Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments

The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter （OM）, total nitrogen （TN）, total phosphorus （TP）, pH and redox potential （Eh）. Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341 f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis （DGGE）. The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. and some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core.

Variations in Laboratory-Scale Actinomycete Communities Exposed to Cadmium as Assessed by Denaturing Gradient Gel Electrophoresis Profiles

The actinomycete populations and functions in cadmium (Cd) contaminated soil were investigated by the cultivationindependent molecular methods.The genomic DNA was extracted and purified from soil adulterated with various concentrations of Cd in the laboratory.The partial 16S rDNA genes were amplified by polymerase chain reaction (PCR) using specific primers bound to evolutionarily conserved regions within these actinomycete genes.The diversity in PCRamplified products,as measured by denaturing gradient gel electrophoresis (EGGE),was used as a genetic fingerprint of the population.Principle component analysis and Shannon-Weaver diversity index (H) analyses were used to analyze the DGGE results.Results showed that the two principal components accounted for only a low level of the total variance.The value H in contaminated soil was lower than that in the control at later stages of cultivation,whereas at earlier stages it was higher.Among the six sampling time points,the first,fifth and sixth weeks had the highest values of H.Significantly negative correlations between bioavailable Cd concentration and H values existed in the samples from weeks 2 (R=0.929,P < 0.05) and 4 (R=0.909,P < 0.05).These results may shed light on the effect of Cd on the soil environment and the chemical behavior and toxicity of Cd to actinomycetes.

Plantations of native shrub species restore soil microbial diversity in the Horqin Sandy Land, northeastern China

Caragana microphylla Lam., a leguminous shrub species, plays an important role in revegetation in the degraded ecosystems of the Horqin Sandy Land, Northeastern China. Large areas planted with this shrub have been artificially established as sand binders for soil protection, which might change the composition of soil bacterial communities with the development of sand dune stabilization. In this paper, we investigated the diversity and composition of native soil bacterial communities in the C. microphylla plantation for sand fixation using polymerase chain reaction with denaturing gradient gel electrophoresis（PCR-DGGE） to understand the influence of this plantation on sandy soil ecosystem development. We collected soil samples from plantations with an age sequence of 0, 9, 16, and 26 years, as well as from the natural community, to identify the differences among soil bacterial communities. The result showed that bacterial abundance and community composition in the sandy land were affected by the age of the C. microphylla plantation. Moreover, bacterial diversity decreased with increasing plantation age, and the composition of the bacterial community in the 26-year plantation was similar to that in the natural community. Phylogenetic analysis of bands excised from the DGGE gels showed that members of alpha Proteobacterium, gamma Proteobacterium, Gemmatimonadetes and Chloroflexi were dominant in the sandy land. The stabilization of moving sand dune and development of sand-fixed plantation resulted in an increase of soil fertility, which could drive the structural evolvement of soil bacterial community, and it needs over 20 years for the soil bacterial community to form a stable structure, similar to the case for the natural vegetation.

Microbial Function, Enzymatic Activities and Diversity in an Anaerobic-Anoxic-Aerobic Reactor System

Enzymatic activities of beta-glucosidase (β-GLC), leucine-aminopeptidase (LAP), and alkaline phosphatase (APA), corresponding to nutrient eliminations, and the microbial community structures were analyzed in an anaerobic-anoxic-aerobic reactor system. Results showed that most activity of β-GLC (64.2 μmol/(L·h)) associated with the largest fraction of small-molecular-weight carbohydrates was found in the aerobic reactor, indicating the existence of coupled hydrolysis-uptake mechanism in the aerobic bacteria. Similar activities of LAP presented in the anoxic and aerobic environments, whose increases accompanied by increments in nitrogen uptake rates greatly accelerated the processes of aerobic nitrification and anoxic denitrification. The highest APA activity displayed in the anaerobic reactor, however, dephosphorization performance was mainly achieved under aerobic condition. Microbial community fingerprints generated by polymerase chain reaction amplification and denaturing gradient gel electrophoresis (PCR-DGGE) revealed that Proteobacterium, Actinobacterium, and Nitrospira were the predominant classes in the activated sludge and there was no evidence of community variations among each function reactor in the system with biomass recycling.

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis(DGGE)

To study the structure of microbial communities in the biological hydrogen produc- tion reactor and determine the ecological function of hydrogen producing bacteria, anaerobic sludge was obtained from the continuous stirred tank reactor (CSTR) in different periods of time, and the diversity and dynamics of microbial communities were investigated by denaturing gra- dient gel electrophoresis (DGGE). The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day, and the ethanol type fermentation was established. After 28 days the structure of microbial community became stable, and the climax community was formed. Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria (Clostridium sp. and Ethanologenbacterium sp.), β- proteobacteria (Acidovorax sp.), γ-proteobacteria (Kluyvera sp.), Bacteroides (uncultured bacte- rium SJA-168), and Spirochaetes (uncultured eubacterium E1-K13), respectively. The hydrogen production rate increased obviously with the increase of Ethanologenbacterium sp., Clostridium sp. and uncultured Spirochaetes after 21 days, meanwhile the succession of ethanol type fer- mentation was formed. Throughout the succession the microbial diversity increased however it decreased after 21 days. Some types of Clostridium sp. Acidovorax sp., Kluyvera sp., and Bac- teroides were dominant populations during all periods of time. These special populations were essential for the construction of climax community. Hydrogen production efficiency was de- pendent on both hydrogen producing bacteria and other populations. It implied that the co- metabolism of microbial community played a great role of biohydrogen production in the reactors.

Application of PCR-DGGE in Research of Bacterial Diversity in Drinking Water

Objective To analyze the structure of bacteria in drinking water by molecular biological techniques, Methods DNA of bacteria in drinking water was directly extracted without culture. 16S ribosomal DNA fragments, including V-6, -7, and -8 regions, were amplified with universal primers （EUBf933CJC and EUBr1387） and analyzed by DGGE. Results DGGE indicated that amplification products could be separated, The results showed that DGGE could be used in the separation of different microbial 16SrRNA genes extracted from drinkng water. Though there were special bacteria in different water samples, the predominant bacteria were essentially the same. Three sequences of the reclaimed specific bands were obtained, and phylogenetic tree of these bands was made. Conclusion Bacterial diversity in drinking water is identified by molecular biological techniques.

Phytoplankton diversity in the East China Sea and Yellow Sea measured by PCR-DGGE and its relationships with environmental factors

Relationships between phytoplankton community composition and environmental variables in the East China Sea (ECS) and Yellow Sea (YS) were investigated using geochemical and molecular microbiology methods. The diversity of phytoplankton was characterized using cultivation-independent PCR-based denaturing gradient gel electrophoresis (DGGE). Groups resulting from unweighted pair-group method with arithmetic averages clustering of the DGGE profiles showed good consistency with the eco-environmental characteristics of the sea area they belonged to. Additionally, the clustering results based on DGGE fingerprinting and those based on morphological compositions were practically identical. The relationship of phytoplankton diversity to environmental factors was statistically analyzed. Temperature, dissolved inorganic nitrogen (DIN), and silicate-Si were found significantly related to the phytoplankton community composition. Canonical correspondence analysis (CCA) was performed to reveal the relationship between community composition and these three environmental factors. Generally, values of the ECS are clearly separated from those of the YS in the CCA biplot, due to mainly the effect of temperature and DIN.

Monitoring impact of mefenacet treatment on soil microbial communities by PCR-DGGE fingerprinting and conventional testing procedures

The effect of acetanilide herbicide mefenacet on soil microbial communities was studied using paddy soil samples with different short-term treatments. The culturable bacteria （plate counts）, dehydrogenase activity and changes in community structure （denaturing gradient gel electrophoresis （DGGE） analysis） were used for biological community assessments. Mefenacet was a significant stimulus to cultural aerobic bacteria and dehydrogenase activity while Sphingobacterium multivorum Y1, a bacterium efficiently degrading the mefenacet, only induced the increasing colony-forming unit （CFU） of bacteria but little effect on dehydrogenase activity during the whole experiment. The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analyzing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the mefenacet-treated and non-treated soils were not significantly different. But supplement of S. multivorum Y1 could increase the diversity of the microbial community in the mefenacet-polluted paddy soil. This work is a new attempt to apply the S. multivorum Y1 for remediation of the mefenacet-polluted environments.

The key procedures and advancement in microbial communities analysis of DGGE

Recently, denaturing gradient gel electrophoresis （DGGE） is well established molecular tool in microbial ecology that allows the study of diversity and dynamics of microbial communities. The technique has been gained the preference since reliable facilitative and inexpensive. In order to promote the DGGE further development and make it become a general analysis technique earlier, the key procedures and the advances in microbial communities analysis of DGGE were introduced in this paper.

Phylogenetic diversity and specificity of bacteria associated with Microcystis aeruginosa and other cyanobacteria

Interactions between bacteria and cyanobacteria have been suggested to have a potential to influence harmful algal bloom dynamics; however, little information on these interactions has been reported. In this study, the bacterial communities associated with five strains of Microcystis aeruginosa, three species of other Microcystis spp., and four representative species of non-Microcystis cyanobacteria were compared. Bacterial 16S rDNA fragments were amplified and separated by denaturing gradient gel electrophoresis （DGGE） followed by DNA sequence analysis. The similarities among bacterial communities associated with these cyanobacteria were compared to the digitized DGGE profiles using the cluster analyses. The bacterial community structure of all cyanobacterial cultures differed. Cluster analysis showed that the similarity values among M. aeruginosa cultures were higher than those of other cyanobacterial cultures. Sequence analysis of DGGE fragments indicated the presence of bacteria including, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria in the cyanobacterial cultures. Members of the Sphingomonadales were the prevalent group among the Microcystis-associated bacteria. The results provided further evidence for species-specific associations between cyanoabcteria and heterotrophic bacteria, which are useful for understanding interactions between Microcystis and their associated bacteria.

Microbial Community Dynamics During Biogas Slurry and Cow Manure Compost

This study evaluated the microbial community dynamics and maturation time of two compost systems： biogas slurry compost and cow manure compost, with the aim of evaluating the potential utility of a biogas slurry compost system. Denaturing gradient gel electrophoresis （DGGE）, gene clone library, temperature, C/N ratio, and the germination index were employed for the investigation, cow manure compost was used as the control. Results showed that the basic strip and dominant strips of the DGGE bands for biogas slurry compost were similar to those of cow manure compost, but the brightness of the respective strips for each system were different. Shannon-Weaver indices of the two compost systems differed, possessing only 22% similarity in the primary and maturity stages of the compost process. Using bacterial 16S rRNA gene clone library analysis, 88 bacterial clones were detected. Further, 18 and 13 operational taxonomic units （OTUs） were present in biogas slurry and cow manure compost, respectively. The 18 OTUs of the biogas slurry compost belonged to nine bacterial genera, of which the dominant strains were Bacillus sp. and Carnobacterium sp.; the 13 OTUs of the cow manure compost belonged to eight bacterial genera, of which the dominant strains were Psychrobacter sp., Pseudomonas sp., and Clostridium sp. Results demonstrated that the duration of the thermophilic phase （more than 50°C） for biogas slurry compost was 8 d less than the according duration for cow manure compost, and the maturation times for biogas slurry and cow manure compost were 45 and 60 d, respectively. It is an effective biogas slurry assimilate technology by application of biogas slurry as nitrogen additives in the manufacture of organic fertilizer.

Bacterial diversity, composition and temporal-spatial variation in the sediment of Jiaozhou Bay, China

Studies on the diversity and distribution of bacterial populations will improve the overall understanding of the global patterns of marine bacteria and help to comprehend local biochemical processes and environments. We evaluated the composition and the dynamics of bacterial communities in the sediment of Jiaozhou Bay (China) using PCR-denaturing gradient gel electrophoresis (DGGE). Sediment samples were collected from 10 different sites in May, August, and November 2008 and in February 2009. There was significant temporal variation in bacterial community composition at all sites. However, the spatial variation was very small. The DGGE analyses of bacterial communities were used to divide the 10 stations into three types. Canonical correspondence analysis (CCA) revealed that the changes in bacterial communities were driven by sediment properties. Sequence analysis of DGGE band-derived 16S rRNA gene fragments revealed that the dominant bacterial groups in the sediment were of the classes γ-proteobacteria and δ-proteobacteria and phyla Bacteroidetes and Nitrospirae. Our results provide considerable insight into the bacterial community structure in Jiaozhou Bay, China.

PCR-DGGE技术分析韩式大酱与中式大酱中微生物多样性

微生物在大酱发酵过程中起到至关重要的作用,并与大酱的风味与质量密切相关,因此研究大酱中微生物的多样性有重要意义。该研究选择加工工艺不同的韩式大酱与中式大酱为对象,采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术,通过PCR扩增、切胶回收、PCR测序等分析不同大酱中的微生物多样性。结果表明,大酱中的微生物由于制作工艺的不同存在明显差异。在韩式大酱中,细菌如芽孢杆菌属(Bacillus)、不动杆菌属(Acinetobacter)、四联球菌属(Tetragenococcus)、嗜盐单胞菌属(Halomonas),真菌如根毛霉属(Rhizomucor)、青霉菌属(Penicillium)、毛霉属(Mucor)、外瓶霉属(Exophiala)、曲霉属(Aspergillus)等分布广泛。在中式大酱中,乳酸菌如片球菌属(Pediococcus)、乳杆菌属(Lactobacillus)、明串珠菌属(Leucanostoc)、肠杆菌属(Enterobacter),酵母如鲁氏接合酵母(Zygosaccharomyces rouxii)分布较多。与中式大酱相比,韩式大酱中的真菌种类更丰富。研究结果为进一步探讨传统发酵大酱品质提供了理论依据。

Diversity of the endophytic filamentous fungal leaf community at different development stages of eucalyptus

Fungal endophytic species inhabiting the leaves of eucalypts are capable of utilising leaf sugars and can influence both plant growth and health. Endophytic fungal symbionts can use simple soluble sugars in leaves as their main carbon source. This study set out to determine the diversity and distribution of the endophytic filamentous fungal leaf community in the hybrid Eucalyptus urograndis due to its economic importance. The fungal leaf community was characterised using denaturing gradient electrophoresis (DGGE) and correlated with levels of leaf nutrients and sugars throughout plant development. Sequencing of DGGE bands revealed the presence of Basidiomycota and Ascomycota phyla. Fourteen species and three genera of filamentous fungi were identified, and the population structure was affected by the plant developmental stage. Levels of K, Cu, N and Mn influenced communities from the clonal garden, whereas leaves in the field had higher glucose, fructose and sucrose. Many fungi were found to be specific to a certain development stages: Diplomitoporus crustulinus, Podosphaera tridactyla and Aspergillus restrictus to the clonal garden stage;Chaetomella acutiseta and Ascotricha chartarum to the shading stage;Erratomyces patelii and Saxomyces sp. to the shading output stage;Lepidostroma sp. and Saxomyces sp. to the dispatch stage;and Mycosphaerella populicola to the field stage. Teratosphaeria toledana and Teratosphaeria acidotherma were found at more than one developmental stage. Cladosporium sp. and Rhodosporidium fluviale colonized and persisted in plants at the dispatch and field stages. This is the first report of P. tridactyla, A. restrictus, E. patelii, Saxomyces and Lepidostroma sp. as endophytes in eucalipt.

Effects of Heavy Metal Pollution of Apple Orchard Surface Soils Associated with Past Use of Metal-Based Pesticides on Soil Microbial Biomass and Microbial Communities

Apple orchard surface soils in Japan are polluted with copper (Cu), lead (Pb), and arsenic (As) due to long-term use of metal-based pesticides. We investigated the effects of heavy metals accumulated in the surface soils in apple orchards on the microbial biomass and the microbial communities. Soil samples were taken from a chestnut orchard (unpolluted control) and five apple orchards with different degrees of heavy metal pollution. Total concentrations of Cu, Pb, and As in soil ranged from 29 to 931 mg/kg, 35 to 771 mg/kg, and 11 to 198 mg/kg, respectively. The amount of microbial biomass carbon expressed on a soil organic carbon basis decreased with increasing concentrations of heavy metals. Thus, the heavy metals that accumulated in apple orchard surface soils had adverse effects on the soil microbial biomass. The analysis of phospholipid fatty acid (PLFA) composition indicated that the microbial community structure had changed because of the pesticide-derived heavy metals in soil. The relative abundance of gram-positive bacterial marker PLFAs increased and that of fungal marker PLFA decreased with increasing concentrations of heavy metals in soil. Denaturing gradient gel electrophoreses targeting the 16S ribosomal RNA gene of bacteria and the 18S ribosomal RNA gene of fungi also showed shifts in the composition of bacterial and fungal communities induced by soil pollution with heavy metals. However, the diversity of microbial communities was not significantly affected by the heavy metal pollution. This was attributable to the adaptation of the microbial communities in apple orchard surface soils to heavy metals derived from previously used pesticides.

PCR-DGGE技术用于湖泊沉积物中微生物群落结构多样性研究

采用PCR-DGGE分子指纹图谱技术比较南京市玄武湖、奠愁湖和太湖不同位置的表层沉积物微生物群落结构，研究结果表明，三湖泊沉积物微生物的16SrDNA的PCR扩增结果约为626bp，为16S rDNA V3～V5区特异性片段。玄武湖和莫愁湖表层沉积物中大约有20种优势菌群，且同一湖泊不同采样点DGGE图谱的差异性不大，细菌群落结构具有较高的相似性，而太湖样品DGGE条带的数目和位置表现出明显差异，且不同采样点图谱的差异性较大。三湖泊除具有特征性的微生物种属外，还分布约5个相同的细菌种群，可能与沉积物的理化性质和水生植被的影响相关。对DGGE图谱中7条主带进行回收、扩增和测序，结果显示其优势菌群具有不同的序列组成，其中5个序列与Genebank中已登录的细菌种群的同源性≥99％，2个序列的同源性为96％和93％，其中2个相似的细菌类群目前尚未获得纯培养。

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术 ( DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组 DNA,并以此基因组 DNA为模板 ,选择特异性引物 F357GC和 R518对 1 6Sr RNA基因的 V3区进行扩增 ,长约 2 30 bp的 PCR产物经变性梯度凝胶电泳 ( DGGE)进行分离后 ,得到不同数目且分离效果较好的电泳条带。结果说明 ,DGGE能够对土壤样品中的不同微生物的 1 6Sr RNA基因的 V3区的 DNA扩增片断进行分离 ,为这些 DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比 ,变性梯度凝胶电泳 ( DGGE)技术能够更精确的反映出土壤微生物多样性 ,它是一种有效的微生物多样性研究技术。

PCR-DGGE方法分析原油储层微生物群落结构及种群多样性

使用基于 16 S r DNA的 PCR- DGGE(变性梯度凝胶电泳 )图谱分析结合条带割胶回收 DNA进行序列分析 ,对新疆克拉玛依油田一中区注水井 (12 # 9- 11)和与该注水井相应的两个采油井 (12 # 9- 9S、13# 11- 8)井口样品微生物群落的多样性进行了比较并鉴定了部分群落成员。 DGGE图谱聚类分析表明注水井与两油井微生物群落的相似性分别为 30 %和 2 0 % ,而两油井间微生物群落结构的相似性为 5 4 %。DGGE图谱中优势条带序列分析表明注水井样品和油井样品中的优势菌群为未培养的环境微生物 ,它们与数据库中 α、γ、δ、ε变形杆菌 (Proteobacteria)和拟杆菌 (Bacteroidetes)有很近的亲缘关系。 DGGE与分子克隆相结合的分子生物学方法在研究微生物提高原油采收率 (MEOR)机理 ,以及指导

DG-DGGE分析产氢发酵系统微生物群落动态及种群多样性

应用双梯度-变性梯度凝胶电泳(DG-DGGE)对生物制氢反应器微生物种群的动态变化及多样性进行监测。间隔7d从反应器取厌氧活性污泥,以细菌16SrDNA通用引物进行V2～V3区域PCR扩增,长约450bp的PCR产物经DGGE分离后,获得污泥微生物群落的16SrDNA指纹图谱。污泥接种到反应器后微生物群落中既有原始种群的消亡和增长,也有次级种群的强化和演变。反应器在运行初期群落演替迅速,15d时微生物群落结构变化最大。群落结构的相似性随着演替时间的增加而逐渐升高,种群动态变化后形成稳定的群落结构。29d时微生物多样性基本保持不变,微生物优势种属达到19个OTU。在细菌竞争和协同作用制约下,种群多样性降低后趋于稳定,形成顶级群落。有些种群在群落结构中一直存在,是群落建成的原始种群,原始种群与次级种群在代谢过程中具有协同作用,表现出群落的综合生态特征。

DGGE污泥堆肥工艺微生物种群结构分析

用DGGE指纹图谱技术对污泥堆肥工艺中的细菌种群动态变化及多样性进行了研究.结果表明,生物法污泥堆肥周期小于8d.对污泥堆肥各工艺环节样品进行DGGE指纹图谱和相似性系数Cs值分析,发现随着反应的持续进行,微生态结构的Cs值越来越高,说明微生物种群结构愈趋稳定.证实污泥微生态能迅速进行优胜劣汰的筛选,调整内部细菌种群结构,从而达到适应环境的目的.在发酵过程中形成的优势细菌种群能长时间保持稳定.