PCR-DGGE技术解析A~2/O工艺好氧单元中微生物群落结构

应用PCR-DGGE方法,追踪了汉沽工业废水处理中好氧工艺的活性污泥系统中微生物群落结构动态变化过程及其微生物群落结构组成。研究结果表明：系统中的微生物群落结构随水质变化而变化,随着培养时间的延长,微生物群落结构趋于稳定,分别属于5大类群,与γ、δ、α、ε变形杆菌（Proteobacterias）、芽孢杆菌（Bacilli）的亲缘关系较近。其中γ变形杆菌是该废水处理过程中的主要菌群,包括Pseudomonas sp.、Rheinheimera sp.、Citrobacter sp.、Klebsiella sp.、Enterbacte-riaceae、Stenotrophomonas maltophilia、Acinetobacter。在整个系统中uncultured Pseudomonas sp.、Halobacillus sp.、Pseudomonassp.、Pseudomonas stutzeri、Acinetobacter sp.可稳定存在于系统中,为该污水处理系统中的优势微生物。因此,提高Halobacillussp.、Pseudomonas sp.、Pseudomonas stutzeri、Acinetobacter sp.菌属在系统中的数量和质量,有利于提高废水生化处理的效果。     

16S rDNA克隆文库分析含硫污水生物反应器中生物群落

利用生物脱硫技术处理炼油厂含硫污水,硫化物选择性地氧化成单质硫。在进水硫化物605.3 mg·L~(-1)的条件下,硫化物去除率90.7%,单质硫生成率达到80.8%。提取反应器中的DNA样本,构建16S rDNA克隆文库对反应器中的生物群落进行分析。对51个阳性克隆子(长度约为1 400 bp)进行测序,测序结果在NCBI数据库利用Blast比对并构建系统发育树。测序结果表明,生物脱硫反应器细菌种群可分为10个类群,数量最多的是γ-变形菌(γ-Proteobacteria)占76.6%,其余类群共占23.4%。γ-Proteobacteria类群中数量最多的是具有脱硫功能的Halothiobacillus sp.(66.7%)、Thiovirga sp.(25.6%)以及Pseudomonas sp.(7.7%)。系统发育树分析结果表明,生物脱硫系统中形成了以化能自养型脱硫菌为主的微生物群落结构,异养菌数量较少。     

ABR-MBR工艺反硝化除磷微生物群落特征分析

为了揭示ABR-MBR组合工艺中反硝化除磷微生物种群演替规律,采用Miseq高通量测序技术考察了该工艺在不同运行阶段除磷功能区的微生物群落结构。结果表明,硝化液回流比逐步从150%提升至300%可促进反硝化除磷菌大量富集,促进系统的启动和稳定运行;系统在运行过程中始终保持较高的微生物多样性;优势微生物种群均以变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)为主,最大丰度分别为55.13%和7.76%,且变形菌门功能性微生物主要集中在γ-变形菌纲(Gamaproteobacteria);功能性除磷菌属主要为气单胞菌属(Aeromonas),假单胞菌属(Pseudomonas)和黄杆菌属(Flavobacterium);其中在逐步提升硝化液回流比过程中气单胞菌属(Aeromonas)被大量富集,其在γ-变形菌纲(Gamaproteobacteria)的相对丰度由5.30%上升至41.49%并在系统后续运行中维持主导地位。系统除磷效果与功能性除磷微生物相对丰度的变化密切相关。系统中微生物种群的多样性和功能微生物的结构稳定性为ABR-MBR工艺的稳定运行和高效处理提供了保证。     

处理高污染河水垂直流人工湿地微生物群落特性

为了研究处理高污染河水垂直流人工湿地基质和根际微生物群落特性,采用PCR-DGGE技术对其进行分析,使用通用引物(BAC 338F GC和BAC 805R)对16S rDNA V3~V5区基因片段进行扩增。结果表明,湿地系统中基质微生物群落多样性随基质深度的增加呈现先增大后减小,根际微生物群落多样性逐层递减,同一垂直深度根际微生物群落多样性均高于基质;根际微环境刺激了严格好氧菌Acinetobacter tjernbergiae(不动杆菌属的)出现,证明根系具有泌氧功能;聚类分析表明湿地系统中微生物群落相似度较高,并随深度增加依次递减;测序结果表明湿地系统中拟杆菌门和变形菌门在系统发育树中分别显示出高度序列同源性,湿地系统上、中层广泛分布着好氧菌Flavobacterium sp.(黄杆菌属)和Sporocytophaga sp.(生孢噬纤维菌属),并出现了大量的脱硫功能菌。     

脱硫MEC的硫价态变化与微生物群落演替规律

采用微生物电解池(microbial electrolysis cell,MEC)进行沼气脱硫研究,探究了阳极的S2-去除率、硫价态的变化及微生物群落演替规律.结果 表明,该方法中S2-去除率可达到90％以上,SO42-为阳极硫氧化反应的主要产物.微生物群落分析结果表明,Pseudomonas为脱硫优势微生物,在阳极生物膜与溶液中的丰度分别为7.6％和15.8％;电活性细菌Geobacter在阳极生物膜与溶液中的丰度分别为11.3％和5.26％,参与了Pseudomonas的种间电子传递,从而提高硫氧化效率.本研究结果可为MEC沼气脱硫工艺提供参考.     

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.     

以聚丁酸丁二醇酯为碳源去除含盐水体硝酸盐的研究

以可生物降解聚合物为碳源和生物膜载体可以解决异养反硝化有机碳源的添加不足或过量的问题.在序批式反应条件下,以聚丁酸丁二醇酯(PBS)为碳源和生物膜载体,对含盐水体异养反硝化过程中的细菌群落特征进行了研究.结果表明,试验条件下硝酸盐可以得到很好的去除,虽然有亚硝酸盐的明显积累,但最终被降低.硝酸盐的存在会降低含PBS水体中溶解性有机物的含量.应用变性梯度凝胶电泳和16S rDNA的方法鉴别到的细菌包括:Pseudomonas stutzeri,Pseudomonas sp.,Alteromonas sp.,Marinobacter salsuginis,Thalassospira xianheensis,Itellibacter vladivostokensis,Euplotopsis encysticus,Alcanivorax venustensis,Halomonas sp.,Agrobacterium tume aciens,Pannonibacter phragmitetus,Vitellibacter vladivostokensis.试验结果表明,反硝化条件下PBS具有较好的可生物降解性和明显的NO3--N去除能力,是比较理想的低C/N含盐水体异养反硝化碳源.     

固定化耐低温混合菌对焦化土壤多环芳烃的降解研究

研究了固定化耐低温真菌-细菌混合菌在低温环境下,对焦化厂污染土壤中的菲(Phe)和苯并[b]荧蒽(BbF)降解的动态变化,利用高通量测序技术分析了降解过程中微生物群落多样性变化。结果表明:在低温条件下固定化混合菌对土壤中Phe、BbF的去除率远高于游离混合菌与固定化单菌,在60d的降解周期下,固定化混合菌对土壤中Phe和BbF的降解率分别可达59.61%和45.24%。处理前,土壤中细菌与真菌初始Shannon多样性指数分别为2.79和0.33,细菌远高于真菌,土壤中土著微生物以细菌为主,高丰度的细菌抑制了真菌的生长代谢。处理后,细菌的Shannon多样性指数下降至1.33,真菌的Shannon多样性指数增加至1.01,Phe和BbF的降解与细菌多样性呈负相关,且细菌多样性的降低减少了其对真菌的抑制作用。对比分析了处理前后土壤中微生物群落组成的变化,结果表明:投加固定化混合菌后,固定化混合菌中的假单胞菌(Pseudomonas sp.)SDR4和高山被孢霉(Mortierella alpina)JDR7在低温下生长代谢良好,并成为降解过程中的优势菌,其物种相对丰度分别提高至79.84%与58.63%。固定化混合菌对低温环境有良好的耐性,固定化混合菌的投加提高了菌株对多环芳烃(PAHs)的生物利用有效性,改变了土壤中微生物群落的结构和丰度,可应用于低温土壤PAHs的原位修复。     

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。     

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。     

A2/O工艺缺氧池中反硝化聚磷菌的比例、特性研究及菌株鉴定

用释磷/聚磷装置和微生物筛选、分离方法研究A2/O工艺缺氧池污泥,确定缺氧池中反硝化聚磷菌(DPB)的比例,筛选、分离得反硝化聚磷单菌株且对单菌株聚磷特性进行研究.结果表明,缺氧池中DPB占聚磷菌(PAO)的比例约为21.5%.从缺氧池分离得到的肠杆菌科、气单胞菌属和假单胞菌属都是DPB,而不动杆菌属仅是好氧PAO,葡萄球菌属和微球菌属仅是一种专职的反硝化菌.反应过程中同时存在O2和NO3时,肠杆菌科优先利用水中的O2进行聚磷;在缺氧环境中,肠杆菌科在COD为30mg/L时的聚磷效果优于COD为180 mg/L时的聚磷效果.可见DPB的反硝化和聚磷的特性与电子受体的存在形式和COD有密切关系.因此,改良传统A2/O工艺和研发同步反硝化聚磷装置时,必须控制缺氧反硝化聚磷单元中混合液的DO和COD.     

人工湿地废水生态处理系统的作用机制

人工湿地废水生态处理新技术的高效去污能力基于独特而复杂的作用机制 ,其中最重要的是湿地基质、大型植物和微生物的高度协同作用。基质在为植物和微生物提供生长介质的同时 ,也能够通过沉淀、过滤和吸附等作用直接去除污染物。大型湿地植物既是微生物的“固定化载体” ,氧气的生产和运输“机器” ,同时还能够稳固湿地床表面 ,起到冬季保温和支撑冰面的作用。微生物是消除污染物的作用者 ,它能够在去除氮、磷等营养物的同时把有机质作为丰富的能源转化为营养物质和能量     

Contributions of fermentative acidogenic bacteria and sulfate-reducing bacteria to lactate degradation and sulfate reduction

The roles of fermentative acidogenic bacteria and sulfate-reducing bacteria (SRB) in lactate degradation and sulfate reduction in a sulfidogenic bioreactor were investigated by traditional chemical monitoring and culture-independent methods. A continuously stirred tank reactor fed with synthetic wastewater containing lactate and SO(2-)(4) at 35 degrees C, 10h of hydraulic retention time was used. The results showed that sulfate removal efficiency reached 99%, and sulfide and acetate were the main end products after 20 d of operation. 16S rRNA gene based clone libraries and single-strand conformation polymorphism profiles demonstrated that the proportion of SRB increased from 16% to 95%, and that Desulfobulbus spp., Desulfovibrio spp., Pseudomonas spp. and Clostridium spp. formed a stable, dominant community structure. The decreasing COD/SO(2-)(4) ratio had little effect on the community pattern except that Pseudomonas spp. and Desulfobulbus spp. increased slightly. The addition of molybdate to the influent significantly changed the microbial community, sulfate removal efficiency and the pattern of end products. Clostridium spp., Bacteroides spp. and Ruminococcus spp. became the dominant community members. The main end products switched from acetate to ethanol and then to propionate with the oxidation-reduction potentials increasing from -420 to -290 mV. A lactate degradation pathway was deduced: lactate served as the electronic donor for Desulfovibrio spp., or was fermented by Clostridium spp. and Bacteroides spp. to produce propionate or ethanol, which were subsequently utilized by Desulfobulbus spp. and Desulfovibrio spp. The acidotrophic SRB oxidized part of the acetate finally.     

Isolation of a selected microbial consortium capable of degrading methyl parathion and p-nitrophenol from a contaminated soil site

A bacterial consortium with the ability to degrade methyl parathion and p-nitrophenol, using these compounds as the only carbon source, was obtained by selective enrichment in a medium with methyl parathion. Samples were taken from Moravia, Medellin; an area that is highly contaminated, owing to the fact that it was used as a garbage dump from 1974 to 1982. Acinetobacter sp, Pseudomonas putida, Bacillus sp, Pseudomonas aeruginosa Citrobacter freundii, Stenotrophomonas sp, Flavobacterium sp, Proteus vulgaris, Pseudomonas sp, Acinetobacter sp, Klebsiella sp and Proteus sp were the microorganisms identified within the consortium. In culture, the consortium was able to degrade 150 mg L?1 of methyl-parathion and p-nitrophenol in 120 h, but after adding glucose or peptone to the culture, the time of degradation decreased to 24 h. In soil, the consortium was also able to degrade 150 mg L?1 of methyl parathion in 120 h at different depths and also managed to decrease the toxicity.     

混合型污水处理厂原位生物强化脱氮中试及微生物群落结构分析研究

针对混合型污水处理厂进水水质波动大、C/N低、总氮稳定达标困难等共性问题,开发出适宜于低C/N、贫营养等苛刻环境条件的高效脱氮菌剂.现场中试结果表明:经过脱氮菌强化后,生化系统脱氮效率提升8.5百分点,出水平均总氮较强化前下降3 mg/L,出水水质稳定性明显提升,生物强化效果显著.在不补加碳源条件下,深度处理出水总氮完...     

采油废水厌氧处理系统的微生物群落特征

为了深入认识石油烃的厌氧降解过程,利用分子生物学技术分析了大庆油田采油废水处理系统厌氧池和进水中的微生物群落特征。基于DGGE和克隆文库的分析结果均表明,厌氧生物膜中存在的古菌源自于采油废水。厌氧生物膜和采油废水中的古菌主要是产甲烷菌,包括嗜甲基的Methanomethylovorans thermophila和利用氢和甲酸的Methanolinea tarda。值得注意的是,氢营养型的M.tarda在厌氧生物膜中得到了富集。进水和厌氧生物膜中的细菌群落结构明显不同。进水中的主要细菌类群为Epsilonproteobacteria,而生物膜中的主要类群为Nitrospira和Deltaproteobacteria。在厌氧生物膜中发现许多与产甲烷古菌(尤其是氢营养型产甲烷菌)协同降解石油烃类物质的细菌相关克隆:其中一个克隆与Syntrophus具有较高的同源性,该类菌是产甲烷菌介导的厌氧烃降解微生物区系中的关键细菌;许多Deltaproteobacteria克隆属于group TA类群,该类群细菌主要参与芳香族化合物产甲烷菌介导的厌氧降解过程。这些结果表明,在大庆油田采油废水厌氧处理系统中已经建立起由产甲烷菌所介导的厌氧石油烃降解的微生物区系。     

Enumeration and factors influencing the relative abundance of a denitrifier, Pseudomonas sp. JR12, entrapped in alginate beads

The relative abundance of the denitrifier, Pseudomonas sp. JR12, was examined in an alginate-based entrapment complex under non-sterile, denitrifying conditions. Immuno-labeling of the Pseudomonas inoculant followed by flow cytometry (FCM) was used for determination of the relative abundance of this bacterium under the various incubation conditions. Additional information on the relative abundance of the inoculant was obtained by a quantitative enzyme-linked immunosorbent assay (ELISA) and results obtained by FCM and ELISA were compared. Ambient nitrate levels controlled the successful, long-term proliferation of the inoculant. At low ambient nitrate levels, Pseudomonas sp. remained the dominant microorganism during incubation. Higher ambient nitrate concentrations, attained by either decreasing the inoculum size of Pseudomonas sp. or raising inlet nitrate concentrations of the medium supplied to the incubation vessels, resulted in a gradual shift toward other, nitrite-accumulating denitrifiers. Thus far, most studies on the use of entrapped microorganisms for bioremediation purposes have been conducted under controlled laboratory conditions. Based on this study, conducted under non-sterile laboratory conditions, it is concluded that in-situ bioremediation using entrapped target microorganisms is bound to fail without a proper understanding of the factors that cause the target microorganism to out-compete undesired microbial invaders. Furthermore, based on the close agreement between the two detection methods used, it is concluded that flow cytometry provides a rapid and accurate tool for the detection of the relative abundance of immuno-labeled target organisms in heterogeneous microbial populations.     

Progress in decontamination by halophilic microorganisms in saline wastewater and soil

Environments with high-salt concentrations are often populated by dense microbial communities. Halophilic microorganisms can be isolated from different saline environments and different strains even belonging to the same genus have various applications. Wastewater and soil rich in both organic matter and salt are difficult to treat using conventional microorganisms typically found in wastewater treatment and soil bioremediation facilities. Studies on decontaminative capabilities and decontamination pathways of organic contaminants (i.e., aromatic compounds benzoate, cinnamate, 3-phenylpropionate, 4-hydroxybenzoic acid), heavy metals (i.e., tellurium, vanadium), and nutrients in the biological treatment of saline wastewater and soil by halophilic microorganisms are discussed in this review.     

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。