibrio desulfuricans F8. The DSR cloned from F8 strain was 2740 bp in length consisting of three ORF, DSRA, DSRB and DSRD as a single operon (DSRABD) regulated by the same operator. DSRA contained typical conservative box of sulfate—sulfite reducing enzyme (SiteⅠand SiteⅡ), which could bind siroheme and [Fe4S4]. DSRB retained a [Fe4S4] binding site, with an uncomplimentary structure for siroheme binding. There was no conservative box in DSRD. Sequence analysis of DSR will provide a theoretical basis for quantitative detection, metabolic pathway modification through gene engineering, and sulfate reducing bacteria (SRB) suppression.     

阿尔金山盐湖极端嗜盐古生菌的分离及16S rDNA序列分析

为了研究分析新疆阿尔金山国家自然保护区阿牙克库木湖嗜盐古生菌物种资源,对分离纯化到的极端嗜盐古生菌AJ2和AJ4,采用PCR方法扩增出其16S rRNA基因(16S rDNA),并测定了基因的核苷酸序列.基于16Sr DNA序列的同源性比较和系统发育学分析,发现菌株AJ2是Natrmema属中一个与其他成员不同的新种,命名为Natrinema ajinwuensis sp.nov.;菌株AJ4是Haloarcula属中成员argentinensis的一个亚种,取名为Haloar-cula argentinensis subsp.ajinwuensis.菌株AJ2和AJ4的16S rDNA序列已被GenBank数据库收录,其序列号分别为AY208972和AY208973.     

利用16SrDNA序列分析和Biolog快速鉴定方法鉴定产脂肪酶菌株

采用16S r DNA序列分析和Biolog快速鉴定两种方法对分离得到的7株产脂肪酶菌株进行了分类鉴定.16S rDNA序列分析结果表明,7株产脂肪酶菌株中TCCC 11087、TCCC 11167、TCCC 11170、TCCC 11180,TCCC 11181属于不动杆菌属,株TCCC 11092、TCCC 11168属于假单胞菌属.Biolog快速鉴定结果显示,TCCC 11087、TCCC 11167、TCCC 11170.TCCC 11180、TCCC 11181 属于溶血不动杆菌,TCCC 11092属于假单胞菌属,TCCC 11168属于荧光假单胞菌.说明两种鉴定方法得到的结果一致,但16S rDNA序列分析方法只能鉴定到属,而Bioiog快速鉴定方法能对多数菌株鉴定到种.     

一株耐盐菌EC51的生长特性及其系统发育分析

从大连市普兰店盐场盐池底部的淤泥中筛选出一株耐盐菌EC51,研究了分离菌株的形态和生长特性,该菌株为革兰氏阳性杆状菌.以其16S rDNA 序列相似性为基础构建系统发育树.结果表明,EC51与Bacillus Pumilus的相似性达到99%.耐盐菌EC51的渗透压补偿溶质（Ectoine）生成情况实验表明,用含2 mol/L NaCl的肉汤培养基,在30 ℃条件下培养48 h,诱导生成Ectoine为197.8 mg/L.