用于脱氮除磷的好氧颗粒污泥培养及微生物结构特征研究

1、学校代码： 10289分类号： Q89密 级： 公开学 号：092300008用于脱氮除磷的好氧颗粒污泥培养及微生物结构特征研究江苏科技大学 硕 士 学 位 论 文 用于脱氮除磷的好氧颗粒污泥培 养及微生物结构特征研究 研究生姓名 林婷 导师姓名 唐玉斌 申请学位类别 理学硕士 学位授予单位 江 苏 科 技 大 学 学科专业 生物化学与分子生物学 论文提交日期 2012年 4月20日 研究方向 环境微生物分子生物学 论文答辩日期 2012年 6月 1日 与生物化学 答辩委员会主席 吴福安 评 阅 人 林婷江苏科技大学 2012年 6月 5日分类号： Q89密 级： 公 开学 号： 092300

2、008理学 硕士学位论文用于脱氮除磷的好氧颗粒污泥培养及微生物结构特征研究学生姓名 林 婷指导教师 唐玉斌 教授江苏科技大学二O 一二年六月A Thesis Submitted in Fulfillment of the Requirementsfor the Degree of Master of ScienceMicroorganism structural characteristics research and cultivation of aerobic granular sludge which is used fornitrogen and phosphorus removalS

3、ubmitted byLin TingSupervised byProfessor Tang Yu-binJiangsu University of Science and TechnologyJune, 2012江苏科技大学学位论文原创性声明本人郑重声明：所呈交的学位论文，是本人在导师的指导下，独立进行研究工作所取得的成果。除文中已经注明引用的内容外，本论文不包含任何其他个人或集体已经发表或撰写过的作品成果。对本文的研究做出重要贡献的个人和集体，均已在文中以明确方式标明。本人完全意识到本声明的法律结果由本人承担。学位论文作者签名：年 月 日江苏科技大学学位论文版权使用授权书本学位论文作者完全

4、了解学校有关保留、使用学位论文的规定，同意学校保留并向国家有关部门或机构送交论文的复印件和电子版，允许论文被查阅和借阅。本人授权江苏科技大学可以将本学位论文的全部或部分内容编入有关数据库进行检索，可以采用影印、缩印或扫描等复制手段保存和汇编本学位论文。本学位论文属于：(1保密，在年解密后适用本授权书。(2不保密。学位论文作者签名： 指导教师签名：年 月 日 年 月 日摘 要摘 要好氧颗粒污泥的成功培养及其易于沉降、能耐受高冲击负荷等显著优点，使其迅速成为当前水处理领域的一个研究热点。好氧颗粒污泥内部可形成缺氧区，使其在去除化学需氧量(COD的同时具备高效同步脱氮除磷功能。本论文以序批式间歇反应

5、器(Sequencing Batch Reactor, SBR 为反应器，利用人工模拟废水培养好氧颗粒污泥，考察颗粒污泥成型过程中污染物去除效果，通过荧光原位杂交染色(FISH技术分析好氧颗粒污泥硝化菌及聚磷菌的分布，并应用变性梯度凝胶电泳(DGGE技术研究好氧颗粒污泥微生物群落结构及变化特征，得出以下主要结论：（1）高负荷运行条件有利于好氧颗粒污泥的形成，在3个不同运行周期（4h ，6h ，12h ）的反应器中，运行周期为4h 的反应器最早出现了成熟的好氧颗粒污泥。随着进水COD 浓度增加，反应器内生物量迅速增加，污泥粒径由0.01mm 增加至1cm 左右，污泥由接种时的橙黄色絮状，逐渐变化

6、为结构密实、规则的黑色球状或椭球状污泥。培养过程中，随着好氧颗粒污泥的形成，污泥的沉降速率逐渐增加，由最初的0.3cm/s增加至10cm/s，污泥浓度在第140d 后稳定在7500mg/L左右，污泥容积指数为35mL/g。（2）成熟的好氧颗粒污泥在高负荷运行条件下，对COD 、总磷、总氮及氨氮均有很好的去除效果。颗粒污泥成熟后，进水COD 、总磷、总氮及氨氮的浓度为1500mg/L、7.5mg/L、80mg/L和60mg/L，出水COD 始终维持在100mg/L以下，总磷、总氮和氨氮分别低于0.8mg/L、30mg/L和8mg/L，去除率分别为94.5%、89.4%，62.92%和89.4%。

7、（3）扫描电镜观察可发现，成熟的颗粒污泥微生物主要由球菌、杆菌和丝状菌等构成，颗粒污泥内外存在许多孔隙，可以为外部基质向颗粒内部传输以及内部微生物生长代谢产物向外逸出提供通道。FISH 检测结果表明，颗粒污泥中存在较多的硝化菌和聚磷菌。硝化细菌在污泥颗粒内外表层皆有分布，而聚磷菌则主要聚集在颗粒污泥的外部和部分大孔径的孔隙处。（4）利用PCR-DGGE 技术分析好氧颗粒污泥颗粒化过程中微生物结构特征变化，结果表明，不同时期颗粒污泥微生物群落结构存在差异，40d 至120d 时亚硝化单胞菌属和丝状菌大量富集，而丝状菌在颗粒污泥解体后(140d数量明显减少，副球菌属等兼性厌氧菌，在颗粒形成后(80

8、d大量富集。成熟颗粒污泥中优势微生物种群为副球菌属(Paracoccus marcusii ，胶团杆菌(Zoogloea ramigera ，红环菌科(Rhodocyclaceae bacterium ，亚硝化单胞菌(Nitrosomonas ，变形菌门(Proteobacterium ，陶厄氏菌属(Thauera 等。关键词: 好氧颗粒污泥；变性梯度凝胶电泳(DGGE；脱氮除磷；种群结构I江苏科技大学理学硕士学位论文IIABSTRACTABSTRACTWith successful cultivation and its significant advantages, aerobic gra

9、nular sludge quickly became a hot research of current water treatment. The particles can form hypoxia area inside that makes it possible to achieve simultaneous denitrification and phosphorus absorption. To further research the removal efficiency of pollutents, this paper used three sequencing batch

10、 reactors (SBR to cultivate aerobic granular sludge with artificial simulation wastewater. In this paper, we focused on using fluorescence in situ hybridization dyeing (FISH to analyze the distribution of nitrobacterium and polyphosphate accumulationg bacteria, and utilizing denaturing gradient gel

11、electrophoresis (DGGE to study on microbial community structure and function of granular sludge and came to the following conclusions.（1）High load was conducive to the formation of aerobic granular sludge. Compared with three reactors which were operating in different cycles (4h, 6h, 12h, the aerobi

12、c granular sludge cycle was first mature in which run for 4h as its operation cycle. With influent COD concentration increased, the biomass enlarged rapidly in three reactors, when the sludge particle size increased from 0.01mm to 1cm as well as the flocculent orange inoculation sludge gradually cha

13、nged to spherical or ellipsoidal-shaped black dense sludge with rules of structure. With the formation of aerobic granular sludge, sludge sedimentation rate gradually increased from 0.3cm/s to 10cm/s, and sludge concentration was steady at about 7500mg/L after 140d, when SVI value was 35mL/g.（2）Thou

14、gh running under high load conditions, mature aerobic granular sludge still had high degradation efficiency of COD, TP, TN, and ammonia nitrogen. Influent concentrations of COD, and TP, TN and NH 3-N were controlled at 1500mg/L, 7.5mg/L, 80mg/L and 60mg/L, and effluent concentrations of COD was alwa

15、ys below 100mg/L, when that of TP, TN and NH3-N, can respectively be less than 0.8mg/L, 30mg/L and 8mg/L, as the removal rates of them were 89.4%, 62.92% and 94.5%, respectively.（3）It could be found by Scanning electron microscopic observation that mature granular sludge microbes were mainly made up

16、 of cocci, bacilli and filamentous bacteria, and the particles had many pores inside. External media could be transferred to the particles inside, as well as internal growth and metabolism of microorganisms can escape to the outside, which were all because of the pores provide access. FISH test resu

17、lts showed that, many phosphorus-accumulating bacteria and nitrifying bacteria are survival inside of the granular sludge. Nitrifying bacteria were distributed both inside and outside surface of the sludgeIII江苏科技大学理学硕士学位论文particles, and phosphorus-accumulating bacteria were concentrated on external

18、side of granular sludge and in some of the large diameter pores.（4）When using PCR-DGGE technology to analyze changes of the microbial structure features in aerobic granular sludge, the results indicated that, in different periods, microbial community structure of the sludge particles were different.

19、 From 40d to 120d, Nitrosomonas and filamentous bacteria were in a large number, but the number of filamentous bacteria in sludge particles reduced when the particles disintegrated (140d. Paracoccus marcusii, as both aerobic and anaerobic bacteria, assembled when sludge particles began to form (80d.

20、 Mature dominant microorganism populations in granular sludge were Paracoccus marcusii, Zoogloea ramigera, Rhodocyclaceae bacterium, Nitrosomonas , Proteobacterium , Thauera , etc.Keywords: aerobic granular sludge; denaturing gradient gel electrophoresis (DGGE; removal of nitrogen and phosphorus; mi

21、crobial community structureIV目录目 录摘 要 . I ABSTRACT . III 目 录 . V第1章 绪论 . 11.1 好氧颗粒污泥培养技术. 11.1.1 好氧颗粒污泥的研究现状 . 11.1.2 好氧颗粒污泥颗粒化的主要影响因素 . 21.1.3 好氧颗粒污泥形成机理 . 31.1.4 好氧颗粒污泥的特点 . 31.1.5 好氧颗粒污泥的应用 . 41.2 荧光原位杂交染色. 51.3 PCR-DGGE技术研究微生物的结构特征. 61.4 本课题研究目的与研究内容. 61.4.1研究目的与意义 . 61.4.2 研究内容 . 6第2章 实验材料与方法 .

22、 . 92.1 实验装置与运行方式. 92.1.1 实验装置 . 92.1.2 运行方式 . 92.2 接种污泥. 102.3 颗粒污泥培养方法. 102.4 分析测试项目与方法. 102.4.1 常规分析项目 . 102.4.2 扫描电镜观察 . 102.4.3 荧光原位杂交染色 . 122.4.3 污泥微生物群落DNA 的提取 . 132.4.4 PCR-DGGE . 13第3章 好氧颗粒污泥的培养及表征 . . 163.1 好氧颗粒污泥的培养. 16V江苏科技大学理学硕士学位论文3.1.1 好氧颗粒污泥培养过程中COD 的变化 . 163.1.2颗粒污泥培养过程中氨氮和总氮的变化 . .

23、 183.1.3 培养过程中总磷的变化 . . 223.2 好氧颗粒污泥脱氮除磷机理分析 . 243.2.1 同步硝化反硝化 . . 243.2.2 反硝化除磷 . . 253.3 好氧颗粒污泥的表征 . 273.3.1 污泥浓度及沉降性能 . . 273.3.2 污泥粒径分布变化 . . 303.3.3 颗粒污泥的外观和生物相 . . 313.3.4 扫描电镜成像分析成熟的颗粒污泥 . . 343.4 本章小结 . 35第4章 好氧颗粒污泥种群结构变化 . . 384.1基于 DGGE 技术的微生物结构特征分析 . 384.1.1 好氧颗粒污泥形成过程污泥微生物群落 DNA 琼脂糖电泳 .

24、384.1.2好氧颗粒污泥GC 夹引物PCR 扩增的琼脂糖电泳 . . 394.1.3 好氧颗粒污泥DGGE 图谱分析 . . 394.2 系统进化树的构建 . 434.3 主成分分析 . 454.4 聚类分析 . 464.5 本章小结 . 48结论 . . 50参考文献 . . 52附录：各条带的16S rDNA序列(或ITS 序列 . . 56攻读学位期间发表的学术论文 . . 60致 谢 . . 62 VICATALOGUECATALOGUEABSTRACT (Chinese . I ABSTRACT (English . . III CATALOGUE . . VChapter 1 I

25、ntroduction . 11.1 Technology of aerobic granular sludge . . 11.1.1 Status in quo of aerobic granular sludge . 11.1.2 Effect elements in granulation process . . 21.1.3 Mechanization of granulation . . 31.1.4 Characteristics of aerobic granular sludge . 31.1.5 Applications of aerobic granular sludge

26、. 41.2 Fluorescence in situ hybridization dyeing . 51.3 Search of microorganism stucture using PCR-DGGE . . 61.4 Aims and content of this rearch . 61.4.1 Aims and significances . 61.4.2 Content . 6Chapter 2 Materials and methods . . 92.1 Device and way of operation . 92.1.1 Device . . 92.1.2 Way of

27、operation . 92.2 Inoculate sludge . 102.3 Cultivation way of granular sludge . . 102.4 Analysis items and methods . . 102.4.1 Analysis items . . 102.4.2 Observation with SEM . 102.4.3 FISH . 122.4.3 Extraction of DNA in sludge . 132.4.4 PCR-DGGE . 13Chapter 3 Cultivation of aerobic granular sludge a

28、nd its characterization . 163.1 Cultivation of aerobic granular sludge . . 163.1.1 Change of COD in cultivation process of aerobic granular sludge . 16VII江苏科技大学理学硕士学位论文3.1.2 Changes of NH3-N and TN in cultivation process of aerobic granular sludge . 183.1.3 Change of TP in cultivation process of aer

29、obic granular sludge . . 223.2 Nitrogen and phosphorus removal mechanism of aerobic granular sludge . . 243.2.1 Simultaneous nitrification and denitrification . . 243.2.2 Denitrification and phosphorus removal . 253.3 Characterization of areobic granular sludge . 273.3.1 MLSS and sedimentation perfo

30、rmance . 273.3.2 Sludge particle size and the distribution . 303.3.3 Appearance of granular sludge and biota . 313.3.4 SEM image analysis of maturation of granular sludge . 343.4 Conclutions of this chapter . 35Chapter 4 Changes in population structure of aerobic granular sludge . . 384.1 Characteri

31、stic analysis of microbial structure based on DGGE technique . . 384.1.1 DNA of the aerobic granular sludge during the formation process . . 384.1.2 DGGE analysis of 16S rDNA V3 fragments PCR products amplified . 394.1.3 Analysis of aerobic granular sludge DGGE fingerprinting . . 394.2 Construction of phylogenetic trees . 434.3 Principal component analysis . 454.4 Cluster analysis . 454.5 Conc