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.     

基于高通量测序的OMS-2对序批式反应器系统微生物群落的影响

随着纳米材料的广泛应用,越来越多的纳米材料随着废水进入污水处理厂,纳米材料对污水生物处理系统的潜在影响越来越受到重视。探讨了氧化锰八面体分子筛(manganese oxide octahedral molecular sieve, OMS-2)纳米颗粒对序批式反应器(sequencing batch reactor,SBR)中活性污泥微生物群落结构的影响;以活性艳红X-3B溶液模拟印染废水,将不同浓度的OMS-2混入稳定运行的SBR中,采用Illumina MiSeq高通量测序分析技术,对不同SBR中微生物分布规律进行了研究。结果表明:SBR添加0.25 g·L~(-1)的OMS-2后,其COD去除率和脱色率分别提升了6%和13.6%;Illumina MiSeq高通量测序显示,在混入0.25 g·L~(-1)的OMS-2后,SBR内污泥菌群中拟杆菌门(Bacteroidetes)和变形菌门(Proteobacteria)的微生物DNA序列操作分类单元(operational taxonomic units,OTU)分别增加了16.8%和96.4%,这2类菌种可能提升了SBR降解有机污染物的能力;不同浓度的OMS-2改变了菌群的多样性和结构,低浓度的OMS-2可以提升微生物菌群的多样性和改变菌群的结构。X射线光电子能谱(XPS)分析表明,OMS-2在SBR中存在锰(Ⅳ)/锰(Ⅲ)转变为锰(Ⅱ)的氧化还原反应,该过程可能影响了菌群的组成。研究为纳米材料的实际应用和环境风险提供了参考。     

运行方式对低基质厌氧氨氧化系统脱氮及菌群结构的影响

为了考察运行方式对厌氧氨氧化系统脱氮性能及菌群结构的影响,建立一套厌氧移动床生物膜反应器,在(25±1)℃恒温、低基质(TN≤60 mg·L~(-1))条件下,分别以连续式和间歇式方式运行,采用高通量测序,基于直系同源蛋白簇基因(COGs),对16S rRNA扩增子测序结果进行功能预测,来表征微生物菌群结构和微生物功能的变化。结果表明:系统总氮负荷为(227±13) mg·(L·d)~(-1)时,间歇式运行脱氮效率(90.6%)优于连续式运行效率(74.6%),生物膜厌氧氨氧化细菌的相对丰度高于悬浮污泥;反应器由连续式变为间歇式运行后,主要功能菌属Ca. Brocadia丰度降低,同时,具有部分反硝化作用的Pseudomonas菌丰度出现明显升高。进一步分析可知,在适量的有机物条件下,间歇式运行能够获得更好的厌氧氨氧化与反硝化协同处理效果。本研究结果可为污水处理厂的实际运行提供参考。     

MBBR一体式耦合短程硝化-厌氧氨氧化处理污泥水

采用移动床生物膜反应器(MBBR)处理已回收磷后的实际污泥水,在进水平均氨氮浓度为167.51 mg·L~(-1)、HRT为22.24 h、DO为0.5 mg·L~(-1)和温度为24～26℃的条件下实现了一体式短程硝化-厌氧氨氧化过程的耦合,对氨氮和总无机氮的最大去除率可达96%和79.7%。但是,一体式反应器受DO浓度影响较大,维持稳定的DO浓度对于系统的氮去除非常重要。荧光原位杂交(FISH)及高通量测序结果表明,MBBR的生物膜及活性污泥中Nitrosomonas菌分别占总菌数的10.46%和21.46%,厌氧氨氧化菌的优势菌种Candidatus Kuenenia在生物膜和活性污泥中分别占总菌数的4.13%和0.71%。因此,MBBR中活性污泥主要完成亚硝化,生物膜主要完成厌氧氨氧化,常温条件下,两者在一个反应体系中共同完成了对污泥水中氮的高效自养脱除。以上结果表明了一体式反应器处理实际污泥水的可行性,可为该工艺在实际工程中的应用提供参考。     

饮用水管网生物膜细菌群落特征及其对腐蚀的影响

通过454高通量测序对北方A和B两实际饮用水管网系统中生物膜细菌群落特征进行表征,并研究了其对管网腐蚀产物组成的影响。结果表明,A管网生物膜细菌群落丰度和多样性高于B管网生物膜。在门水平上,两管网生物膜细菌群落主要为变形菌门Proteobacteria,但B管网生物膜中的相对丰度(67.10%)高于A(46.50%)。在纲水平上,A管网生物膜主要为β-变形菌纲Betaproteobacteria,B管网生物膜中主要为β-变形菌纲Betaproteobacteria和δ-变形菌纲Deltaproteobacteria。在属水平上,A管网生物膜中腐蚀相关菌群主要为硝酸盐还原菌和铁还原菌,其腐蚀产物主要为α-Fe OOH和Fe_3O_4,而B管网生物膜中硫酸盐还原菌特别是脱硫弧菌属Desulfovibrio含量较高,其可能与管垢中绿锈含量高有关。     

北方某污水厂MBBR工艺升级改造后的高效脱氮除磷效果

为研究北方某污水厂经过MBBR提标改造后,在秋冬季进水碳源较低的条件下生化段脱氮除磷率高于理论值的原因,采用沿程水质测定法及小试实验的方法验证其脱氮除磷效果,并通过基于16S rRNA的高通量测序对好氧段微生物菌群进行分析。结果表明,系统在好氧区存在显著的TN去除,去除率约占15%～20%,在缺氧区存在显著的TP去除,去除率高达63.04%,显示系统内发生了同步硝化反硝化(SND)和反硝化除磷现象。通过小试实验验证了好氧SND现象主要来自于悬浮载体,得益于悬浮载体生物膜功能菌分层分布;反硝化除磷现象则得益于系统较长的缺氧停留时间及较短的泥龄。系统中SND和反硝化除磷的存在是系统在低碳源消耗条件下取得高效脱氮除磷效果的主要原因;微生物菌群分析验证了SND现象主要来源于悬浮载体;悬浮载体上硝化菌群相对丰度为28.56%,是污泥的14倍,反硝化菌相对丰度约8.34%,为SND效果的发生提供了微观保证;污泥中存在Candidatus Accumulibacter、Acinetobacter和Tetrasphaera,为该污水厂存在反硝化除磷及高效除磷现象提供了微观证据。     

贫营养条件下2种MBR污泥微生物的菌群对比

为了揭示贫营养环境下MBR污泥微生物群落结构的演替和菌群变化的异同,取洗浴再生水、工业再生水MBR的污泥进行周期培养,利用PCR-DGGE和克隆测序技术获得了DNA指纹图谱并建立系统发育树。研究表明,微生物群落结构在贫营养条件下演替明显,洗浴水污泥微生物形成新的优势菌群(Uncultured Pseudomonas)而工业水只维持了原有的部分菌群(Uncultured Sphaerotilus)。2种污泥培养过程中种群多样性变化突出且差异显著。同时洗浴水污泥菌群相似性在培养第8天时发生突变而工业水总体变化平缓。克隆测序表明2种MBR污泥中既有与贫营养环境适生的共性种属又有与各自来源相对应的特性种属。菌群特异性与废水来源紧密相关,是造成2种污泥对贫营养环境适应能力不同的根本原因。     

2种磷酸盐生长环境对同步去除与富集磷酸盐工艺的影响

针对同步去除与富集磷酸盐溶液的问题,研究了在低磷环境和低磷高磷交替环境下悬浮填料生物膜反应器的除磷能力和释磷能力,采用扫描电子显微镜(SEM)和高通量测序对第0、45和95天的污泥进行了表征。结果表明:低磷环境下好氧出水磷酸盐浓度稳定在0.5 mg·L~(-1)以下,厌氧阶段的最大释磷量为6.05 mg·L~(-1);在低磷高磷交替环境中,好氧出水磷酸盐浓度基本在0.5 mg·L~(-1)以下,富磷溶液浓度最高可达63 mg·L~(-1)。SEM结果表明,同步去除与富集磷酸盐的悬浮填料生物膜反应器中的主要微生物是杆状菌。高通量测序结果表明:第0、45和95天的变形菌门(Proteobacteria)的相对丰度分别为48.3%、57.1%和89.1%,占主导地位;而红环菌科(Rhodocyclaceae)的相对丰度分别为18.1%、19.0%和30.8%,是反应器中的优势菌科;动胶菌属(Zoogloea)是同步去除与富集磷酸盐的悬浮填料生物膜工艺中的主要功能菌。在悬浮填料生物膜工艺中,低磷高磷交替的生长环境下培养的聚磷生物膜能够使好氧出水的磷酸盐浓度达到国家排放标准,并在厌氧阶段得到高浓度的磷酸盐富集溶液,且这种生长环境更适合聚磷微生物的生长。     

不同供氧策略和氨氮浓度下SBR中微生物群落的演变

以供氧策略和进水氨氮浓度为控制因素,应用PCR-DGGE技术并结合系统处理效果研究SBR内活性污泥微生物群落结构的演替情况。结果表明,在一定范围内,进水氨氮浓度越小越有利于COD和氨氮的去除,同时污泥性状主要受供氧时间的影响,运行前40 d,MLSS值均在4 000 mg·L~(-1)以上波动。微生物群落结构受供氧的影响演替剧烈,多样性指数在时间上呈先增后减的变化趋势,曝停比为4 h∶2 h的间歇曝气B方案下菌种最丰富,多样性指数达1.092、1.079。切胶测序结果显示,SBR系统内微生物菌种大部分为未培养菌种,其中酸杆菌属和绿弯菌属占据优势地位,这些菌种在污水生物处理中对污泥性能和系统的运行有重要的影响。     

复合生物活性填料对活性污泥生物特性的影响

为了考察生物活性填料对活性污泥生物特性的影响,采用电子扫描显微镜、激光粒度仪和Miseq高通量测序技术,分别对SBR中污泥的形态、粒径和微生物群落分布进行了研究。结果表明,投加生物填料可以降低污泥的产率,促进污泥沉降,增强脱氮除磷效能;投加生物活性填料的SBR相比对照组SBR的污泥结构紧密,污泥颗粒分布均匀,且颗粒粒径大;内置填料和外置填料的活性污泥粒径在28.988~124.573μm的颗粒占据了大部分的比例,而对照组的活性污泥粒径在0.554~23.538μm的颗粒占据了大部分的比例;3个微生物群落主要分布在Sphingobacteriales、Xanthomonadales、Burkholderiales、Rhodocyclales、Flavobacteriales和Spirochaetales等6个目内;内置填料活性污泥微生物群落(17.12%)和外置填料活性污泥微生物群落(23.17%)中的黄色单胞菌目,要比对照组活性污泥微生物群落(5.51%)的比例高,说明通过复合生物活性填料的作用,能够改善活性污泥微生物群落的除磷能力,这与反应器实际运行效果和污泥粒径分析研究内容一致。     

Bacterial community structure and diversity responses to the direct revegetation of an artisanal zinc smelting slag after 5 years

This comparative field study examined the responses of bacterial community structure and diversity to the revegetation of zinc (Zn) smelting waste slag with eight plant species after 5 years. The microbial community structure of waste slag with and without vegetation was evaluated using high-throughput sequencing. The physiochemical properties of Zn smelting slag after revegetation with eight plant rhizospheres for 5 years were improved compared to those of bulk slag. Revegetation significantly increased the microbial community diversity in plant rhizospheres, and at the phylum level, Proteobacteria, Acidobacteria, and Bacteroidetes were notably more abundant in rhizosphere slags than those in bulk waste slag. Additionally, revegetation increased the relative abundance of plant growth-promoting rhizobacteria such as Flavobacterium, Streptomyces, and Arthrobacter as well as symbiotic N₂ fixers such as Bradyrhizobium. Three dominant native plant species (Arundo donax, Broussonetia papyrifera, and Robinia pseudoacacia) greatly increased the quality of the rhizosphere slags. Canonical correspondence analysis showed that the differences in bacterial community structure between the bulk and rhizosphere slags were explained by slag properties, i.e., pH, available copper (Cu) and lead (Pb), moisture, available nitrogen (N), phosphorus (P), and potassium (K), and organic matter (OM); however, available Zn and cadmium (Cd) contents were the slag parameters that best explained the differences between the rhizosphere communities of the eight plant species. The results suggested that revegetation plays an important role in enhancing bacterial community abundance and diversity in rhizosphere slags and that revegetation may also regulate microbiological properties and diversity mainly through changes in heavy metal bioavailability and physiochemical slag characteristics.

     

Studies on the restoration succession of PFU microbial communities in a pilot-scale microcosm

In order to imitate the restoration succession process of natural water ecosystem, a laboratory microcosm system of constant-flow-restoration was designed and established. A eutrophycation lake, Lake Donghu, was selected as the subject investigated. Six sampling stations were set on the lake, among which the water of station IV was natural clean water, and others were polluted with different degrees. Polyurethane foam unit microbial communities, which had colonized in the stations for a month, were collected from these stations and placed in their respective microcosms, using clean water of station IV to gradually replace the water of these microcosms. In this process, the healthy community in clean water continuously replaced the damaged communities in polluted water, the restoration succession of the damaged communities was characterized by weekly determination of several functional and structural community parameters, including species number (S), diversity index (DI), community pollution value (CPV), heterotrophy index (HI), and similarity coefficient. Cluster analysis based on similarity coefficient was used to compare the succession discrepancies of these microbial communities from different stations. The ecological succession of microbial communities during restoration was investigated by the variable patterns of these parameters, and based on which, the restoration standards of these polluted stations were suggested in an ecological sense. That was, while being restored, the water of station 0 (supereutrophycation) should be substituted with natural clean water by 95%; station I (eutrophycation), more than 90%; station II (eutrophycation), more than 85%; station III (eutrophycation), about 85%; station V (mesoeutrophycation), less than 50%. The effects of the structural and functional parameters in monitoring and assessing ecological restoration are analyzed and compared.     

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。     

Functional biodiversity of microbial communities in aerobic selector slaughterhouse wastewater.

The biodiversity of the microbial communities of an aerobic selector and a conventional system treating slaughterhouse wastewater were evaluated using the Biolog technique (Biolog Inc., Hayward, California). Principal components analysis of patterns and level of microbial activity indicate that microbial communities statistically differentiate between the selector and conventional system. Biolog data indicated that there was seasonal variation in the structure and function of the microbial community in conventional samples, which may be a useful indicator of wastewater community disturbance and unsteadiness, while, for the selector system, there were no recognizable statistical differences between winter and summer data communities, Biodiversity indices indicated that the selector system maintained a high functional diversity (Shannon-Weaver diversity index [H'] from 3.8 to 4.6) than the conventional system (H' from 1.8 to 2.8). The high values in the selector system were a reflection of the fact that most of the carbon sources were used, contributing to the very high Shannon indices. In addition, the high substrate equitability values (J) obtained for the selector samples indicated that the microbial communities between the summer and winter samples used the carbon sources in the same proportion. In contrast, differences in the equitability of the microbial communities within the conventional system were observed. This indicated a pattern representing unevenness.     

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...     

Antibiotic resistance genes in an urban river as impacted by bacterial community and physicochemical parameters

Antibiotic resistance genes (ARGs) in urban rivers are a serious public health concern in regions with poorly planned, rapid development. To gain insights into the predominant factors affecting the fate of ARGs in a highly polluted urban river in eastern China, a total of 285 ARGs, microbial communities, and 20 physicochemical parameters were analyzed for 17 sites. A total of 258 unique ARGs were detected using high-throughput qPCR, and the absolute abundance of total ARGs was positively correlated with total organic carbon and total dissolved nitrogen concentrations (P < 0.01). ARG abundance and diversity were greatly altered by microbial community structure. Variation partitioning analysis showed that the combined effects of multiple factors contributed to the profile and dissemination of ARGs, and variation of microbial communities was the major factor affecting the distribution of ARGs. The disparate distribution of some bacteria, including Bacteroides from mammalian gastrointestinal flora, Burkholderia from zoonotic infectious diseases, and Zoogloea from wastewater treatment, indicates that the urban river was strongly influenced by point-source pollution. Results imply that microbial community shifts caused by changes in water quality may lead to the spread of ARGs, and point-source pollution in urban rivers requires greater attention to control the transfer of ARGs between environmental bacteria and pathogens.

     

Taxonomic structure and potential nitrogen metabolism of microbial assemblage in a large hypereutrophic steppe lake

Recent studies have expanded the interests about microbial community and function following the rapid development of high-throughput sequencing techniques in the freshwater ecosystem. In this study, we aimed to attain a deep understanding of microbial community structure and potential nitrogen metabolism in Hulun Lake, a shallow hypereutrophic steppe lake in the Mongolian Plateau in China. The result demonstrated that cyanobacteria were the most dominant phylum. Network analysis showed both intra- and inter-phylum co-occurrence were pervasive, and there were modular structures in the microbial assemblages. The cluster dominated by proteobacteria was mainly negatively connected to the cluster dominated by both proteobacteria and actinobacteria. Cyanobacteria were tightly clustered together and positively connected to these two clusters. The major nitrogen metabolism pathways were glutamine synthetase–glutamate synthase and assimilatory nitrate reduction, indicating the nitrogen was mainly retained in the lake by microbial uptake. Cyanobacteria contributed 43.25% gene reads involved in the overall nitrogen metabolism but mainly contributed to assimilatory nitrate reduction and nitrogen fixation, aggravating the lake eutrophication. This study adds to our knowledge of microbial assemblages and nitrogen metabolism in the shallow hypereutrophic lake and provided an insight understanding for the purposes of lake ecosystem’s protection and efficient management in the Mongolian Plateau.

     

Ni2＋对活性污泥活性及群落多样性的影响

通过检测活性污泥的电子传递体系活性以及生物多样性，研究Ni2＋对活性污泥微生物活性及群落多样性的影响。结果表明：与对照系统相比，5mg／L的Ni2＋对2，3，5-lriphenylteItrazoliumchloride（TTC．ETS）活性未产生显著的影响；但当Ni2＋的浓度进一步增大到10、20和40mg／L后，其对序批式反应器内活性污泥TTC—ETS活性的抑制率分别达到（36．794-11．14）％、（55．88±13．90）％和（70．97±6．78）％。低浓度Ni2＋．能增强活性污泥微生物对碳源的利用，但高于10mg／L的Ni2＋则显著抑制了活性污泥微生物对碳源的利用。各个SBR系统中微生物群落最常见的物种相近，物种丰富度和均一性则均有所不同，其中群落物种丰富度随着Ni2＋浓度的增加而逐渐减小。TTC—ETS活性、平均每孔颜色变化率、Shan—liOn指数和Simpson指数，与Ni2＋的胁迫浓度之间的显著相关性表明，它们均可有效地表征Ni2＋胁迫对活性污泥微生物活性及群落多样性的影响程度。     

A+OSA污泥减量工艺的微生态特性

采用16S rDNA序列与PCR-DGGE(polymerase chain reaction-denaturing gradient gel electrophoresis)分析技术相结合的方法,研究了A+OSA(the anoxic+oxic-settling-anaerobic)污泥减量工艺在不同工况下的减量效果及其微生态特性。结果显示,在自然条件下,A+OSA工艺可有效减少剩余污泥27%左右。分子生物研究表明,解耦联池的插入可以明显改变系统微生物的群落结构,且随着解耦联池水力停留时间的延长,系统中部分微生物被"淘洗",微生物丰富度和多样性指数均有所降低。相似性分析表明,参照系统和A+OSA工艺分属于2个不同的集群,但在A+OSA工艺内部各反应池样品间具有较高的相似性,且各反应池在HRT为5.16 h和7.14 h时,表现为显著相似。通过上述研究可为该工艺优化及调控提供理论指导。     

RAPD marker and substrate utilization pattern applied to study microbial community diversity in the soil affected by agricultural chemicals

Present analyses of random amplified polymorphic DNA (RAPD) and Biolog GN substrate utilization pattern are combined to further study the diversity of microbial communities in four soils affected by agricultural chemicals. The results showed that the four soil microbial communities were apparently distinguishable in the diversity at RAPD level in terms of the richness and modified richness in the summer, which supports our previous report using the same soils in winter. A significant difference for the average well color development (AWCD) at 72 h incubation was found among the soils in winter using Biolog GN substrate utilization pattern, but this difference was not found among the soils in summer. However, Shannon-Weaver indices for microbial communities in the summer soils polluted by agricultural chemicals were significantly higher than those in winter at metabolic level; in contrast, no significant difference existed between the two seasons for microbial communities in the soil without chemical pollution. Present results suggest that the combined approach using RAPD and substrate utilization pattern could be used to effectively quantify microbial community diversity and its changes among the seasons in the soils affected by agricultural chemicals, simultaneously at molecular and physiological levels.