DNA extraction method affects microbial community profiles from soils and sediment

To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles generated. This was reflected both in the number of bands or ribotypes detected from each sample and in subsequent principle coordinate analysis and unweighted-pair group method using arithmetic average analyses. The methods also differed significantly in their robustness, i.e. reproducibility across multiple analyses. Two methods, both based on bead beating, were demonstrated to be suitable for comparative studies of a range of soil and sediment types. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

重金属污染土壤中微生物的多样性

<正>土壤重金属污染是指人类活动将重金属混入到土壤中,致使土壤中重金属含量明显高于原有含量,并造成生态环境质量恶化的现象[1]。污染土壤的重金属主要包括汞(Hg)、镉(Cd)、铅(Pb)、铬(Cr)和类金属砷(As)等生物毒性显著的元素,以及有一定毒性的锌(Zn)、铜(Cu)、镍(Ni)等元素。除了来自农药、废水、污泥和     

盐碱地微生物类群的多样性

土壤是微生物的大本营,由于土壤理化性质的不同,与环境相适应的土壤微生物种类也各不相同。土壤盐碱化是一个世界性的难题,在我国主要分布在淮河以北、西北及新疆、青藏高原等内陆干旱、半干旱地区的河流冲积平原、盆地和湖泊沼泽地区。     

云南腾冲热海两热泉菌藻席细菌多样性的研究

应用显微形态观察和变性梯度凝胶电泳(DGGE)对云南腾冲热海两热泉菌藻席的细菌多样性进行了比较分析.直接从环境样品中提取总DNA,用两套细菌通用引物进行PCR扩增,得到包含V8和V9高变区的16S rDNA片段,进行DGGE分析,结合形态观察,结果显示,热泉菌藻席中存在丰富的细菌多样性,且不同温度范围的菌藻席细菌组成差异显著.     

应用DGGE研究微生物群落时的常见问题分析

变性梯度凝胶电泳(DGGE)是通过核酸片段对微生物群落进行研究,可以监测未培养细菌及其功能基因,被广泛地应用于微生物群落多样性和动态分析,并成为微生物分子生态学研究中的重要手段之一。文中论述了DGGE操作过程中遇到的常见问题,并提出了相应的解决方法。全面分析了样品预处理过程和PCR扩增效果对DGGE分析的影响,探讨了DGGE图谱的优化过程和图谱分析方法,并对DGGE的应用前景进行了综述。     

Wildfire effects on microbial biomass and diversity in pine forests at three topographic positions

The aim of this study was to determine microbial biomass carbon and diversity at three topographic positions (ridge, middle slope and valley bottom) at varying periods after the occurrence of wildfire. The purpose of this study was to enhance knowledge on the microbiological status of post-fire sites for a better estimate of the success of their restoration. Results showed that the biomass carbon and microbial diversity were affected by fire: the averages of the burnt plots were <50% of those of the unburnt plots. In addition, the biomass carbon at the sites studied 3 years after and 25 years after fire, and 5 months after and 9 years after fire, were not significantly different from each other. On the other hand, the site studied 5 months after fire showed the highest microbial diversity, followed by the ones studied 3, 25, 9 and 6 years after fire. In terms of topographic position, the ridge was shown to be the most affected by fire. These results show that fire can adversely affect the microbial biomass carbon and microbial diversity of the soil, the recovery of which may take a long time.     

新疆一号冰川土壤细菌多样性的研究

应用变性梯度凝胶电泳（DGGE）技术分离PCR扩增的16SrDNA来研究土壤微生物的多样性。直接从新疆一号冰川不同海拔高度的土壤样品中提取总DNA。用两套细菌通用引物分别扩增16SrDNA的V3和V6／V9高变区的特异性片段,PCR产物进行DGGE分析。PCR—DGGE图谱表明,PCR产物经DGGE检测到的条带清晰且分离效果好。结果表明,PCR—DGGE是一种快速研究微生物群落结构的有效方法。     

DGGE/TGGE技术及其在微生物分子生态学中的应用

变性梯度凝胶电泳(DGGE)和温度梯度凝胶电泳(TGGE)是近些年微生物分子生态学研究中的热点技术之一。由于DGGE／TGGE技术具有可靠性强、重现性高、方便快捷等优点,被广泛地应用于微生物群落多样性和动态性分析。文章对DGGE／TGGE技术原理与关键环节、局限性和应用前景进行了综述。     

德兴铜矿尾矿重金属污染对土壤中微生物多样性的影响

【目的】为更好地了解重金属污染与微生物多样性之间的相互作用关系,以江西德兴铜矿4#尾砂库为研究对象,采集野外实地样品共16件进行分析(包括尾砂样品以及周围农田和菜地土壤样品)。【方法】一方面对样品中可培养异养细菌进行平板计数,一方面采用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)对样品中可培养和不可培养微生物分子生态多样性进行研究;同时采用PCA(Principle component analysis)方法分析样品理化性质、重金属及主要元素与可培养细菌数量及微生物多样性之间的相互关系。【结果】元素分析结果表明该尾矿区样品受到不同程度重金属Cu、Cd、Zn、Ni、Pb和Cr的污染;可培养异养细菌在尾砂样品中数量最少,在菜地和农田土壤样品中有明显增加;多样性指数(Shannon-Weaver index H)计算结果发现H最大值出现在距离尾矿中等距离、重金属浓度在中等程度的样品中。PCA分析结果表明可培养异养菌数量与理化性质如有机碳、有机质、含水率等相关性较大,重金属影响不明显;而多样性指数H除与上述理化性质相关性较大外,还受到重金属Ag、Zn、As、Pb、Ni、Cr等的影响,而在样品中含量普遍比较高的重金属如Cu、Cd等并不成为影响微生物多样性的主要因素。【结论】从这些长期受重金属污染的野外实地样品来看,以上结果说明不同重金属浓度对微生物多样性的影响可能并不是实验室研究的简单的线性关系。     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study,PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu,Lake Mochou in Nanjing and Lake Taihu in Wuxi.Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted.The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g),while that of sediments in Lake Taihu was relatively low.After DNA purification,the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE.The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments.The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of micro-organisms were identified in the sediment samples of both lakes.These results suggest that the sediment samples of these two city lakes (Xuanwu,Mochou) have similar microbial communities.However,the DGGE profiles of sediment samples in Lake Taihu were significantly differ-ent from these two lakes.Furthermore,the DGGE pro-files of sediment samples in different locations in Lake Taihu were also different,suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou.The differences in microbial diversity may be caused by the different environmental conditions,such as redox potential,pH,and the concentrations of organic matters.Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced.The results of sequencing analysis indicate that five sequences shared 99%-100% homology with known sequences (Bacillus and Brevibacillus,uncultured bacteria),while the other two sequences shared 93%-96% homology with known sequences (Acinetobacter,and Bacillus).The study shows that the PCR-DGGE tech-nique combined with sequence analysis is a feasible and efficient method for the determination of microbial com-munities in sediment samples.     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu, Lake Mochou in Nanjing and Lake Taihu in Wuxi. Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted. The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g), while that of sediments in Lake Taihu was relatively low. After DNA purification, the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE. The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments. The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of microorganisms were identified in the sediment samples of both lakes. These results suggest that the sediment samples of these two city lakes (Xuanwu, Mochou) have similar microbial communities. However, the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes. Furthermore, the DGGE profiles of sediment samples in different locations in Lake Taihu were also different, suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou. The differences in microbial diversity may be caused by the different environmental conditions, such as redox potential, pH, and the concentrations of organic matters. Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced. The results of sequencing analysis indicate that five sequences shared 99%–100% homology with known sequences (Bacillus and Brevibacillus, uncultured bacteria), while the other two sequences shared 93%–96% homology with known sequences (Acinetobacter, and Bacillus). The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples. __________ Translated from Acta Ecologica Sinica, 2006, 26(11): 3610–3616 [译自: 生态学报]     

Analysis of microbial diversity in oligotrophic microbial fuel cells using 16S rDNA sequences

Molecular ecological techniques were applied to analyze the bacterial diversity of two oligotrophic microbial fuel cells (MFCs) enriched using river water or artificial wastewater (AWW) as fuel. Denaturing gradient gel electrophoresis (DGGE) of the PCR amplified 16S rDNA showed that different microbial communities were present in the two MFCs and these were different from the river sediment used to initiate the enrichment. Nearly complete 16S rDNA was amplified and sequenced. Over 80% of the clones were Proteobacteria. Betaproteobacteria were the dominant clones (46.2%) in MFCs fed with river water, and about 64.4% of the clones in MFCs fed with AWW were Alphaproteobacteria. Actinobacteria were found only in the MFC fed with AWW, and Deltaproteobacteria, Acidobacteria, Chloroflexi and Verrucomicrobia in the MFC fed with river water. Many clones were related to uncultured bacteria, some with homology less than 95%, indicating that many novel bacteria were enriched in the oligotrophic MFCs.     

Microbial structural diversity estimated by dilution-extinction of phenotypic traits and T-RFLP analysis along a land-use intensification gradient

The present work tested whether the relationship between functional traits and inoculum density reflected structural diversity in bacterial communities from a land-use intensification gradient applying a mathematical model. Terminal restriction fragment length polymorphism (T-RFLP) analysis was also performed to provide an independent assessment of species richness. Successive 10-fold dilutions of a soil suspension were inoculated onto Biolog GN(R) microplates. Soil bacterial density was determined by total cell and plate counts. The relationship between phenotypic traits and inoculum density fit the model, allowing the estimation of maximal phenotypic potential (Rmax) and inoculum density (KI) at which Rmax will be half-reduced. Though Rmax decreased with time elapsed since clearing of native vegetation, KI remained high in two of the disturbed sites. The genetic pool of bacterial community did not experience a significant reduction, but the active fraction responding in the Biolog assay was adversely affected, suggesting a reduction in the functional potential.     

变性梯度凝胶电泳在微生物生态学中的应用

变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)是目前在微生物生态学上应用比较广泛的技术之一,具有简便、准确可靠和重复性好等优点。对DGGE的原理、流程、各项技术要点和在微生物生态学上的应用等方面进行了详细地论述,同时归纳和总结了DGGE的优缺点和局限性。     

温泉微生物多样性与酶类分析

温泉微生物多样性包括物种多样性、遗传多样性、生理多样性和生态多样性。温泉微生物多样性受地理位置、温度和酸碱度、水化学成分(硫、硼、铁、砷等离子和化合物)及氧气和光照等生态因子的影响。目前已经从温泉微生物中筛选出了许多具有热稳定性的酶。本文分析了温泉微生物多样性及其影响因素,并讨论了具有广泛应用价值的温泉微生物酶,为拓展我国温泉微生物多样性研究、深入认识和开发温泉微生物资源提供基础。     

Microbial community composition and functional diversity in the rhizosphere of maize

This study investigates the small-scale stratification of bacterial community composition and functional diversity in the rhizosphere of maize. Maize seedlings were grown in a microcosm with a horizontal mesh (53 M) creating a planar root mat and rhizosphere soil. An unplanted microcosm served as control. Thin slices of soil were cut at different distances from the mesh surface (0.2–5.0 mm) and analysed for bacterial community composition by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) of 16S rDNA and tested for activities of different enzymes involved in C, N, P and S cycling. Bacterial community composition and microbial functional diversity were affected by the presence of the maize roots. The bacterial composition showed a clear gradient up to 2.2 mm from the root surface, while no such gradient was observed in the unplanted pot. Invertase and phosphatase activities were higher in the close vicinity of maize roots (0.2–0.8 mm), whereas xylanase activity was unaffected. This study shows that the changes in bacterial community composition and functional diversity induced by roots may extend several millimetres into the soil.     

气调包装生鲜冷却牛肉贮藏中微生物多样性分析

【目的】分析普通包装和气调包装(65%O2和35%CO2)生鲜冷却牛肉在贮藏(4°C)过程中的微生物多样性。【方法】通过16S rDNA V3区PCR-DGGE(变性梯度凝胶电泳)方法和16S rDNA克隆分析法研究生鲜冷却牛肉中微生物菌落结构及菌相变化规律。【结果】初始菌相主要有嗜冷杆菌属(Psychrobacter)和假单胞菌属(Pseudomonas)。贮藏过程中,普通包装和气调包装生鲜冷却牛肉中优势菌均为Brochothrix和Pseudomonas。气调包装冷却牛肉中细菌种类较少,两种包装生鲜牛肉在贮藏前期菌相变化明显。【结论】不同包装冷却牛肉中微生物菌落结构有较大差异,气调包装中CO2对Pseudomonas等细菌起到了一定的抑制作用。     

Efficiency of DNA extraction methods on the evaluation of soil microeukaryotic diversity

The evaluation of microbial molecular diversity has been mainly based on the extraction of total DNA from environmental samples. The indirect extraction methods, which have been used for prokaryotes, have never been used to recover soil microeukaryotic DNA. We evaluated the efficiency of an improved indirect DNA extraction protocol developed herein and the direct lysis (the sodium dodecyl sulfate (SDS)-based method and commercial DNA extraction kit) on estimating the molecular diversity of soil microbial eukaryotes. DNA quality and quantity as well as denaturing gradient gel electrophoresis (DGGE) profiles were determined using three soil samples from different stations. The indirect method detected the highest DGGE bands in spite of the low DNA yield. The commercial kit detected a lower number of DGGE bands than the indirect method. The SDS-based method produced the lowest DGGE bands and DNA purity but the highest yield. Using the indirect method, we further evaluated the effect of freezing and air-dried preservations on estimating the microeukaryotic diversity. In spite of the low DNA yield obtained from the air-dried preservation, no significant differences were found in either the number of DGGE bands or the DNA purity between two manners. Our results indicate that the improved indirect method could obtain a high purity of intracellular DNA and high efficiency in the estimation of molecular diversity of soil microbial eukaryotes.