Digitization of DGGE (denaturing gradient gel electrophoresis) profile and cluster analysis of microbial communities

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA profiles were objectively digitized using an image analyzer; the individual microbial species in a community can thus be precisely quantified. The similarity between various microbial communities was compared to the digitized DGGE profiles using the cluster analyses technique. The microbial community in a biofilm was considerably different from that in suspended sludge obtained from the same system.     

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。     

Spatial variation in microbial community structure, richness, and diversity in an alluvial aquifer

Relatively little is known regarding the spatial variability of microbial communities in aquifers where well fouling is an issue. In this study 2 water wells were installed in an alluvial aquifer located adjacent to the North Saskatchewan River and an associated piezometer network developed to facilitate the study of microbial community structure, richness, and diversity. Carbon utilization data analysis revealed reduced microbial activity in waters collected close to the wells. Functional PCR and quantitative PCR analysis indicated spatial variability in the potential for iron-, sulphate-, and nitrate-reducing activity at all locations in the aquifer. Denaturing gradient gel electrophoresis analysis of aquifer water samples using principal components analyses indicated that the microbial community composition was spatially variable, and denaturing gradient gel electrophoresis sequence analysis revealed that bacteria belonging to the genera Acidovorax , Rhodobacter , and Sulfuricurvum were common throughout the aquifer. Shannon's richness (H') and Pielou's evenness (J') indices revealed a varied microbial diversity (H' = 1.488-2.274) and an even distribution of microbial communities within the aquifer (J' = 0.811-0.917). Overall, these analyses revealed that the aquifer's microbial community varied spatially in terms of composition, richness, and metabolic activity. Such information may facilitate the diagnosis, prevention, and management of fouling.     

DGGE/TGGE技术在土壤微生物分子生态学研究中的应用

传统的微生物生态学研究方法只限于环境样品中极少部分(0.1%￣1%)可培养的微生物类群,极大程度地限制了对土壤微生物群落结构的研究。综述了以16S rDNA为主要研究对象的DGGE/TGGE(Denaturing gradientgel electrophoresis,DGGE/Temperature gradient gel electrophoresis,TGGE)技术原理,以其为主要手段结合PCR扩增、克隆建库、序列测定以及种系分析对土壤微生物的群落结构和多样性研究的最新动态。DGGE/TGGE技术极大地推动了土壤微生物分子生态学的发展,同时也为实际问题的诊断、作物生长跟踪监测等提供了技术支撑,在土壤微生物分子生态学研究和生产实践中起着越来越重要的作用。     

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。     

利用时间进程法优化活性污泥DG-DGGE图谱

目的:为了探讨电泳时间对双梯度-变性梯度凝胶电泳(DG-DGGE)分析活性污泥样品时的影响。方法:提取污泥DNA后,以通用引物338f/534r扩增16S rDNA序列,采用时间进程法优化PCR扩增产物的DG-DGGE分离效果。结果:采用不同电泳时间进行DGGE分析时,DGGE图谱存在显著的差异。16S rDNA V3区(200 bp)在凝胶梯度6%~12%,变性剂梯度30%~60%时,在200V电压下,最佳电泳时间为5h。     

Sheep-urine-induced changes in soil microbial community structure

Soil microbial communities play an important role in nutrient cycling and nutrient availability, especially in unimproved soils. In grazed pastures, sheep urine causes local changes in nutrient concentration which may be a source of heterogeneity in microbial community structure. In the present study, we investigated the effects of synthetic urine on soil microbial community structure, using physiological (community level physiological profiling, CLPP), biochemical (phospholipid fatty acid analysis, PLFA) and molecular (denaturing gradient gel electrophoresis, DGGE) fingerprinting methods. PLFA data suggested that synthetic urine treatment had no significant effect on total microbial (total PLFA), total bacterial or fungal biomass; however, significant changes in microbial community structure were observed with both PLFA and DGGE data. PLFA data suggested that synthetic urine induced a shift towards communities with higher concentrations of branched fatty acids. DGGE banding patterns derived from control and treated soils differed, due to a higher proportion of DNA sequences migrating only to the upper regions of the gel in synthetic urine-treated samples. The shifts in community structure measured by PLFA and DGGE were significantly correlated with one another, suggesting that both datasets reflected the same changes in microbial communities. Synthetic urine treatment preferentially stimulated the use of rhizosphere-C in sole-carbon-source utilisation profiles. The changes caused by synthetic urine addition accounted for only 10-15% of the total variability in community structure, suggesting that overall microbial community structure was reasonably stable and that changes were confined to a small proportion of the communities.     

Biodiversity analysis of microbial community in the chem-bioflocculation treatment process

Total DNA was directly extracted from environmental samples and amplified with polymerase chain reaction (PCR) technique. The PCR products were fingerprinted via denaturing gradient gel electrophoresis (DGGE). Significant differences were observed in the microbial community structures between traditional treatment process and chem-bioflocculation process. The microbial community structure shift at different sampling locations in chem-bioflocculation process and on two typical operational conditions was studied. 16S rDNA V3 regions of some dominant species were sequenced and the species were identified. The microbial communities were stable in both the chem-bioflocculation process and the activated sludge process under various experimental conditions presented in this work. The attached growth treatment process was less stable when operational conditions changed.     

Bacterial dynamics in steady-state biofilters: beyond functional stability

The spatial and temporal dynamics of microbial community structure and function were surveyed in duplicated woodchip-biofilters operated under constant conditions for 231 days. The contaminated gaseous stream for treatment was representative of composting emissions, included ammonia, dimethyl disulfide and a mixture of five oxygenated volatile organic compounds. The community structure and diversity were investigated by denaturing gradient gel electrophoresis on 16S rRNA gene fragments. During the first 42 days, microbial acclimatization revealed the influence of operating conditions and contaminant loading on the biofiltration community structure and diversity, as well as the limited impact of inoculum compared to the greater persistence of the endogenous woodchip community. During long-term operation, a high and stable removal efficiency was maintained despite a highly dynamic microbial community, suggesting the probable functional redundancy of the community. Most of the contaminant removal occurred in the first compartment, near the gas inlet, where the microbial diversity was the highest. The stratification of the microbial structures along the filter bed was statistically correlated to the longitudinal distribution of environmental conditions (selective pressure imposed by contaminant concentrations) and function (contaminant elimination capacity), highlighting the central role of the bacterial community. The reproducibility of microbial succession in replicates suggests that the community changes were presumably driven by a deterministic process.     

PCR-based denaturing gradient gel electrophoretic evaluation of changes in the non-methanogenic population of stressed upflow anaerobic sludge blanket granules

Summary The performance of upflow anaerobic sludge blanket (UASB) bioreactors is influenced by the composition of the substrate and the microbial species present in the granules. The aim of this study was to determine if a change in the structure of the non-methanogenic microbial community takes place when UASB brewery granules are subjected to the sudden addition of different carbon sources at different concentrations. A shift in the microbial community did occur when the granules were subjected to lactate medium. The granules that were stressed with glucose medium did not show changes in the microbial consortium regardless of the increase in the glucose concentrations. The polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) method was successfully applied to show changes in the structure of the microbes present in UASB granules that were cultivated under different environmental conditions.     

Molecular Characterization of a Bacterial Consortium Enriched from an Oilfield that Degrades Phenanthrene

Characterization of functional and phylogenetic genes was carried out on a bacterial consortium, enriched from a water treatment system of an oilfield, that could use phenanthrene as the sole carbon source. The mixed culture degraded 130 mg phenanthrene l⁻¹ in 16 days, which is significantly faster than previously reported pure cultures. The existence of catabolic genes (nahAc, C23O) in the mixed culture was quantitated by most probable number PCR. The plasmid encoding phenanthrene catabolic genes increased relative to the chromosome genes. Heterogeneous bacteria were present according to both PCR denaturing gradient gel electrophoresis and cloning methods, suggesting the possible existence of cooperation between different biochemical PAH-transforming pathways. Revisions requested 15 December 2005; Revisions received 23 January 2006     

Screening and Isolation of PHB-Producing Bacteria in a Polluted Marine Microbial Mat

The characteristics of microbial mats within the waste stream from a seafood cannery were compared to a microbial community at a pristine site near a sandy beach at Puerto San Carlos, Baja California Sur, Mexico. Isolation of poly-beta-hydroxybutyrate (PHB)-producing bacteria, recognition of brightly refractile cytoplasmatic inclusions, lipophilic stains with Sudan Black and Nile Red, and chemical extraction of PHB were used as a culture-dependent strategy for the detection of PHB-producing bacteria. The culture-independent approach included denaturing gradient gel electrophoresis of phylotypes of 16S rRNA of microbial communities from environmental samples. Significant differences in community structure were found among the polluted and pristine sites. These differences were correlated with the physicochemical characteristics of the seawater column. At the polluted site, the seawater was rich in nutrients (ammonia, phosphates, and organic matter), compared to the pristine location. Partial sequencing of 16S rDNA of cultures of bacteria producing PHB included Bacillus and Staphylococcus at both sites; Paracoccus and Micrococcus were found only at the polluted site and Rhodococcus and Methylobacterium were found only at the pristine site. Bands of the sequences of 16S rDNA from both field samples in the denaturing gradient gel electrophoresis (DGGE) analyses affiliated closely only with bacterial sequences of cultures of Bacillus and Staphylococcus. High concentrations of organic and inorganic nutrients at the polluted site had a clear effect on the composition and diversity of the microbial community compared to the unpolluted site.     

Role of vermicompost chemical composition, microbial functional diversity, and fungal community structure in their microbial respiratory response to three pesticides

The relationships between vermicompost chemical features, enzyme activities, community-level physiological profiles (CLPPs), fungal community structures, and its microbial respiratory response to pesticides were investigated. Fungal community structure of vermicomposts produced from damaged tomato fruits (DT), winery wastes (WW), olive-mill waste and biosolids (OB), and cattle manure (CM) were determined by denaturing gradient gel electrophoresis of 18S rDNA. MicroResp? was used for assessing vermicompost CLPPs and testing the microbial response to metalaxyl, imidacloprid, and diuron. Vermicompost enzyme activities and CLPPs indicated that WW, OB, and DT had higher microbial functional diversity than CM. The microbiota of the former tolerated all three pesticides whereas microbial respiration in CM was negatively affected by metalaxyl and imidacloprid. The response of vermicompost microbiota to the fungicide metalaxyl was correlated to its fungal community structure. The results suggest that vermicomposts with higher microbial functional diversity can be useful for the management of pesticide pollution in agriculture.     

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R515对16S rRNA基因的V3区进行扩增,长约230bp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16S rRNA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。     

Molecular Characterization of Microbial Population Dynamics during Sildenafil Citrate Degradation

Little is known about pharmaceutical and personal care products pollutants (PPCPs), but there is a growing interest in how they might impact the environment and microbial communities. The widespread use of Viagra (sildenafil citrate) has attracted great attention because of the high usage rate, the unpredictable disposal and the unknown potential effects on wildlife and the environment. Until now information regarding the impact of Viagra on microbial community in water environment has not been reported. In this research, for the first time, the genetic profile of the microbial community, developing in a Viagra polluted water environment, was evaluated by means of the 16S and 18S rRNA genes, for bacteria and fungi, respectively, amplified by polymerase chain reaction (PCR) and separated using the denaturing gradient gel electrophoresis (DGGE) technique. The DGGE results revealed a complex microbial community structure with most of the population persisting throughout the experimental period. DNA sequences from bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria and fungi found previously mainly in polluted environmental and treating bioreactors. Biotransformation ability of sildenafil citrate by the microbial pool was studied and the capability of these microorganisms to detoxify a polluted water ecosystem was assessed. The bacterial and fungal population was able to degrade sildenafil citrate entirely. Additionally, assays conducted on Daphnia magna, algal growth inhibition assay and cell viability determination on HepG2 human cells showed that biotransformation products obtained from the bacterial growth was not toxic. The higher removal efficiency for sildenafil citrate and the lack of toxicity by the biotransformation products obtained showed that the microbial community identified here represented a composite population that might have biotechnological relevance to retrieve sildenafil citrate contaminated sites.     

Soil Microbial Community Response to Nitrobenzene Exposure for a Spartina Wetland

To explore the response of the soil microbial community to nitrobenzene (NB) exposure in a Spartina marsh, a short-term (45 d) mesocosm study was conducted at three NB concentrations of (10, 50, and 100) mg kg^?1. Dry soil, sterile and unsterile controls were also compared. The ability of the microbes to biodegrade NB was studied in an effort to predict the outcome of NB in the mesocosm. The results indicated that a microbial community is capable of doing so. Microbial enumeration and enzyme assays showed that the fluctuations in microbial communities and polyphenol oxidase activities are related to the initial NB concentration. Moreover, cluster analyses through denaturing gradient gel electrophoresis (DGGE) revealed very similar patterns (95.5%) throughout the 45 d term, indicating that the microbial community regenerates when NB is exhausted. Although volatilization and photolysis were the major processes responsible for the reduction in NB in contaminated mesocosms and the microbial community regenerated at the end of incubation, the data indicate potential ecological risks in outfall areas even if the discharged wastewater complies with the national wastewater discharge standards.     

黄翅大白蚁后肠降解滤纸微生物群落的分析简

黄翅大白蚁（ Macrotermes barneyi）具有高效降解木质纤维素的能力,其后肠内存在着丰富的共生微生物。采用活性电泳和变形梯度凝胶电泳的方法对黄翅大白蚁后肠降解滤纸微生物群落进行分析。活性电泳实验证实了此微生物群落纤维素酶的存在（内切葡聚糖酶、β葡萄糖苷酶和木聚糖酶）,变形梯度凝胶电泳实验鉴定出微生物组的群落结构,即7种细菌和3种真菌。本研究初步阐明了黄翅大白蚁后肠内与滤纸降解相关的微生物种类,为进一步了解黄翅大白蚁纤维素的降解机制以及生物质资源的高效利用提供了理论基础。     

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。     

不同日粮饲养黄牛的胃部微生物群落多样性比较分析

为分析不同饲料喂养对黄牛胃中微生物群落结构的影响,分别用芒草单一喂养和农家混合饲料喂养两年的黄牛为实验组和对照组。以瘤胃、蜂巢胃、重瓣胃和皱胃四个胃中的微生物为研究对象,采用原位包埋裂解法和脉冲场电泳（pulsed field gel electrophoresis,PFGE）提取微生物总 DNA,脉冲场电泳（pulsed field gel elec-trophoresis,PFGE）。使用细菌16S rRNA 引物341F ／534R 及真菌18S rDNA 引物 NS1／GCFungi 进行降落 PCR 扩增。变性梯度凝胶电泳（denaturing gradient gel electrophoresis,DGGE）对扩增产物进行区分,使用硝酸银染色。电泳扫描结果通过 Quantity one 软件进行分析,SPSS 软件进行数据统计。针对实验组和对照组瘤胃样品共性条带和特性条带测序并比对。结果表明：实验组与对照组细菌群落结构变化较大,UPGAM聚类图上被分为两支,相似性只有0．35。且香农多样性指数及条带丰度都明显少于对照组。真菌 DGGE 图谱条带差别不大,聚类图上显示实验组四个样品与对照组四个样品相似性在0．43～0．68之间。多样性及条带丰度在实验组与对照组之间差异0．0027～0．5999。测序结果,细菌与数据库中未培养细菌种类较为接近,而真菌中部分与已知菌种接近。芒草单一饲养对牛胃中的微生物群落结构具有极大的影响,对细菌的影响尤为明显。     

不同富集和分离培养条件下的含酚废水处理生物膜微生物群落结构与活性比较分析

采用Biolog和变性梯度凝胶电泳(DGGE)技术研究了不同苯酚浓度培养对焦化废水处理厂反硝化池生物膜样品中微生物群落结构和代谢类型的影响。DGGE结果表明, 不同浓度苯酚和不同培养方式富集培养后, 细菌16S rDNA的部分条带分布谱形发生改变, 还有部分条带只受到了苯酚浓度变化的影响; 富集培养过程中由于碳源组成相对焦化废水简单, DGGE条带所代表的优势微生物多样性有所降低。Biolog试验结果表明, 生物膜样本的细菌群落代谢能力最强; 低浓度苯酚富集后的样品能利用的底物碳源类型最丰富。对Biolog试验结果的主成分分析显示, 相同浓度苯酚富集培养后的细菌群落代谢功能多样性相似, 但从DGGE结果看出其结构组成产生了变化。富集培养使样品微生物群落的代谢功能发生改变, 低浓度的苯酚富集增加了群落中微生物的代谢类型。而不同条件获得的分离物其苯酚降解能力的初步分析也表明, 富集与分离条件对苯酚降解菌的分离能力和得到的菌株特性具有差别。