生物造粒流化床微生物群落结构及其动态变化

为了研究生物适粒流化床污水处理反应器颗粒污泥中微生物群落结构及其动态变化,分别从生物造粒流化床10、60、110 cm处取颗粒污泥,通过细胞裂解直接提取颗粒污泥细菌基因组DNA.以细菌和古细菌16S rRNA基因通用引物530F/1490R,对活性污泥中提取的细菌基因组DNA进行PCR扩增,长约1 kb的PCR扩增产物纯化后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱.结果显示,生物造粒流化床反应器颗粒污泥中的微生物群落非常丰富,在10 cm处微生物的种属达到23种,60 cm处为21种,110 cm处为20种;生物造粒流化床不同高度都有一些各自的特有种属和共有种属,反应器不同高度的微生物群落演替不明显,微生物群落相似性为83.1%,群落结构较为稳定.     

生物造粒流化床污水处理反应器的微生物多样性

为了研究生物造粒流化床污水处理反应器颗粒污泥的微生物种群多样性,分别从生物造粒流化床10、60和110cm处取颗粒污泥,通过细胞裂解直接提取颗粒污泥细菌基因组DNA,PCR扩增后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱,对特征条带进行序列测定及序列同源性分析。16S rRNA序列分析表明,获得的18个OTUs均属于细菌域,其中61%属于变形菌,17%属于放线菌,11%属于低G C革兰氏阳性菌,11%属于其它未知细菌。     

生物造粒流化床污水处理反应器中微生物的分布特征

对生物造粒流化床污水处理反应器10cm、60cm、110cm处好氧细菌总数以及反硝化菌、反硫化菌分别进行计数,同时,用光学显微镜、扫描电子显微镜以及石蜡切片技术对粒状污泥中细菌的分布情况进行研究。结果发现,流化床中好氧细菌非常丰富,在反应器10cm处,每克污泥微生物的数量可达1.6×10⁸个,说明好氧细菌在生物造粒流化床有机物生物降解中起主导作用;同时,流化床中也有一定数量的兼性厌氧菌存在,并且随着流化床床体的升高有增加的趋势,这与溶解氧(DO)随流化床床体高度的增加而迅速降低有关;随着回流比的增加,溶解氧增高,相应的好氧细菌有所增加而兼性厌氧菌减少;对于颗粒污泥,其表面和内部微生物分布数量有很大的差异。     

PCR-DGGE技术分析染整废水微生物群落多样性

旨在揭示水解酸化-生物接触氧化工艺处理染整废水过程中的微生物多样性.取初级沉淀池,水解酸化池,生物接触氧化池和二沉池的活性污泥,通过细胞裂解直接提取基因组DNA,以细菌通用引物进行16S rRNA基因V3区域PCR扩增,将PCR产物进行变性梯度凝胶电泳,获得微生物群落的DNA特征指纹图谱,并对条带进行统计分析和切胶测序,进行了同源性分析并建立了系统发育树.研究表明,整个水处理过程中含有丰富的微生物群落,其中初级沉淀池、水解酸化池、二沉池和生物接触氧化池的污泥样品分别测出36条带、42条带、30条带和29条带.不同区段微生物群落间相似度最高达68％,最低达42.4％,说明群落间演替明显,不同工艺区段既存在共同的微生物种属也存在特异微生物种属.     

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。     

厌氧颗粒污泥微生物总DNA的提取

目的:为了从分子水平上了解厌氧颗粒污泥中微生物的种类和数量,研究一种高效提取环境微生物DNA的方法。方法:厌氧颗粒污泥样品经液氮速冻、沸水浴融化、溶菌酶处理和SDS裂解后,琼脂糖凝胶电泳检测所提取的DNA,以提取的总DNA为模板,进行细菌核糖体小亚基16S rDNA基因V8、V9区的PCR扩增。结果:经检测,其DNA片段约为20 kb,样品D260nm/D280nm值为1.88,扩增结果理想,与OMEGA公司提供的试剂盒提取效果基本一致。结论:为薯类酒糟厌氧发酵污泥中微生物群落的分子生态学研究提供了一种简便、可靠的DNA提取方法。     

煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较

目的:在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法:分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果:相同处理量(108个)的革兰氏阳性菌(1株)、革兰氏阴性菌(4株)、古菌(1株)经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260/OD280的值更接近1.8-2.0(纯DNA的OD260/OD280在1.8-2.0之间),前者所提DNA回收率最大可达后者的16.7倍;煮沸裂解法只需较少菌(102个)便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论:两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随菌浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。     

煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较

目的：在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法：分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果：相同处理量（108个）的革兰氏阳性菌（1株）、革兰氏阴性菌（4株）、古菌（1株）经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260／OD280的值更接近1．8-2．0（纯DNA的OD260／OD280在1．8—2．0之间）,前者所提DNA回收率最大可达后者的16．7倍;煮沸裂解法只需较少菌（102个）便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论：两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随茵浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。     

不同粪便DNA提取方法比较分析北大核心CSCD

肠道微生物群落结构和多样性与人体疾病密切相关。然而,相关群落结构分析结果可能受到DNA提取质量等实验因素影响。因此,评估不同DNA提取方法对肠道特定种属的提取效果,对于全面、准确获取人体肠道微生物谱,深入探究肠道微生物群落结构具有指导意义。本研究旨在借助实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT qPCR)技术,以DNA提取纯度、浓度,以及对肠道中特定种属微生物基因组DNA的提取丰度为指标,对5种DNA提取方法进行比较分析。结果表明,试剂盒Q的提取效果最佳,特别是对乳杆菌属和双歧杆菌属等革兰氏阳性菌的提取效果较好。N试剂盒的平均DNA提取浓度较Q试剂盒低,但在纯度方面,二者无显著性差异。与其他3种商用试剂盒(M、PSP、TG)相比,N方法对肠道内指定微生物基因组的提取效果仅次于Q试剂盒,位居第二。相比之下,M试剂盒提取所得DNA,质量较高,但浓度偏低,对于肠道内革兰氏阳性菌的提取效果不很理想。TG试剂盒和PSP试剂盒提取所得DNA在浓度、质量以及细菌丰度方面均不及其他验证的试剂盒。综上,Q试剂盒可作为肠道微生态研究相关实验中获取高质量基因组DNA的提取方法。本研究结果为肠道微生态研究相关实验中基因组DNA提取方法的选择提供参考依据。     

小鼠胚胎胃肠道细菌基因组DNA提取方法比较

目的:筛选能均衡地提取小鼠胚胎胃肠道微生物区系各种细菌总DNA的方法.方法:分别采用反复冻融法、CTAB -SDS法、柱式基因组DNA提取试剂盒法提取小鼠胚胎胃肠道细菌基因组DNA,对其进行琼脂糖凝胶电泳、紫外分光光度计测定、PCR扩增等质量检测.结果:CTAB -SDS方法提取的基因组DNA纯度较高,OD260/OD280平均值最高,为1.845,电泳条带清晰,能满足下游的PCR扩增等分子操作.结论:确定CTAB -SDS方法为提取小鼠胚胎胃肠道细菌基因组DNA的最佳方法,为研究不同种动物胚胎的肠道菌群的结构和多样性奠定了基础.     

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.     

Perturbation‐independent community development in low‐temperature anaerobic biological wastewater treatment bioreactors

The reproducibility and stability of low‐ temperature anaerobic wastewater treatment systems undergoing transient perturbations was investigated. Three identical anaerobic expanded granular sludge bed‐based bioreactors were used to degrade a volatile fatty acid and glucose‐based wastewater under sub‐ambient (15°C) conditions. The effect of a variety of environmental perturbations on bioreactor performance was assessed by chemical oxygen demand removal. Temporal microbial community development was monitored by denaturation gradient gel electrophoresis (DGGE) of 16S rRNA genes extracted from sludge granules. Methanogenic activity was monitored using specific methanogenic activity assays. Bioreactor performance and microbial population dynamics were each well replicated between both experimental bioreactors and the control bioreactor prior to, and after the implementation of most of the applied perturbations. Gene fingerprinting data indicated that Methanosaeta sp. were the persistent, keystone members of the archaeal community, and likely were pivotal for the physical stability and maintenance of the granular biofilms. Cluster analyses of DGGE data suggested that temporal shifts in microbial community structure were predominantly independent of the applied perturbations. Biotechnol. Bioeng. 2010;105: 79–87. © 2009 Wiley Periodicals, Inc.     

用PCR指纹图技术分析焦化废水接触氧化池中悬浮污泥和生物膜的微生物种群组成

目的:应用分子生物学方法,以处理焦化工业废水(A2/O生物膜工艺)中的悬浮污泥和生物膜的微生物群落作为研究对象,分析不同环境微生物群落的组成差异.方法:首先提取群落的总DNA,获得ERIC-PCR和LP-RAPD指纹图谱并进行对比分析,然后结合群落探针杂交的技术,检查同样迁移率的条带的序列同源性,运用UVIBAND/MAP软件比较所得群落指纹图谱的相似性指数,从而可以得到群落差异的量化结果.结果:焦化废水接触氧化池中,悬浮污泥和生物膜的微生物群落组成存在相当大的差异.结论:通过这种差异的比较分析,有可能让我们更准确地了解氧化池中微生物的群落组成情况,有利于分析其与系统功能的关系.     

Molecular method to assess the diversity of Burkholderia species in environmental samples

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.     

喹啉与吲哚驯化的反硝化反应器的微生物群落结构分析比较

[目的]本研究旨在比较分析分别以喹啉和吲哚为底物,在相同条件下驯化的两个反硝化生物反应器的微生物群落结构.[方法]采用相同的种子污泥和相同的驯化条件,经过大约6周的驯化后,两个反应器均达到稳定而高效的污染物去除能力,通过16S rDNA克隆文库技术对两个反应器的微生物群落结构进行研究.[结果]研究发现,微生物群落结构表现出很大的差异.喹啉驯化的群落中所有的OTU都属于Betaproteobacteria,而吲哚驯化的群落中Betaproteobacteria占56.3%,吲哚驯化的群落具有更高的多样性.两个群落的优势OTU也不同,喹啉驯化群落中Thauera及其它Rhodocyclaceae科的微生物占整个群落的73%,而吲哚驯化群落中优势OTU为Comamonadaceae科、Alcaligenaceae科和Rhodocyclaceae科等类型的微生物,其中Comamonadaceae科的一个OTU占整个群落的28.7%.[结论]不同的驯化底物对微生物群落的组成具有较强的选择作用.首次报道并比较了可高效降解喹啉和吲哚的反硝化生物反应器的微生物群落结构.     

盐城滨海滩涂湿地典型植物群落土壤微生物组成与结构特征

土壤微生物是生态系统维持正常结构与功能的重要组成部分,为探究盐城滩涂典型湿地土壤微生物群落结构特征,以江苏盐城滩涂互花米草、藨草、盐地碱蓬、芦苇及淤泥质光滩5种典型群落为对象,采用16S rRNA高通量测序技术分析0—10 cm(表层)、10—30 cm(中层)、30—60 cm(深层)土壤微生物多样性及群落结构。结果表明：(1)几种植物群落间,土壤微生物群落结构差异较大,主要体现在细菌群落结构的差异性,古菌群落结构差异相对较小。光滩与植物群落间,在土壤细菌种类及相对丰度上差异相对较大,互花米草群落与本土植物群落间,在微生物群落的细菌种类组成上存在较大差异;藨草群落土壤表层微生物群落结构与互花米草群落相似,深层与盐地碱蓬、芦苇群落相似。(2)同一群落不同层次土壤微生物群落结构相似,差异小于不同群落间土壤微生物群落的结构差异性;不同群落对应层次间,表深层土壤中五种群落土壤微生物多样性存在显著差异,中层土壤中五种群落微生物多样性差异不显著。总体上,植物群落类型对土壤微生物群落结构的影响大于土壤深度;与本土植物群落相比,互花米草群落土壤主要优势门微生物种类差异较小,但部分优势门微生物相对丰度...     

Microbial community succession in a bioreactor modeling a souring low-temperature oil reservoir subjected to nitrate injection

Injection of up-flow packed-bed bioreactors with excess volatile fatty acids and limiting concentrations of nitrate and sulfate gave complete reduction of nitrate from 0 to 5.5 cm and complete or near-complete reduction of sulfate from 3.2 to 11.5 cm along the bioreactor flow path. Most of the biomass (85%) and most of the genes for nitrate reduction (narG, 96%; napA, 99%) and for sulfate reduction (dsrB, 91%) were present near the inlet (0–5.5 cm) of the 37-cm-long bioreactor. Microbial community analysis by a combination of denaturing gradient gel electrophoresis and pyrosequencing of 16S rRNA amplicons indicated that nitrate-reducing Arcobacter and Pseudomonas species were located from 0 to 3.2 cm and throughout, respectively. Desulfobulbus species were the main sulfate reducers present and acetotrophic methanogens of the genus Methanosaeta predominated at 20–37 cm. Overall, the results indicated a succession of microbial communities along the bioreactor flow path. In the absence of nitrate, the sulfate reduction zone moved nearer to the bioreactor inlet. The sulfide concentration in the bioreactor effluent was temporarily lowered after nitrate injection was re-started. Hence, the bioreactor sulfide output could be disrupted by pulsed, not by constant nitrate injection, as demonstrated also previously in a low-temperature oil field.