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101.
Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil 总被引:1,自引:0,他引:1
Alexandre Jousset Enrique Lara Marcell Nikolausz Antonis Chatzinotas 《The Science of the total environment》2010,408(5):1221-1225
Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to greatly simplify and accelerate such studies. In this present work a ciliate-specific fingerprint method based on the amplification of a phylogenetic marker gene (i.e. the 18S ribosomal RNA gene) with subsequent analysis by denaturing gradient gel electrophoresis (DGGE) was developed and used to monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. The semi-nested approach generated ciliate-specific amplification products from all soil samples and allowed to distinguish community profiles from a PAH-polluted and a non-polluted control soil. Subsequent sequence analysis of excised bands provided evidence that polluted soil samples are dominated by organisms belonging to the class Colpodea. The general DGGE approach presented in this study might thus in principle serve as a fast and reproducible diagnostic tool, complementing and facilitating future ecological and ecotoxicological monitoring of ciliates in polluted habitats. 相似文献
102.
Isolation and identification of the autochthonous starter from a naturally fermented meat allows control of the fermentation process and promises microbiological safety for this specialty. Thus the purpose of this study was to identify the lactic acid bacteria and coagulase-negative cocci present in a traditional Taiwanese naturally fermented ham (TNFH) and to study the microbial dynamics at different ripening stages; the approach was a combination of conventional microbiological cultivation, polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequencing. In total, twelve different strains of lactic acid bacteria and three Staphylococcus strains were identified in the TNFH samples, whereas only 5 dominant strains were observed in the TNFH samples when the DGGE as a culture-independent method was applied. The bacterial ecology on the surface of the samples was mainly characterized by the stable presence of Lactobacillus sakei and Staphylococcus saprophyticus; nonetheless Leuconostoc mesenteroides and Carnobacterium divergens were the most abundant bacteria found in the final product. These results are also agreed with the findings of the culture-independent method. In addition, Microbacterium spp., Carnobacterium spp., Enterobacter spp., Brochothrix spp., Enterococcus spp., and Bacillus spp. were also present at the beginning of the ripening, but few bacteria were found at the center of the TNFH samples during the early ripening stages. However, after 30 days of ripening, the microbial ecology at the center of the TNFH samples paralleled that of the surface. Finally, as far as we have been able to determine, our report is the first to investigate the microbiological dynamics in fermented meat products using combination of cultivation, the Harrison disc method, DGGE and DNA sequencing as the culture-dependent method. Our report is also the first to show the presence of Staphylococcus arlettae in a fermented sausage and ham product. 相似文献
103.
Katrien Broekaert Marc Heyndrickx Lieve Herman Frank Devlieghere Geertrui Vlaemynck 《Food microbiology》2013
The dominant microbiota of brown shrimp (Crangon crangon) were systematically identified during storage under different conditions. Freshly caught shrimp were processed on board the fishing vessel under the best possible hygienic conditions (IDEAL), unpeeled and manually (sterile) peeled, then stored on ice and at 7.5 °C until microbiologically spoiled. Results were compared with industrially processed (INDUSTRIAL) shrimp. Isolates grown on various media were identified by 16S rRNA and gyrB gene sequencing. We examined the total microbiota and microbial population shifts of shrimp under various storage conditions using denaturant gradient gel electrophoresis (DGGE). The microbiota differed somewhat during storage and among the various storage conditions; however, members of the genera Psychrobacter and Pseudoalteromonas were found to dominate the microbiota of all shrimp samples regardless of processing procedures or storage conditions. Most isolates could be identified by gyrB gene sequencing as Psychrobacter immobilis or Psychrobacter cibarius. Also Pseudoalteromonas nigrifaciens, Pseudoalteromonas elyakovii or Pseudoalteromonas paragorgicola dominated the microbiota of brown shrimp during storage. Also species from the genera Planocuccus, Exiguobacterium, Carnobacterium, Pseudomonas, Chryseobacterium and Staphylococcus were detected during storage of brown shrimp. 相似文献
104.
The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy® Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended. 相似文献
105.
The goal of this study was to investigate the bacterial community in activated sludge with an anaerobic side-stream reactor (ASSR), a process permitting significant decrease in sludge production during wastewater treatment. The study operated five activated sludge systems with different sludge treatment schemes serving as various controls for the activated sludge with ASSR. Bacterial communities were analyzed by denaturing gradient gel electrophoresis (DGGE), sequencing and construction of phylogenetic relationships of the identified bacteria. The DGGE data showed that activated sludge incorporating ASSR contained higher diversity of bacteria, resulting from long solids retention time and recirculation of sludge under aerobic and anaerobic conditions. The similarity of DGGE profiles between ASSR and separate anaerobic digester (control) was high indicating that ASSR is primarily related to conventional anaerobic digesters. Nevertheless, there was also unique bacteria community appearing in ASSR. Interestingly, sludge in the main system and in ASSR showed considerably different bacterial composition indicating that ASSR allowed enriching its own bacterial community different than that from the aeration basin, although two reactors were connected via sludge recirculation. In activated sludge with ASSR, sequences represented by predominant DGGE bands were affiliated with Proteobacteria. The remaining groups were composed of Spirochaetes, Clostridiales, Chloroflexi, and Actinobacteria. Their putative role in the activated sludge with ASSR is also discussed in this study. 相似文献
106.
本文研究了香蕉抗性淀粉对高脂饮食菌群人源化小鼠(HFA)肠道菌群的调控效果。将30只肠道菌群人源化昆明小鼠随机分为3组(10只/组),分别饲喂普通饮食、高脂饮食和高脂饮食+抗性淀粉。提取第0周和第8周新鲜粪便DNA,比较用16S r DNAV6-V8区段特异性引物扩增产物,以PCR-DGGE技术手段检测抗性淀粉组、高脂饮食组和普通饮食组肠道菌群差异。第0周,三组小鼠菌群总菌丰富度,多样性指数和均匀度无显著差异(p0.05),小鼠和粪便供者之间菌群相似性为36%;三组小鼠肠道菌群相似性达到60-93%,PCA分析显示三组小鼠的数据点都聚在一起,说明在干预前无明显差异,适用于分析抗性淀粉对肠道菌群的影响;膳食干涉第8周后小鼠肠道菌群的多样性指数和丰富度数值,抗性淀粉组都极显著低于高脂组和普通饲料组(p0.01),PCA分析也显示三组小鼠肠道菌群各组的数据点都分布在不同区域,说明抗性淀粉对高脂饮食HFA小鼠肠道菌群结构产生显著影响。 相似文献
107.
Kris Audenaert Klaas D'Haene Kathy Messens Tony Ruyssen Peter Vandamme Geert Huys 《Food microbiology》2010
The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 °C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants. 相似文献
108.
109.
强化A2O工艺中的反硝化除磷比例,是提高该工艺处理低C/N比污水时脱氮除磷效率的有效途径。采用52.5 L的A2O反应器处理实际生活污水,研究了系统在不同的反硝化除磷比例情况下微生物的种群变化及关系。试验结果表明,随着水质条件及运行状态的改变,系统反硝化除磷的比例也在变化,同时微生物种群结构表现为一种动态的演替过程,工艺条件与微生物的种群结构具有很强的映射关系。测序结果表明,具有传统除磷功能的Acinetobacter在系统反硝化除磷得到强化的时候会逐渐被淘汰,而Uncultured Chlorobi bacterium会逐渐得到增殖,可能是系统中具有反硝化除磷功能的微生物。 相似文献
110.
Bacterial community composition and structure of biofilms developing on nanofiltration membranes applied to wastewater treatment 总被引:6,自引:0,他引:6
Ivnitsky H Katz I Minz D Volvovic G Shimoni E Kesselman E Semiat R Dosoretz CG 《Water research》2007,41(17):3924-3935
The structure and microbial communities of biofilms developing on cross-flow nanofiltration (NF) membranes at different temperatures (20, 25 or 34 degrees C) and operation lengths (8h-24days) were studied. Feedwater comprised tertiary quality wastewater effluent or synthetic media mimicking effluents of intermediate quality. After each run, the membranes were autopsied for bacterial enumeration, bacterial community composition and microscopy visualization (SEM, CLSM and AFM/NSOM). Community composition was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) coupled with sequence analysis of 16S rRNA gene fragments from dominant bands. Deposition of polysaccharides and initial bacterial colonization were observed within 8h, whereas developed biofilms markedly affecting membrane permeability were evident from days 2-3 onwards. Regardless of applied conditions, the heterotrophic plate counts in the biofilm were 3-4x10(6)CFU/cm(2) and the thickness of the biofouling layer was 20-30microm. From a total of 22 sequences obtained from 14 independent experiments, most species identified were Gram negative (19 of 22 sequences). Proteobacteria were found to be a prevalent group in all cases (16 of 22 sequences) and among it, the beta-subclass was the most predominant (8 sequences), followed by the gamma-subclass (5 sequences). Pseudomonas/Burkholderia, Ralstonia, Bacteroidetes and Sphingomonas were the dominant groups found in most cases. Even though the microbial population might be important with respect to biofouling patterns, membrane permeability decline seems to be more substantially influenced by the formation and accumulation of exopolymeric substances (EPS). 相似文献