紫花苜蓿根际氢氧化细菌的分离与鉴定

利用气体循环培养体系从陕西乾县HUP-豆科植物紫花苜蓿(Medicago sativa)根际土壤中分离获得37株细菌.菌株氧化氢能力测定结果表明,8株菌氧化氢和自养生长能力较强,初步确定为氢氧化细菌类群;根据其形态特征、培养特征和生理生化特性,鉴定为7个不同属:假单胞菌属(Pseudomonas)、邻单胞菌属(Plesiomonas)、脂肪杆菌属(Pimelobacter)、黄色杆菌属(Xanthobacter)、勒米诺氏菌属(Leminorella)、地杆菌属(Terrabacter)和稀有杆菌属(Rarobacter);其中氧化氢能力最强的优势菌株WMQ-7 16S rDNA序列(GenBank登录号为EU807744)长度为1 451bp,GC含量为53.8%,其核苷酸序列与假单胞菌属同源性高于99%,在系统发育树上位于同一分支,将WMQ-7菌株鉴定为假单胞菌属(Pseudomonas).图2表5参16     

不同生境产脂肪酶菌株的筛选及多样性

为发掘不同特性脂肪酶微生物资源,采用纯培养方法从青海高寒草地、雅安山区、成都平原分离得到产脂肪酶菌株54株.通过对菌株的DNA进行ERIC-PCR指纹图谱分析,按聚类树划分为5个操作分类单元.16S rDNA序列测定和聚类分析显示,分离菌株分布于芽孢杆菌属(Bacillus)、克雷伯氏菌属(Klebsiella)、假单胞菌属(Pseudomonas)、葡萄球菌属(Staphylococcus)、肠杆菌属(Enterobacter).多样性分析表明,与青海高寒草地、雅安山区相比,成都平原产脂肪酶菌株遗传多样性更为丰富.在54株菌中都出现了300 bp和1 300 bp两个特异且稳定出现的条带,这两个特异条带可能是产脂肪酶菌株的SCAR标记条带.     

石油降解菌株G5 16S rDNA全序列的系统学分析

用PCR方法扩增了一株石油降解菌株G5的16S rRNA基因全序列，并对其进行了克隆和测序．对该序列在GenBank中的BLAST结果表明，所有与该序列高度同源的序列都是假单胞菌的16S rRNA基因．其中假单胞菌的代表菌株Pseudomonas aeruginosa，P．fluoroscens，P .putida，P.syringae的16S rRNA基因序列与G5的16S rRNA基因序列同源性分别为93．4％，98．4％，96．3％，97．5％．对G5和其他39株假单胞菌的16S rRNA基因序列进行聚类分析，获得的系统发育树与RDP(Ribosomal Database Project)报道的系统发育树基本一致，其中菌株G5与5株P．chlororaphis聚类在一起．图2参7     

深海沉积物宏基因组文库中产蛋白酶克隆的筛选及性质分析

提取东太平洋深海沉积物宏基因组DNA,构建了未培养微生物宏基因组文库.该文库平均插入片段30 kb以上,含有7 000个克隆,含外源DNA总长度约为210 Mb.从文库中筛选到18个产胞外蛋白酶的克隆,16S rRNA分析表明,它们分别来源于假单胞菌(Pseudomonas)、寡养单胞菌(Stenotrophomonas)和弧菌(Vibrio)3个菌属.根据活性大小及来源菌株的16S rRNA序列比较,选择10个蛋白酶进行酶学性质分析,结果表明,它们的最适作用pH值在7～10之间,最适作用温度在10～40℃之间.其中Pro20蛋白酶最适作用温度最低,为10℃;Pro1蛋白酶具有最适作用温度低(20℃)、pH耐受性好、热稳定性较好等特性,具有良好的应用开发潜力.图6参16     

土壤甲胺磷抗性细菌种群特征脂肪酸生物标记的分析（Characteristics of PLFA Biomarkers for Acephatemet Resistant Bacteria Isolated from the Soils）

应用高浓度甲胺磷培养基,对农药厂排污口土壤微生物进行分离,鉴定出21株细菌,分属13个细菌属.对甲胺磷抗性细菌脂肪酸的分析,共测定到38个脂肪酸生物标记(PLFA),这些生物标记分为4种类型,即1)高频次分布的生物标记,普遍存在于细菌类群,属于细菌总体类群(Bacteriaingeneral)的生物标记;2)中频次分布的生物标记,在细菌种出现概率中等,可以用于代表细菌属类群(Bacteriumgenus)识别生物标记;3)低频次分布的生物标记,在细菌中的分布概率较小,可以用于指示特定细菌种间差异的生物标记;4)微频次分布的生物标记,这种生物标记仅在一种细菌种类出现,是细菌种特征生物标记.利用脂肪酸生物标记分析同属细菌不同种的差异,可将假单胞杆菌属分为2类,第1类包括了铜绿假单胞菌、荧光假单胞菌、丁香假单胞菌,其特征为17:0CYCLO生物标记含量小于3.60%;第2类包含了伞菌假单胞菌、威隆假单胞菌、恶臭假单胞菌、温哥华假单胞菌,其特征为17:0CYCLO生物标记含量大于5.99%.利用脂肪酸生物标记的差异对甲胺磷抗性细菌种的聚类分析,能有效地将细菌类群分为3类,微生物群落中存在着稳定的生物标记和受环境影响的生物标记,论文提供了一种脂肪酸生物标记的分析方法,结合细菌的生物学特性研究,可以解释微生物在环境中的变化,对于土壤微生物群落的研究具有重要意义.     

大庆油田聚合物驱后油藏微生物多样性研究

从大庆油田4个聚合物驱后的采油井中提取总DNA,扩增其16S rDNA片段,克隆到大肠杆菌,然后用ARDRA法对所得的298个克隆中的插入片段进行分析,建立了16S rDNA克隆文库.采用文库库容值(C)、香农-威纳指数(H)和均匀度指数(E)对文库微生物多样性进行了评价.结果表明,4个样品中共得出22个操作分类单元(OUT).其中4个OUT(在整个群落中占78.18%,9个OUT只有一个克隆.对22个OUT的代表克隆序列、GenBank数据库比对和系统发育分析表明,聚驱后油井中的细菌基本属于α变形菌纲(24.83%)、γ变形菌纲(23.49%)、δ变形菌纲(35.56%)和未培养微生物(16.78%).与石油烃降解和产生物表面活性剂相关的假单胞菌(Pseudomonas)和不动杆菌(Acinetobacter),在文库中占的比例分别是17.79%和6.02%.     

黄河三角洲湿地土壤微生物群落结构分析

采用末端限制性片段长度多态性分析(T-RFLP)技术和16S rDNA克隆文库的方法,分析了黄河三角洲滨海湿地土壤不同深度细菌和古菌的群落结构.研究表明,随着深度的增加,细菌群落的多样性下降,而古菌群落多样性则有上升的趋势,且土壤的细菌和古菌群落结构都呈现出规律的层状分布.该土壤包括各种硫酸盐还原菌、产甲烷古菌、光合细菌等丰富的细菌和古菌资源.图5参27     

34株假单胞菌的泛基因组分析

假单胞菌属在各种环境广泛分布且具有重要的生态应用价值,然而当前研究对其环境适应性遗传基础的解析尚不充分.选择33株有效发表且具有全基因组序列以及一株新分离的假单胞菌的全基因组进行泛基因组学分析.结果表明,选择的假单胞菌属菌株泛基因组含有173 271条蛋白序列,共有30 847个同源基因家族,包括1 505个核心基因,基因组呈开放型.通过分析不同基因组的基因组演化特征,发现通过基因水平转移获得的基因占整个基因组的比例为32%-48%,其平均基因岛数量为53个.发现假单胞菌属泛基因组的开发性与其频繁的基因水平转移事件和快速的基因进化有关.进一步分析表明,不同基因组中的功能基因家族按照菌株的环境分布进行聚类,表明对特殊环境的适应决定了假单胞菌的遗传特征.本研究初步揭示了假单胞菌属细菌遗传特征的共性与特性,结果可为解析假单胞菌属环境适应性的遗传基础与功能多样性的演化发生机制提供研究线索.(图8表1参37)     

耐盐异养硝化菌驯化方法及分离菌株鉴定

采用高C/N、逐渐降低DO、菌体对数生长期前期转接以及增加培养液中海水比例的方法完成了耐盐异养硝化菌的富集和驯化,并从中分离筛选出两株高效异养硝化菌qy37和gs2.通过对两株菌的形态观察、生理生化试验以及16S rRNA序列分析,确定菌株qy37和菌株gs2分别为假单胞菌属(Pseudomonas)和盐单胞菌属(Halomonas).     

四川盆地大豆根瘤内生细菌的分离鉴定及促生效果

以四川盆地大豆根瘤为材料,采用划线法分离内生细菌、16S rDNA PCR-RFLP分析其遗传多样性,并结合菌株促生特性和盆栽试验筛选优良促生菌.从分离获得的130株内生细菌中选取了40株细菌作为供试菌株,16S rDNA序列表明分属于芽孢杆菌属(Bacillus)、肠杆菌属(Enterobacter)、假单胞菌属(Pseudomonas)、中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium).以大豆为供试作物筛选出了12株具有促生能力的细菌,所有菌株均能分泌吲哚-3-乙酸(IAA),浓度达到0.353-32.404μg/mL;7株能产铁载体,活性单位为7.35%-34.31%;有11株具有溶磷能力,溶磷量达到4.26-10.6μg/mL;6株具有固氮能力.接种12株供试菌株后,玉米的农艺性状、植株全氮和全磷含量均优于单施化肥处理,其中菌株DA16-5效果最好,表现出良好的促生潜力.综上,四川盆地大豆根瘤内生菌遗传多样性丰富并且普遍具有促生能力,是重要的生物资源.     

Spatial characteristics analysis by PCR-DGGE for bacterial communities in surface sediments of Tangxi River,Hefei City北大核心CSCD

Microbes play an important role in material circulation and pollutant release in urban sediments, and its community structure can provide an important basis for evaluating the pollution load and ecosystem health of an urban river. In this study, bacterial communities in sediments from different locations of Tangxi River in Anhui Province were analyzed and compared, with an objective of evaluating the effects of aquatic environment on spatial characteristics of bacterial communities and the feasibility of using bacterial community composition as an indicator of urban river health. PCR-DGGE was applied to analyze the bacterial communities; fifteen major bands of 16S rDNA genes fragments from DGGE profiles of sediment samples were further eluted from gel, reamplified and sequenced. The sequences of these fragments were compared with the database in GenBank (NCBI). The collected samples were clustered based on UPGM Aanalysis. In addition, relationships between bacterial communities and environmental factors were analyzed by Monte Carlo test and redundancy analysis (RDA). The DGGE profiles indicated that upstream surface sediments had about 30 bands, but the differences in brightness were not significant. Although midstream surface sediments had only 18 bands on average, the brightness of some bands was relatively high, indicating that some dominant species of bacteria existed in these sediments. The amounts of bands in downstream sediments were between those of upstream and midstream, with some bands of high brightness. The NCBI comparison results showed that ten sequences shared 98-100% homology with known sequences, one with 97%, and the other four with uncultured bacteria. Shannon index (H) of bacterial diversity from upstream surface sediments was 3.31 on average, which was significantly (P < 0.05) higher than that of midstream surface sediments, and slightly higher than that of downstream surface sediments. UPGMA results showed both the distribution of bacteria communities and the diversity were strongly related with the sampling locations (Eigenvalue = 0.188, P = 0.134). Monte Carlo test and RDA analysis showed that the ion exchangeable form of nitrogen (IEF-N) was the main factor influencing the spatial characteristics of bacterial communities in the sediments. The aquatic plants had a significant effect on the richness of bacterial communities in the sediments, and the effectiveness of plant species was more notable than that of the vegetation coverage. The above results indicated that bacterial community structure and diversity in the surface sediments can well reflect the degree of urbanization development and effectiveness of ecological restoration in Tangxi River region.     

化学-生物絮凝污水处理工艺中微生物群落结构变化分析

利用分子生物学技术，直接从化学-生物絮凝工艺的活性污泥样品中提取DNA，对16S rDNA V3区进行PCR扩增，结合DGGE（变性浓度梯度凝胶电泳），分析了活性污泥中微生物群落结构，并对Shannon多样性指数进行分析讨论，通过研究指出系统中细菌数量的增加或减少．测定了活性污泥中部分菌种的16S rDNA V3区片段序列，通过NCBI（美国国立生物技术信息中心）基因库比对，初步确定细菌的属．结果表明，PCR—DGGE结合测序技术是一种完全可行的快速进行环境样品微生物研究的分析方法．图3表4参13     

Modified dehydrogenase enzyme assay for evaluation of the influence of Hg,Cd, and Zn on the bacterial community structure of a wastewater treatment plant

In this study, a traditional assay was modified to evaluate the effect of Hg, Cd, and Zn on the bacterial community of a sequencing batch reactor and activated sludge plants and heavy metal-resistant bacterial species were determined. After the isolation of metal-resistant bacteria, their 16S rRNA gene fragments were sequenced. The BLAST program was used to compare the resulting 16S rRNA sequences with those in GenBank database to identify the isolated bacterial species. Hg was found to be the most toxic metal for both bacterial communities investigated. Sequence batch reactor bacteria were comparatively more resistant to Hg, Cd, and Zn than those from activated sludge. The resistant strains were close to the members of genus Pseudomonas, Kocuria, Stenotrophomonas, Enterococcus, and Staphylococcus. The modified dehydrogenase enzyme assay seems to be simple, robust, and competent for evaluation of the impact of metals on bacterial activity. Sequencing batch reactor systems should be preferred over activated sludge when wastewaters containing hazardous metals are to be treated.     

Monitoring microbial diversity and natural product profiles of the sponge<Emphasis Type="Italic"> Aplysina cavernicola</Emphasis> following transplantation

In order to assess the stability of the microbial community of the sponge Aplysina cavernicola under in situ conditions, sponges were transplanted from their original location (>40 m depth) to shallower, more light-exposed sites (7-15 m depth). Transmission electron microscopy revealed that the microbial community remained visually unchanged and free of cyanobacteria over the experimental time period of 3 months. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA gene sequences allowed a distinction between the variable and permanent fraction of the bacterial community. Comparative sequence analysis of four variable DGGE bands revealed high sequence similarity to representatives of the Alpha- and Gammaproteobacteria and the phylum Bacteroidetes, which have been recovered previously from Mediterranean seawater as clone sequences or by cultivation. Seven (out of 12) permanent DGGE bands showed high sequence similarity to a sponge-specific, monophyletic 16S rRNA gene sequence cluster within the Acidobacteria division, and to a sequence cluster of uncertain affiliation. These sequence clusters represent members of a common microbial community that is shared among distantly related sponges from different, non-overlapping geographic regions. Four additional permanent DGGE bands showed high sequence similarity to a Betaproteobacterium, Burkholderia cepacia, which is not typically known as a marine bacterium. High-performance liquid chromatography analyses of sponge tissues revealed no changes in metabolite pattern, indicating that these compounds are expressed constitutively irrespective of the variations resulting from the transplantation experiment.     

Comparison on bacterial community of rhizosphere and bulk soil of poplar plantation based on pyrosequencing北大核心CSCD

By the 454 pyrosequencing technology, this research compared the bacterial communities in poplar plantation rhizosphere and bulk soil for an accurate understanding of bacterial community colonization in the two soil environments. The species annotation showed that rhizosphere soil contained 145 bacterial genera and bulk soil contained 141 bacterial genera, with 8 common genera shared by both at a relative abundance of more than 4%. The 8 genera in common were Acidobacterium GP1, Acidobacterium GP3, Acidobacterium GP6, Gemmatimonas, Bradyrhizobium, Burkholderia, Streptomyces and Acidobacterium GP4. The relative abundance of the same bacterial community was significantly different between rhizosphere and bulk soil environments. Alpha diversity analysis showed that the bacterial community diversity of rhizosphere soil was higher than that of bulk soil, but the difference was not significant. The results of bacterial communities sorting could reflect the variation of soil bacterial communities from rhizosphere to the bulk and the spatial variation among different sampling points, indicating a contribution of about 21.2% variance of bacterial communities by the effect of rhizosphere. Beta diversity analysis showed great difference between rhizosphere and bulk soil samples in bacterial community composition. There were 15 genera specific to rhizosphere soil and 11 to bulk soil. The abundance of 23 genera, mainly cellulose degrading bacteria and nitrogen-fixing bacteria, changed significantly. Selectivity of root to rhizosphere microorganisms is an important mechanism leading to significant differences in the rhizosphere microbial community composition and structure, which may significantly impact the carbon and nitrogen cycles of the root-soil interface.     

Biogeography of bacterioplankton in lakes and streams of an Arctic tundra catchment

Bacterioplankton community composition was compared across 10 lakes and 14 streams within the catchment of Toolik Lake, a tundra lake in Arctic Alaska, during seven surveys conducted over three years using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified rDNA. Bacterioplankton communities in streams draining tundra were very different than those in streams draining lakes. Communities in streams draining lakes were similar to communities in lakes. In a connected series of lakes and streams, the stream communities changed with distance from the upstream lake and with changes in water chemistry, suggesting inoculation and dilution with bacteria from soil waters or hyporheic zones. In the same system, lakes shared similar bacterioplankton communities (78% similar) that shifted gradually down the catchment. In contrast, unconnected lakes contained somewhat different communities (67% similar). We found evidence that dispersal influences bacterioplankton communities via advection and dilution (mass effects) in streams, and via inoculation and subsequent growth in lakes. The spatial pattern of bacterioplankton community composition was strongly influenced by interactions among soil water, stream, and lake environments. Our results reveal large differences in lake-specific and stream-specific bacterial community composition over restricted spatial scales (<10 km) and suggest that geographic distance and connectivity influence the distribution of bacterioplankton communities across a landscape.     

云南玉溪烟区轮作与连作土壤细菌群落多样性比较研究

为评价云南玉溪烟区轮作和连作对土壤细菌群落多样性影响,从土壤中直接提取土壤细菌总DNA,用细菌16S rDNA特异性引物进行PCR扩增和变性梯度凝胶电泳(DGGE),每个处理样品3次重复,在DGGE图谱中相似性较高,基本聚在一起,从整体证明了试验操作方法较为精确.从泳道分析图可知,轮作土壤细菌群落各泳道条带数量13～29条不等,平均为21条.连作土壤细菌群落各泳道条带数量从4～20条不等,平均为12条.从每条泳道的条带数方面和光密度值分别进行细菌群落多样性各指标的比较,结果表明,轮作处理细菌群落丰富度指数均大于连作处理,轮作香农-威纳和辛普森指数(1/D)均大于连作,表明轮作微生物多样性较连作高,轮作方式可以提高植烟土壤细菌群落的多样性.     

长链烷烃降解菌alkB片段的分离和鉴定

分离并鉴定了长链烷烃降解菌Pseudomonasaeruginosa1785和P.marginata766烃羟化酶基因alkB片段.根据烃羟化酶的保守氨基酸序列,设计兼并引物,扩增P.aeruginosa1785和P.marginata766的alkB片段,获得了目标产物.经DNA测序和氨基酸序列分析,证实目标片段编码的肽段含有烃羟化酶的特征基序.由此确认采用该方法分离到了长链烷烃降解基因的alkB同源体片段.DNA序列比对结果表明,P.aeruginosa1785和P.marginata766的alkB片段与P.aeruginosaPAO1的alkB1和alkB2的相似性分别达到95.7%和94.8%.这些alkB片段可用于分析烃降解微生物群落结构.图3表2参11     

厌氧发酵过程pH对微生物多样性和产物分布的影响

采用PCR-DGGE技术,研究了不同pH条件下蔬菜类有机垃圾厌氧发酵过程中的微生物多样性,探讨了微生物群落结构与发酵产物分布的关系.Shannon指数分析表明,pH=7和pH=8时的微生物多样性较高,随时间变化规律相似,而pH=5时的微生物多样性较低.UPGMA聚类分析和PCA分析结果也表明,pH=7和pH=8时的微生物群落结构相似,pH=5与之显著不同.在不同pH条件下的优势菌属都是乳酸细菌和梭菌。微生物多样性的变化与发酵产物分布具有一定相关性,pH通过影响微生物群落结构的变化最终影响发酵产物的分布.图4表2参27     

Quantification of polyphenols during retting and characterization of bacteria from the Kadinamkulam Backwaters, Kerala

The retting environment which provides a competitive niche for specialized microbes is speculated to harbour a variety of microbes with high biodegradation potential. In this context, an effort has been made to isolate and identify bacterial species having high tolerance to phenol In vitro. Maximum polyphenol (1.897 mg l(-1)) as observed during the initial period of retting, which decreased as retting proceeded. Based on biochemical characterization, the isolated bacterial strains were identified as Micrococcus sp., Moraxella sp. strain MP1, Moraxella sp. strain MP2 and Moraxella sp. strain MP3, Pseudomonas sp. strain PP1 and Pseudomonas sp. strain PP2, Amphibacillus sp., Brucella sp. strain BP1 and Brucella sp. strain BP2, Aquaspirillum sp., Escherichia coli strain EP1 and Escherichia coli strain EP2, Campylobacter sp., Aeromonas sp., Neisseria sp., Vibrio sp., Erwinia sp. and Mesophilobacter sp. These strains were found to tolerate maximum concentration of phenol viz. 200 to 1000 mg l(-1). Plasmid analysis of phenol resistant bacterial isolates showed that almost all the cultures had at least one plasmid of size > 1Kb. Studies on the protein profile of isolated bacterial cultures showed the presence of proteins with molecular sizes ranging from 10 to 85 KDa with exception of Mesophilobacter and Neisseria having still high molecular weight protein (95 KDa). Bacterial strains isolated from coir-ret-liquor showed tolerance to high phenol concentration.