PCR-DGGE技术在水解酸化-缺氧法处理采油废水的微生物研究中的应用

采用PCR-DGGE技术直接从水解酸化和缺氧反应器中的污泥样品提取DNA,测定部分菌种的16S rDNA V3区片段序列,通过NCBI基因库比对,初步确定不同生物反应器内优势菌种,并进行了多样性指数分析.结果表明,水解酸化反应器中的生物膜与缺氧反应器中悬浮污泥微生物种群结构存在较大的差异,显示了在不同环境条件下,微生物群落结构的连续动态变化过程.     

膜生物反应器中不同阶段微生物群落结构演变的研究

为了研究膜生物反应器（membrane bioreactor, 简称MBR）中微生物群落结构的演变情况,对不同阶段运行状况下(包括培养驯化阶段、初期增长阶段、污泥流失阶段和恢复成熟阶段4个阶段)污泥中的微生物进行了考察。从MBR污泥中提取细菌总基因组DNA,进行聚合酶链式反应变性梯度凝胶电泳(PCR-DGGE)图谱的直观分析,以及总细菌的Shannon多样性指数、各污泥样品的相似性分析和聚类分析。研究表明,反应器中的微生物群落比较丰富。不同的阶段状况下,微生物的群落结构变化比较明显,能够比较好地反映MBR的运行状况和系统处理效能的关系,并且发现微生物的群落结构能够随着反应器的运行状况的变化做出调整恢复。     

用于填埋场恶臭气体控制的微生物除臭剂筛选及其除臭机制研究

从上海市老港垃圾填埋场的污泥和垃圾渗滤液中分离筛选得到数株具有较强除臭能力的菌株,并制成复合微生物除臭剂,考察了该除臭剂对污泥和垃圾释放NH3以及恶臭气体浓度等的去除效果,并结合聚合酶链式反应(PCR)-变性梯度凝胶电泳(DGGE)技术对该除臭剂的微生物群落结构进行分析,初步探究其除臭机制。结果表明:(1)实验室小试时,48h,复合微生物除臭剂对污泥释放NH3的去除率维持在30%~45%,对垃圾释放NH3的去除率维持在25%~35%,去除效果好于EM菌剂。(2)污泥中转码头100t污泥中试试验表明,在复合微生物除臭剂投加量为0.5t条件下,24h后复合微生物除臭剂对NH3的去除率达到37.5%,恶臭气体浓度下降了19.1%。(3)PCR-DGGE结果显示,复合微生物除臭剂的优势菌种为产碱杆菌、芽孢杆菌和显核菌,与EM菌剂差异较明显,且Shannon-Wiener多样性指数明显高于EM菌剂。其除臭机制可能为化学吸收与微生物降解、抑制的联合作用除臭。     

污泥好氧颗粒化过程中微生物群落结构的演变与分析

为了揭示颗粒污泥形成过程中微生物群落结构多样性的演变过程,以人工配水为进水,在SBR中采用厌氧/好氧循环的手段成功培育出具有聚磷特性的颗粒污泥,利用基于16S rDNA的PCR-DGGE技术获得了微生物群落的DNA特征指纹图谱,对条带进行了统计分析和切胶测序,并建立了系统发育树。结果表明,污泥沉降性能的改善要先于颗粒污...     

溶解氧对晚期垃圾渗滤液短程硝化及微生物群落结构变化的影响

以上海老港垃圾填埋场配套污水处理设施中的污泥为菌种源,在序批式活性污泥反应器(SBR)中对晚期垃圾渗滤液进行短程硝化处理,调节SBR中溶解氧浓度,考察溶解氧对渗滤液短程硝化的影响,分析不同溶解氧条件下污泥微生物群落结构的变化.结果表明,低溶解氧(0.2～0.5 mg/L)条件下,SBR可以获得较高的短程硝化效率,反应17h后,SBR内亚硝态氮/氨氮(质量比)为1.05,氨氮负荷可达到1.5 kg/(kg·d)(以每千克污泥悬浮固体每天承担的氨氮计),出水可以满足后续厌氧氨氧化处理的要求.从污泥变形梯度凝胶电泳(DGGE)图谱中可以看出,SBR微生物群落结构中主要优势种有uncultured Bacteroidetes bacterium、uncultured bacterium、uncultured Candidatus Amoebophilus sp.等.随着溶解氧含量的升高,SBR内微生物群落结构的多样性有所升高,但溶解氧对微生物群落结构影响有限.     

SBR中SRT对总细菌群落结构的影响研究

为了揭示序批式反应器不同污泥停留时间(SRT)下总细菌群落结构的异同及SRT变化对总细菌群落结构的影响,应用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)进行研究。通过克隆测序发现,不同的SRT条件下生物多样性和种群结构会有所差异,既存在各SRT条件下相同的优势菌群(Escherichia coli和Aeromonas sp.),也存在某些SRT下特有的优势菌群(Uncultured Peptostreptococcaceae),SRT为40 d时检测到以降解硫酸盐获得能源的优势微生物。研究还表明,SRT为40 d时多样性指数取得最大值,各SRT条件下微生物的种群相似性差别较大。     

以游泳馆污水为处理对象的SBR中不同污泥负荷下氨氧化菌群落的演变

为了研究污泥负荷对SBR系统内活性污泥微生物中氨氧化菌群落结构的影响,应用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,对不同污泥负荷条件下SBR处理经投加葡萄糖调节的游泳馆污水的活性污泥中氨氧化菌进行了分析。研究结果表明,氨氧化菌的群落结构在不同污泥负荷条件下变化明显,在有机碳源较低的情况下生长旺盛,随着污泥负荷的提高其DGGE图谱条带数量逐渐减少,亮度逐渐减弱;在高污泥负荷环境下,氨氧化菌受到严重抑制,多样性指数大幅下降,并从系统中消失。SBR系统内氨氧化菌大部分为不可培养的变形菌,最常见的氨氧化菌是β变形菌中的亚硝化螺菌和亚硝化单细胞菌。     

硫自养高氯酸盐还原菌培养与驯化

考察了以污水处理厂的好氧污泥作为接种污泥,以S0作为电子供体培养驯化硫自养高氯酸盐还原菌的过程,并利用PCR-DGGE技术对不同阶段的污泥样品进行群落分析。结果表明,接种污泥在经过短暂的适应期后能具有较高的还原ClO-4的性能,随着驯化时间的延长,降解速率显著提高。培养驯化过程中微生物种群结构发生了演替变化,菌种Dechloromonas sp.CL、Quadrisphaera granulorum、Comamonadaceae bacterium 32-4、Acidovorax caeni、filamentous bacterium Plant1Iso8、Candidatus Nitrospira defluvii存在整个驯化培养阶段。随着培养驯化时间的延长,菌种Bacteroidetes bacterium S22-33、Herbaspirillum huttiense逐渐消失,出现新菌种Methyloversatilis universalis、beta proteobacterium HTCC379、beta proteobacterium CDB21和Clostridium bifermentans,并逐渐成为优势菌种。     

2种好氧颗粒污泥反应器工艺处理城市污水

以低浓度城市污水作为原水,絮状污泥作为接种污泥,分别采用序批式生物反应器(SBR)与气升式间歇反应器(SBAR)培养好氧颗粒污泥.分别考察2种工艺中好氧污泥颗粒化过程中的污泥特性以及对污染物的去除效果,结果表明2种工艺均成功培养出稳定的好氧颗粒污泥.SBR工艺中好氧颗粒污泥的污泥容积指数(SVI)、挥发性组分所占比例(...     

不同污泥负荷下SBR处理游泳馆污水的微生物群落的演变

污泥负荷直接影响微生物的生长模式,当污泥负荷发生变化时,短时间内微生物群落结构将发生明显变化。为了研究污泥负荷冲击对SBR系统内活性污泥微生物群落结构的影响,应用聚合酶链式反应-变性梯度凝胶电泳(PCRDGGE)技术,对不同污泥负荷冲击时,SBR处理游泳馆污水中的活性污泥微生物进行了考查。研究表明,在不同污泥负荷冲击的条件下,以MBR污泥为接种污泥,SBR工艺处理游泳馆污水系统内活性污泥微生物群落结构变化明显,多样性指数随着污泥负荷升高而逐渐增加并趋于稳定,但污泥冲击负荷过高多样性指数反而下降,SBR系统内微生物菌种大部分为未经培养菌种,肠杆菌属、甲苯单胞菌属以及γ-变形菌纲细菌等。微生物通过对不同负荷阶段环境条件的适应及演变,逐渐形成了适应相应污泥负荷的微生物种群。     

应用于PCR-DGGE分析的活性污泥微生物总DNA提取方法比较

探讨适用于PCR-DGGE分析研究的活性污泥细菌和真菌的DNA提取方法。采用5种方法提取活性污泥微生物基因组DNA,以DNA纯度、含量、片段大小及DGGE条带多样性作为考察指标评价提取方法的优劣,以确定最佳实验方案。紫外吸收法和琼脂糖凝胶电泳结果显示,试剂盒法提取的DNA含量最低,其余4种方法获得的DNA含量无显著差异,就DNA纯度而言,试剂盒法最优;除高温裂解法对真菌细胞壁裂解效果较差外,其他4种方法均能不同程度地裂解细菌和真菌细胞;DGGE结果表明,高温裂解法获得的细菌条带最多,基于SDS的细胞裂解法得到的真菌条带最多。综合分析,高温裂解法更适合于活性污泥中细菌的PCR-DGGE分析,基于SDS的细胞裂解法则更适合于污泥中真菌的PCR-DGGE分析。     

Effect of flocculant on microbial populations and function in Chem-Bioflocculation (CBF) treatment process using PCR-DGGE

The group-specific primers were applied to investigate microbial communities in Chem-Bioflocculation (CBF) reactor by PCR-DGGE method. α-proteobacteria, β-proteobacteria, actinomycetes and acidobacterium, were analysed by using diversity index at different dosage of Polyaluminium Ferric Chloride (PAFC) based on DGGE patterns. Clustering analysis showed that community structure of specific bacteria groups changed when flocculant dosage was adjusted, but flocculant dosage had dissimilar effect on the community structure of different bacteria group. α-proteobacteria possibly was the most influential populations relative to biological function in CBF reactor. The biological function should be inhibited when the flocculant dosage exceeded 80 mg/L.     

有机碳源条件下厌氧氨氧化SBBR中的微生物多样性

研究有机碳源对SBBR厌氧氨氧化菌群等微生物的影响。采用16S rDNA序列与PCR-DGGE分析技术相结合的方法,对稳定运行的反应器内的活性污泥和生物膜样品,进行细菌多样性图谱分析,同时采用巢式PCR-DGGE技术对浮霉状菌属(Planctomycetes)细菌进行分析。结果表明,在有机碳源反应系统细菌条带数和多样性指数均高于无机系统,与活性污泥相比,生物膜表尤为明显。当进水不含有机碳源时,氨氧化细菌(ammonia oxidizing bacteria,AOB),厌氧氨氧化菌(anaerobic ammonia oxidizing bacteria,ANAMMOX)为优势功能菌;当进水含有机碳源时,系统中存在的AOB以亚硝化单胞菌(Nitrosomonas sp.)为优势菌群,同时存在反硝化菌,如索氏菌(Thauera sp.)以及厌氧氨氧化菌,它们共同作用完成N的去除。此外,与无机碳源系统相比,有机碳源的存在,有利于浮霉状菌的积累,但压缩了ANAMMOX的生存空间。本研究可为厌氧氨氧化工艺处理低C/N比有机废水提供了理论依据。     

Fungicide impacts on microbial communities in soils with contrasting management histories

The impacts of the fungicides azoxystrobin, tebuconazole and chlorothalonil on microbial properties were investigated in soils with identical mineralogical composition, but possessing contrasting microbial populations and organic matter contents arising from different management histories. Degradation of all pesticides was fastest in the high OM/biomass soil, with tebuconazole the most persistent compound, and chlorothalonil the most readily degraded. Pesticide sorption distribution coefficient (K(d)) did not differ significantly between the soils. Chlorothalonil had the highest K(d) (97.3) but K(d) for azoxystrobin and tebuconazole were similar (13.9 and 12.4, respectively). None of the fungicides affected microbial biomass in either soil. However, all fungicides significantly reduced dehydrogenase activity to varying extents in the low OM/biomass soil, but not in the high OM/biomass soil. The mineralization of subsequent applications of herbicides, which represents a narrow niche soil process was generally reduced in both soils by azoxystrobin and chlorothalonil. 16S rRNA-PCR denaturing gradient gel electrophoresis (DGGE) indicated that none of the fungicides affected bacterial community structure. 18S rRNA PCR-DGGE analysis revealed that a small number of eukaryote bands were absent in certain fungicide treatments, with each band being specific to a single fungicide-soil combination. Sequencing indicated these represented protozoa and fungi. Impacts on the specific eukaryote DGGE bands showed no relationship to the extent to which pesticides impacted dehydrogenase or catabolism of herbicides.     

The effects of perennial ryegrass and alfalfa on microbial abundance and diversity in petroleum contaminated soil

Enhanced rhizosphere degradation uses plants to stimulate the rhizosphere microbial community to degrade organic contaminants. We measured changes in microbial communities caused by the addition of two species of plants in a soil contaminated with 31,000 ppm of total petroleum hydrocarbons. Perennial ryegrass and/or alfalfa increased the number of rhizosphere bacteria in the hydrocarbon-contaminated soil. These plants also increased the number of bacteria capable of petroleum degradation as estimated by the most probable number (MPN) method. Eco-Biolog plates did not detect changes in metabolic diversity between bulk and rhizosphere samples but denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified partial 16S rDNA sequences indicated a shift in the bacterial community in the rhizosphere samples. Dice coefficient matrices derived from DGGE profiles showed similarities between the rhizospheres of alfalfa and perennial ryegrass/alfalfa mixture in the contaminated soil at week seven. Perennial ryegrass and perennial ryegrass/alfalfa mixture caused the greatest change in the rhizosphere bacterial community as determined by DGGE analysis. We concluded that plants altered the microbial population; these changes were plant-specific and could contribute to degradation of petroleum hydrocarbons in contaminated soil.     

水解溶菌酶污泥减量过程中的微生物群落变化

利用水解溶菌酶对SBR系统中的剩余污泥进行减量。通过与未加水解溶菌酶的相同系统对比,研究了水解溶菌酶作用下的SBR系统中剩余污泥的减量效果与微生物群落结构的变化。结果表明,在50 d的运行期内,水解溶菌酶作用下的SBR系统中剩余污泥减量总计达到76.3%,同时该系统对COD与TN的平均去除率分别为88.2%与53.8%。通过PCR-DGGE分析可知,随着运行时间的增加两系统微生物群落结构的差异逐步明显,SBR系统中原有的部分优势微生物在水解溶菌酶的作用下逐渐减弱。另外,对微生物群落的部分优势细菌进行克隆测序和系统发育树分析,通过鉴定获得的7条细菌的16S rDNA序列,它们分别与放线菌和杆菌同源性在97%以上。     

Variations of bacterial community structure in flooded paddy soil contaminated with herbicide quinclorac

The denaturing gradient gel electrophoresis (DGGE) method was applied to determine the relative genetic complexity of microbial communities in flooded paddy soil treated with herbicide quinclorac (3,7-dichloro-8-quinoline-carboylic acid). The results obtained showed a significant effect of quinclorac on the development of bacterial populations in soils contaminated with different concentrations of the herbicide at the early time after application. In general, however, the number of populations of the same soil sample treated with the same concentration of the quinclorac differed obviously with increasing incubation time within the early 8 weeks. The scale of differences in banding patterns-showed that the microbial community structures of the quinclorac-treated and non-quinclorac-treated soils were not significantly different after 21 weeks of incubation. Quantification, as demonstrated in this paper, was studied by establishing dose-response relationships. Significant pattern variations were quantified. Prominent DGGE bands were excised, cloned and sequenced to gain insight into the identities of predominant bacterial populations. The majority of DGGE band sequences were related to bacterial genera Clostridium, Sphingobacterium, Xanthomonas and Rhodococcus.     

A preliminary in vitro assessment of GroBiotic-A, brewer's yeast and fructooligosaccharide as prebiotics for the red drum Sciaenops ocellatus

This study examined the effects of brewers yeast, fructooligosaccharide (FOS), and GroBiotic-A, a mixture of partially autolyzed brewers yeast, dairy components and dried fermentation products, on the intestinal microbial community of red drum, Sciaenops ocellatus. Gastrointestinal (GI) tracts were aseptically removed from three sub-adult red drum previously maintained on a commercial diet and placed in an anaerobic chamber. Intestinal contents were removed, diluted and incubated in vitro in one of four liquid media: normal diet alone, diet + 2% (w/w) GroBiotic-A, diet + 2% brewers yeast, and diet + 2% FOS. After 24 and 48 h of incubation at 25 degrees C, supernatants were removed for volatile fatty acid (VFA) analysis and DNA was extracted for denaturing gradient gel electrophoresis (DGGE) analysis. Polymerase chain reaction (PCR) was performed on a highly conserved region of M 16S rDNA and the amplicons were subjected to DGGE. The microbial community (MC) fingerprint was used to distinguish microbial populations. The intestinal contents incubated with GroBiotic-A had significantly (P<0.05) higher acetate and total VFA concentrations at 48 h compared to the other treatments. DGGE analysis demonstrated that the microbial community was significantly altered by Grobiotic-A and brewers yeast.     

Variations of Bacterial Community Structure in Flooded Paddy Soil Contaminated with Herbicide Quinclorac

The denaturing gradient gel electrophoresis (DGGE) method was applied to determine the relative genetic complexity of microbial communities in flooded paddy soil treated with herbicide quinclorac (3,7-dichloro-8-quinoline-carboylic acid). The results obtained showed a significant effect of quinclorac on the development of bacterial populations in soils contaminated with different concentrations of the herbicide at the early time after application. In general, however, the number of populations of the same soil sample treated with the same concentration of the quinclorac differed obviously with increasing incubation time within the early 8 weeks. The scale of differences in banding patterns-showed that the microbial community structures of the quinclorac-treated and non-quinclorac-treated soils were not significantly different after 21 weeks of incubation. Quantification, as demonstrated in this paper, was studied by establishing dose-response relationships. Significant pattern variations were quantified. Prominent DGGE bands were excised, cloned and sequenced to gain insight into the identities of predominant bacterial populations. The majority of DGGE band sequences were related to bacterial genera Clostridium, Sphingobacterium, Xanthomonas and Rhodococcus.