PCR-DGGE分析啤酒废水生物处理工艺的微生物区系

应用基于16s rDNA的PER-DGGE(变性梯度凝胶电泳)技术对啤酒废水"水解酸化 SBR"工艺的微生物多样性进行研究.分别取水解酸化池与SBR池中不同深度以及不同处理时段的活性污泥,提取样品总DNA,通过PER扩增、变性梯度凝胶电泳,将16S rDNA(V3区)的PER扩增片段割胶克隆测序确定样品中的微生物群落,与筛选出的高效菌株进行对比分析.结果表明,水解酸化池中的微生物群落随深度的改变,在结构组成和种群数量上均有较大差异,2 m深处微生物群落相对丰富,优势条带突出;SBR池不同深度微生物种群结构一致,沉淀期、进水期和曝气期不同处理时段的微生物种类一致,但优势菌群不同;所测序列v2、23、25、31、h5和15号分别与菌株uncultured Thermotogales sp.Comamonas sp.WT OTU1、Agrobaaerium tumefaciens、Bacillus subtilis、Bdellovibrio bacteriovorus、Comamonas testosteroni有高度同源性,活性污泥样品中的优势条带与高效菌株的序列不同,表明筛选出的高效菌并非为实际处理过程中的优势菌.     

Shinella zoogloeoides BC026对吡啶的降解特性研究

从首钢焦化厂的污水处理系统中分离1株能以吡啶为唯一碳、氮源的细菌BC026,它具有自絮凝特性,对卡那霉素、氨苄青霉素和壮观霉素具有抗性,可在阿须贝无氮培养基中良好生长.通过16S rRNA序列分析和Biolog微生物鉴定系统鉴定,确定该菌为Shinella zoogloeoides.纯菌对单基质的降解实验表明,在30℃、180 r/min和pH为7的条件下,当投菌量为0.1 g/L时,BC026可在17 h内将400 mg/L吡啶完全降解;在吡啶初始浓度为99～1 806 mg/L的无机盐培养基中,BC026均能保持降解活性,较高初始浓度的吡啶对BC026的生长产生一定抑制,但BC026在适应后对吡啶的降解速率较快;降解最适温度为30～35℃,最适pH为8.BC026对吡啶的代谢途径研究表明：降解的第一步是断开吡啶的2条C—N链,生成氨氮和戊二醛,随后戊二醛被氧化为戊二酸,并最终转化为二氧化碳和水;吡啶中的氮有59.5%转化成氨氮.     

应用PCR-DGGE技术研究处理含氨废气的生物滤塔中微生物多样性

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.本文应用DGGE技术对处理含氨废气的生物滤塔中微生物多样性随时间的变化进行了研究.在生物滤塔运行的不同时间采集滤塔中的填料样品,进行了生物滤塔对氨处理效果的分析和DGGE分析.结果显示,污泥和混合填料生物滤塔氨的出气浓度在经过一段时间后,逐渐升高,而堆肥生物滤塔的运行情况较好,对氨的去除效率始终在98%以上.PCR-DGGE图谱显示,不同时间的相同填料中微生物DGGE图谱有着明显的差异性.Shannon指数分析表明,填料中微生物的多样性都随着反应器运行时间的延长而有所减少.运行一个月后,混合填料的微生物多样性指数最低为0.389;其次为污泥填料,其多样性指数为0.473;堆肥填料的微生物多样性程度最高为0.569.生物滤塔对氨的去除效果与填料中微生物多样性Shannon指数之间有一定的相关关系.主成分分析(PCA)显示对于堆肥和污泥来说,填料样品之间微生物群落结构相似性较高,而混合填料样品间的微生物群落结构相似性较低.     

DGGE/TGGE方法在土壤微生物群落研究中的应用

变性梯度凝胶电泳(DGGE)或温度梯度凝胶电泳(TGGE)已成为环境微生物学领城比较微生物群落多样性和检测种群动力学的重要分子手段,本文介绍了DGGE/TGGE方法在土壤微生物群落研究中的应用进展及存在的缺陷,并提出了改进建议.     

南淝河细菌群落结构的研究

为了研究受污染城市河道南淝河在进行修复前微生物群落的多样性,于2009年10月(秋季)和2010年1月(冬季)在南淝河河道中采集水样,直接从水中提取总DNA,用细菌16S rDNA的通用引物对V3区进行PCR扩增,PCR产物经DGGE(变形梯度凝胶电泳)分离后,获得水体细菌群落的DGGE指纹图谱;同时运用平板计数法对水体的异养细菌进行计数。结果表明:不同季节,不同位点南淝河的细菌群落的多样性都很丰富,但不同季节优势种有差异;相同季节临近位点细菌种群结构的相似性较高,且有顺河而下相近位点相似性增高的趋势。南淝河水体异养细菌数量秋季为20.3×103～616.3×103CFU/mL。冬季为2.3×103～53.6×103CFU/mL,冬季异养细菌数量比秋季低一个数量级,并且从上游到下游逐渐增多。但与其他同等富营养化水平的水体相比,其异养细菌含量较低。     

PCR-DGGE技术在城市污水化学生物絮凝处理中的特点

通过PCR-DGGE等分子生物学技术可以不经过常规培养直接从活性污泥和生物膜样品中提取DNA,对16Sr DNA V3区进行PCR扩增,结合DGGE(变性梯度凝胶电泳),从而分析活性污泥与生物膜中微生物种群结构.研究证实,活性污泥培养前后微生物种群结构发生很大的改变.同时对2种污水处理工艺中微生物种群结构进行了对比研究,对同一反应器不同位置微生物分布以及不同工况下的微生物种群结构进行了初步探讨.测定了活性污泥中部分菌种的16S rDNA V3区片段序列,通过NCBI(美国国立生物技术信息中心)基因库比对,初步确定细菌的属.结果显示,PCR-DGGE结合测序技术是一种完全可行的快速进行环境学样品微生物研究的分析方法.     

PCR-DGGE技术解析生物制氢反应器微生物多样性

为了揭示发酵法生物制氢反应器厌氧活性污泥的微生物种群多样性 ,从运行不同时期取厌氧活性污泥 ,通过细胞裂解直接提取活性污泥的基因组DNA .以细菌 16SrRNA基因通用引物F338GC/R5 34进行V3高变异区域PCR扩增 ,长约 200bp的PCR产物经变性梯度凝胶电泳 (DGGE)分离后 ,获得微生物群落的特征DNA指纹图谱 .研究表明 ,不同时期的厌氧活性污泥中存在共同种属和各自的特异种属 ,群落结构和优势种群数量具有时序动态性 ,微生物多样性呈现出协同变化的特征 .微生物多样性由强化到减弱 ,群落结构之间的相似性逐渐升高 ,演替速度由快速到缓慢 .优势种群经历了动态演替过程 ,最终形成特定种群构成的顶级群落 .     

Monitoring microbial community structure and succession of an A/O SBR during start-up period using PCR-DGGE

Polymerase chain reaction (PCR) - denaturing gradient gel electrophoresis (DGGE) protocol was employed for revealing microbial community structure and succession in a sequential anaerobic and aerobic reactor performing enhanced biological phosphorus removal (EBPR) during start-up period.High phosphorus removal was achieved after 15 d.On day 30, phosphorus removal efficiency reached to 83.2% and the start-up was finished.DGGE profiles of periodical sludge samples showed that dominant microbial species were 19 OTUs (operational taxonomy unit ).Unweighted pair-group method using Arithmetic averages (UPGMA clustering analysis revealed that rapid community succession correlated to lower phosphorus removal rate and higher phosphorus removal efficiency reflected on steady community structure.Sequencing results indicated that determined sequences (12 OTUs) belonged to proteobacterium, Actinobacteria, Gemmatimonadales and unaffiliate group.Proteobacterium, Tetrasphaera elongate and Gemmatimonas aurantiaca may act important roles in phosphorus removal.With little amount as known glycogen accumulating organisms, Candidatus Competibacter phosphatis still at accumulating-phase had limited influence on microbial community structure.When climax community was obtained, dominant microbes were 14 OTUs.Microbes in a large amount were uncultured bacterium, Thauera sp., uncultured γ-Proteobacterium and Tetrasphaera elongata.     

中药废水污泥群落结构解析中PCR-DGGE引物的选择与评价

在中药废水生物活性污泥群落DGGE解析过程中,为了探讨16S rDNA通用引物的扩增效率和对污泥细菌群落的表征能力,选用包括简并性引物在内的11对通用引物扩增16S rDNA序列的4个可变区,并应用DGGE图谱评价不同引物的扩增效率和微生物种群多样性.结果表明,采用不同引物对进行DGGE分析时,微生物种群多样性存在着显...     

Structural and metabolic responses of microbial community to sewage-borne chlorpyrifos in constructed wetlands

Long-term use of chlorpyrifos poses a potential threat to the environment that cannot be ignored, yet little is known about the succession of substrate microbial communities in constructed wetlands(CWs) under chlorpyrifos stress. Six pilot-scale CW systems receiving artificial wastewater containing 1 mg/L chlorpyrifos were established to investigate the effects of chlorpyrifos and wetland vegetation on the microbial metabolism pattern of carbon sources and community structure, using BIOLOG and denaturing gradient gel electrophoresis(DGGE) approaches. Based on our samples, BIOLOG showed that Shannon diversity(HV) and richness(S) values distinctly increased after 30 days when chlorpyrifos was added. At the same time, differences between the vegetated and the non-vegetated systems disappeared. DGGE profiles indicated that H Vand S had no significant differences among four different treatments. The effect of chlorpyrifos on the microbial community was mainly reflected at the physiological level. Principal component analysis(PCA) of both BIOLOG and DGGE showed that added chlorpyrifos made a difference on test results.Meanwhile, there was no difference between the vegetation and no-vegetation treatments after addition of chlorpyrifos at the physiological level. Moreover, the vegetation had no significant effect on the microbial community at the genetic level. Comparisons were made between bacteria in this experiment and other known chlorpyrifos-degrading bacteria. The potential chlorpyrifos-degrading ability of bacteria in situ may be considerable.     

不同16SrDNA靶序列对DGGE分析活性污泥群落的影响

为探讨不同通用引物扩增16S rDNA靶序列对活性污泥微生物群落分析的影响,更合理的利用变性梯度凝胶电泳(DGGE)技术分析活性污泥样品.从连续流搅拌槽式反应器(CSTR)中获取活性污泥,以3对通用引物341f/534r、968f/1 401r和341f/926r扩增16S rDNA序列,用DGGE分离PCR扩增产物.研究表明采用不同引物对进行DGGE分析时,群落多样性和动态存在显著的差异.341f/534r和968f/1 401r的靶序列分离效果较好,341f/926r的靶序列分离效果较差.引物341f/534r和341f/926r DGGE图谱显示S2和S3相似性高,引物968f/1 401r DGGE图谱显示S1和S2相似性高.由此可见采用不同引物对进行DGGE分析时,群落结构之间的相似性和动态是不一致的.341f/534r的DGGE图谱中条带丰富,多样性最好,968f/1 401r的DGGE图谱次之,341f/926r DGGE图谱条带最少,多样性也较差.因此,在利用DGGE分析活性污泥样品时采用引物341f/534r和968f/1 401r是比较适宜的.     

不同16SrDNA靶序列对DGGE分析活性污泥群落的影响

为探讨不同通用引物扩增16S rDNA靶序列对活性污泥微生物群落分析的影响,更合理的利用变性梯度凝胶电泳(DGGE)技术分析活性污泥样品.从连续流搅拌槽式反应器(CSTR)中获取活性污泥,以3对通用引物341f/534r、968f/1 401r和341f/926r扩增16S rDNA序列,用DGGE分离PCR扩增产物.研究表明采用不同引物对进行DGGE分析时,群落多样性和动态存在显著的差异.341f/534r和968f/1 401r的靶序列分离效果较好,341f/926r的靶序列分离效果较差.引物341f/534r和341f/926r DGGE图谱显示S2和S3相似性高,引物968f/1 401r DGGE图谱显示S1和S2相似性高.由此可见采用不同引物对进行DGGE分析时,群落结构之间的相似性和动态是不一致的.341f/534r的DGGE图谱中条带丰富,多样性最好,968f/1 401r的DGGE图谱次之,341f/926r DGGE图谱条带最少,多样性也较差.因此,在利用DGGE分析活性污泥样品时采用引物341f/534r和968f/1 401r是比较适宜的.     

Comparison the effects of bioaugmentation versus biostimulation on marine microbial community by PCR–DGGE: A mesocosm scale

In order to better understand the effects of biostimulation and bioaugmentation processes on a marine microbial community, three different mesocosm experiments were planned. Natural seawater(10.000 L) was artificially polluted with crude oil(1 L) and(1) inorganic nutrients(Biostimulating Mesocosm, BM),(2) inorganic nutrients and an inoculum of Alcanivorax borkumensis SK2(Single Bioaugmentation Mesocosm, SBM),(3) inorganic nutrients and inoculums of A. borkumensis SK2 and Thalassolituus oleivorans MIL-1(Consortium Bioaugmentation Mesocosm, CBM). During the experimental period(20 days), samples were taken from each mesocosm and the community structure was analyzed by PCR–DGGE. The 16 S r RNA gene DGGE banding patterns and sequence analysis demonstrated that biostimulation had the lowest effect on microbial biodiversity in the mesocosms; however, the biodiversity of the marine microbial community dramatically decreased in the CBM(Shannon index was 0.6 in T3). The community structures among the three mesocosms were also markedly different,and major bacteria derived from DGGE bands were related to uncultured Gamma Proteobacteria. The biodegradation results show that the Single Bioaugmentation Mesocosm(SBM) system had the highest percentage of degradation(95%) in comparison to the BM mesocosm(80%) and CBM(70%).     

Cd对欧美杂交杨生长紫色土和冲积土微生物多样性的影响

研究了现实环境中cd污染水平对长江流域典型土壤微生物多样性的影响,分析了现实Cd浓度胁迫对盆栽欧美杂交杨（Populus deltoides×Populus nigra）生长的紫色土和冲积土中可培养的微生物数量、生物量和细菌群落多样性的影响．结果表明,cd处理显著增加了栽植杨树的紫色土中可培养细菌和放线菌数量,降低了可...     

超声波促进好氧/缺氧污泥消化过程中细菌群落结构分析

应用基于16S rDNA的PCR-DGGE对超声波-好氧/缺氧污泥消化过程中微生物种群的多样性进行研究.通过SDS细胞裂解法提取不同时期污泥中的基因组DNA,采用通用引物进行V3区域PCR扩增,长约190 bp的PCR产物经DGGE分离后,获得污泥微生物群落的DNA特征指纹图谱,对条带进行切胶测序,使用序列数据进行同源性分析并建立系统发育树.DGGE图谱表明,在反应器运行的不同时期,微生物群落结构发生动态演替.5、10、15、20、25 d微生物相似性与0 d相比分别为61.2%、48.2%、46.4%、42.6%、41.7%,总细菌Shannon指数经历了一个从逐渐减少到趋于稳定的过程,这表明超声波改变污泥内部性质,导致微生物多样性的降低.UPGMA聚类分析将DGGE图谱区分为三大族群并对应于不同运行时期.测序结果表明,超声波-好氧/缺氧污泥消化中微生物群落主要为Firmicute、Genuscitrobacter、Bacilli、α-Proteobacteria、β-Proteobacteria.     

PCR—DGGE技术在废水生物处理中的应用研究

聚合酶链式反应一变形梯度凝胶电泳（PCR—DGGE）作为一种分子指纹图谱能够较准确地反应污水处理过程中微生物群落结构多样性及其动态变化而广泛应用于废水生物处理技术的研究中。PCR—DGGE技术不仅能对样品中微生物群落结构多样性及种群演替进行分析,还能用于污水处理中优势菌群的分离与鉴定,克服了传统方法费时费力、培养条件苛刻等局限性,进而运用该技术构建高效的污染物降解工程菌,提高难降解有机物的去除效率,调节实际工艺参数,提高污水处理效率。     

多环芳烃污染对桑园土壤微生物结构及种群多样性的影响

采用磷脂脂肪酸(PLFAs)分析法和变性梯度凝胶电泳(DGGE)考察了受多环芳烃(PAHs)污染的桑园3个区域的土壤微生物群落结构及种群多样性的变化.PLFAs分析结果表明,区域2中微生物PLFA总量最高,主要为细菌和真菌;聚类分析揭示,土壤中微生物的PLFAs主要分为3大类群;冗余分析表明,土壤PAHs污染程度对土壤微生物群落结构有一定的影响.DGGE指纹图谱分析结果显示,在PAHs污染较高区域,其电泳条带较多,且3个区域中Shannon指数和Simpson优势度差异达显著水平,区域2种群优势度较高;主成分分析表明,不同区域微生物的种群结构存在显著性差异.     

IC反应器处理啤酒废水的效能及其微生物群落动态分析

在35℃下连续运行内循环厌氧反应器(IC),通过不断提高进水容积负荷,研究不同运行负荷下反应器处理模拟啤酒废水的运行情况,并探讨微生物的种群结构与其活性变化之间的相互关系.IC反应器效能分析表明其最大进水容积负荷(以COD计)可达20 kg.(m3.d)-1,COD去除率为85%以上,最大比产甲烷活性(以VSS计)可达210 mL.(g.d)-1;污泥脱氢酶活性与细菌DGGE图谱表明反应器中脱氢酶活性的变化趋势与细菌DGGE条带总光强度的变化趋势一致,DGGE图谱中细菌条带的总光密度值可作为厌氧体系中生物量的一个参考指标;辅酶F420与古细菌DGGE分析表明,污泥中辅酶F420含量与甲烷鬃菌属(Methanosaeta sp.)的相对丰度有一定的相互联系,在低负荷条件下甲烷八叠球菌属(Methanosarcina mazei)优势地位比较显著,随着负荷的提高,甲烷鬃菌属(Methanosaeta sp.)优势地位逐渐显著,且辅酶F420含量随之升高,当最大负荷为20kg.(m3.d)-1时,辅酶F420的含量(以VSS计)达到0.16μmol.g-1,而此时甲烷鬃菌属(Methanosaeta sp.)占据尤为明显的优势地位;聚类分析(UPGAMA)和Shannon指数也表明在不同反应负荷下,微生物的群落结构变化明显.     

短程硝化工艺出水水质及微生物区系分析

实验采用短程硝化工艺处理高氨氮废水,并用PCR-DGGE分析了系统中的微生物区系。结果表明,进水氨氮浓度在50～400mg/L之间梯度增加时,出水氨氮浓度在10mg/L以下;随着氨氮浓度的增加,亚硝态氮积累率逐渐升高,当氨氮浓度达到300mg/L时,积累率达到90%左右。DGGE分析结果表明,随着运行时间的延长,微生物区系多样性减少;氨氮负荷为50mg/L与400mg/L相比,相似性为0.24。     

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.