好氧颗粒污泥的培养过程

附件2论文中英文摘要作者姓名：倪丙杰论文题目：好氧颗粒污泥的培养过程、作用机制及数学模拟作者简介：倪丙杰，男，1981年11月出生，2006年9月师从于中国科学技术大学俞汉青教授，于2009年7月获博士学位。

中文摘要好氧颗粒污泥是活性污泥微生物通过自固定最终形成的结构紧凑、外形规则的生物聚集体。

它具有相对密实的微观结构、优良的沉淀性能、较高浓度的生物体截留和多样的微生物种群。

因此，好氧颗粒污泥技术作为一种新型的废水生物处理形式，在城市污水和工业废水处理中具有非常广阔的应用前景。

好氧颗粒污泥的形成过程非常复杂，它的作用机制涉及到微生物的生长与竞争、氧的传质、底物的扩散及微生物产物的形成等各个方面。

本论文系统地研究了好氧颗粒污泥的形成过程、作用机制和数学模拟，探索了颗粒污泥在好氧和缺氧条件下的胞内储存过程机理，深入阐明了颗粒污泥中胞外聚合物和溶解性微生物产物的形成规律，并首次成功地以低有机物浓度城市污水为基质在中试规模反应器中培养出性能优良的好氧颗粒污泥。

主要研究内容和研究结果如下：1. 分别采用豆制品加工废水和混合酸废水在SBR反应器中培养好氧颗粒污泥，基于实验结果和形成机理的分析，实现了好氧颗粒污泥形成过程的定量描述。

粒污泥在形成过程中粒径逐渐增大，沉降速度提高到40 m h-1，污泥体积指数SVI减小至20 mL g-1，COD去除效率高于98%；模型能够很好地定量描述好氧颗粒污泥的形成过程及基质在颗粒内部的扩散；好氧颗粒污泥的形成过程可分为适应期、快速生长期和成熟期。

2. 通过好氧颗粒污泥的间歇实验，探索了颗粒中自养微生物和异养微生物的生长与竞争；根据实验结果修正了ASM3模型，用以描述好氧颗粒SBR反应器中自养菌和异养菌的同时生长，并分析两类微生物对于溶解氧的竞争和在颗粒中的空间分布；发现氨氮和COD 在颗粒SBR反应器运行周期的前1.5小时内分别被自养菌和异养菌消耗完毕，且异养菌消耗更多的溶解氧；自养菌主要位于颗粒的外层，而异养菌则分布于颗粒的外层或者中心。

3. 通过McCarty建立的生物热能学方法估算出活性污泥的微生物产率，分析了反应器中关键化学和生物组分的总体计量学；与生化反应和电子平衡相耦合，建立了分析反应器中各种重要化学组分的动态变化的热力学新方法，并同修正后的ASM1模型相结合，解析了豆制品废水活性污泥处理系统中C16H24O5N4、CH2O、细胞(C5H7O2N)、H+、NH4+、HCO3-和CO2浓度的动态变化规律。

4. 通过不同初始条件下的颗粒污泥储存实验，探索了好氧条件下颗粒污泥的胞内储存机制和电子转移途径，实现了好氧颗粒污泥胞内储存过程的准确描述。

当系统中外源底物过剩时，底物被微生物快速利用并形成一定量的储存物质；在外源底物消耗完之后，微生物则会利用胞内储存物质进行生长。

5. 采用好氧和缺氧条件下的呼吸速率实验结果对反硝化条件下颗粒污泥的储存机理进行了探索，发现其生化机理过程主要包括反硝化条件下的水解、缺氧同时储存和生长、缺氧维持以及生物衰减。

动力学分析结果表明，好氧颗粒污泥的反硝化胞内储存过程NUR包括4个不同的硝酸盐利用线性阶段；根据缺氧储存机理，建立了缺氧校正系数的估算方法，并解析出好氧颗粒污泥在反硝化条件下的胞内储存过程。

6. 采用凝胶渗透色谱(GPC) 和三维荧光光谱(EEM) 对活性污泥胞外多聚物（EPS）的产生过程进行了表征。

EPS的形成量及其组分分子量分布依赖于外源底物的利用，EPS量在底物利用过程中逐渐增加；EPS中的荧光物质主成分有两种：最大激发发射波长位于280/340 nm的蛋白类物质和最大激发发射波长位于320/400 nm的类富里酸物质。

7. 提出了一套估算活性污泥EPS形成动力学参数的新方法。

在该方法中利用非线性最小二乘法建立目标函数，而采用蒙特卡罗法优化目标函数；参数估计结果表明，此方法能够准确、简便地获得活性污泥微生物降解有机物过程中EPS的形成动力学参数值及其相应的可靠性信息；外源底物电子的分布途径为：61%的电子用于细胞生物量的合成，21%的电子转移到电子受体溶解氧用于生物呼吸产生能量，而剩下的18%的电子则用于EPS的形成。

8. 集成运用凝胶渗透色谱、耗氧速率分析、多糖和蛋白测定、三维荧光光谱、傅立叶变换红外光谱和溶解性有机碳测定等技术，全面分析了活性污泥SMP的分子量分布、化学特性及其分类，对污泥微生物降解底物过程中SMP的形成及其分类划分进行了定性和定量研究。

发现UAP产生于底物利用阶段，其分子量低于290 kDa，而BAP为290-5000 kDa范围内的生物大分子物质，BAP则可进一步分为生长相关BAP (GBAP) 和内源相关BAP (EBAP)。

9. 提出了分析SMP、UAP和BAP产生动力学的新方法，并建立了描述SMP形成过程及其划分的数学模型。

基于底物利用积分方程分别建立了UAP和BAP目标函数，运用规划求解法优化目标函数使其最小化，从而得到活性污泥SMP形成的相关速率系数μH、K S、k BAP 以及产率系数Y H、k UAP的最佳估计值；在此基础上描述了3种SMP的产生与底物利用之间的联系以及SMP的形成机理。

10. 采用实验分析和数学模拟相结合的方法阐明了好氧颗粒污泥系统中EPS、SMP和X STO的形成和消耗过程，在将这3种不同微生物代谢产物的电子转移途径相耦合的基础上，建立一套统一的EPS、SMP和X STO的形成动力学模型，分析了反应器污泥停留时间(SRT) 对EPS、SMP和X STO形成的影响，并提出了基于EPS、SMP和X STO动力学模型调控颗粒反应器的新方法。

11. 对好氧颗粒污泥自养菌微生物产物的产生规律、成分特性及数学模拟进行了系统的研究，阐明了自养菌微生物代谢产物的形成机理，并对自养微生物体系中产生的EPS和SMP 进行了表征；探索了自养微生物体系中异养微生物利用自养微生物代谢产物的生长规律；发现NOB在好氧条件下以亚硝酸盐作为电子供体进行生长，并产生新的NOB细胞、UAP、EPS和硝酸盐；这些自养微生物的代谢产物是自养体系中异养菌细胞合成的唯一电子供体来源。

12. 首次利用低浓度实际城市污水在中试规模反应器中培养出性能优良的好氧颗粒污泥，探索了低浓度有机废水作为底物条件时污泥颗粒化的关键因素, 并且实现了中试颗粒污泥反应器的数学模拟。

好氧颗粒污泥粒径适中、沉降性良好、生物活性高；颗粒污泥主要以丝状菌为骨架，由结构密实地杆状细菌和球菌组成；分析表明体积交换率和沉降时间是低浓度废水条件下SBR反应器中污泥颗粒化的关键参数。

13. 采用好氧颗粒污泥为接种物，在上流式厌氧污泥反应器(UASB) 中实现厌氧氨氧化(ANAMMOX) 过程的快速启动，并对所形成ANAMMOX颗粒污泥的结构和物化特性以及反应器运行工况进行表征；在此基础上，采用16S rRNA基因扩增和PCR-DGGE等分子生物学技术对启动过程中颗粒污泥的微生物种群结构变化进行定量分析，为拓展好氧颗粒污泥的应用领域提供了新的思路。

关键词：好氧颗粒污泥；数学模拟；微生物产物；序批式反应器 (SBR)；中试颗粒污泥反应器Formation process, function mechanism and mathematical modeling ofthe aerobic granular sludgeNi Bing-JieABSTRACTAerobic granular sludge is a type of microbial aggregate through self-immobilization and granulation of the microorganisms in activated sludge. Aerobic granule usually has regular shape, good settling ability, compact structure, high biomass retention, and diversity of microbial population. It ensures a higher substrate removal efficiency, less excess sludge disposal, less area consumption and lower costs for capital construction, compared with activated sludge flocs. Therefore, this process has been regarded as a promising wastewater treatment system. However, aerobic granulation is a very complex phenomenon. There are numerous internal interactions among process variables, such as growth, storage, microbial products formation and endogenous respiration, and sludge characteristics, including biomass detachment, oxygen transfer and diffusion. In this thesis, the formation, the function mechanism and the mathematical modeling of aerobic granular sludge were systematically explored. The storage processes of aerobic granules under both aerobic and anoxic conditions were investigated. The production of extracellular polymeric substances (EPS) and soluble microbial products (SMP) in aerobic granular sludge were also explored. Furthermore, this work was the first attempt to cultivate aerobic granules on low-strength municipal wastewater in a pilot-scale sequencing batch reactor (SBR). Main contents and results are as follows:1. Aerobic granules were successfully cultivated in SBRs fed with both soybean-processing and fatty-acids-rich wastewaters. Based on experimental observations and formation mechanism analysis, the aerobic granulation process in terms of mean radius profiles was quantitatively characterized. In the granulation process, the mean diameter of bioparticles gradually increased. Their settling velocity increased to 40 m h-1, while their sludge volume index (SVI) decreased to 20 mL g-1. The chemical oxygen demand (COD) removal efficiency increased to 98%. The developed model is applicable to describing the aerobic sludge granulation process and substratediffusion within granules appropriately. Three phases in the granule formation process could be clearly distinguished: initial exponential growth phase, linear growth phase afterwards and final stable phase.2. The autotrophic and heterotrophic growth and competition in aerobic granular sludge were explored using a batch experimental approach. The activated sludge model No.3 (ASM3) was modified based on experimental results in order to describe the simultaneous autotrophic and heterotrophic growth in aerobic granules. The distribution within granules and competition for dissolved oxygen of autotrophs and heterotrophs were analyzed. It was found that full oxidation of ammonia and COD by autotrophs and heterotrophs occurred within 1.5 h. The heterotrophs accounted for major oxygen consumption than the autotrophs. The autotrophs were mainly located on the outer layer of granules, whereas the heterotrophs were present in the center of granules, or on the outer layer of granules.3. With the bioenergetic methodology established by McCarty, the microbial yield was predicted and the overall stoichiometrics for biological reactions involving the key chemical and biological species in activated sludge were established. The bioenergetic methodology was integrated with a modified activated sludge model No.1 (ASM1) to formulate a new approach to analyze the activated sludge process, with the treatment of soybean-processing wastewater as an example. This approach was able to approximately describe the treatment of soybean wastewater by activated sludge in terms of the concentration dynamics of C16H24O5N4, CH2O, cell (C5H7O2N), H+, NH4+, HCO3- and CO2.4. The internal storage mechanisms and electron flows from the external substrate occurring in aerobic granule sludge were explored with extensive storage experiments under different initial conditions. The simultaneous growth and storage processes in aerobic granules were accurately modeled. Aerobic granules in an SBR were subjected to alternative feast and famine conditions, and were able to rapidly take up carbon substrate in wastewater and to store it as intracellular storage products when the substrate was in excess. After the depletion of the external substrate, the accumulated storage polymer was utilized for heterotrophic growth.5. The internal storage mechanisms in aerobic granule sludge under anoxic conditions were investigated with respirometric experimental results. Hydrolysis, simultaneous anoxic storage and growth, anoxic maintenance, and endogenous decay were found to be the main bioreactionprocesses governing the anoxic storage in the aerobic granules.Kinetic analysis of nitrate utilization rate (NUR) indicates that the NUR of granules-based denitrification process included four linear phases of nitrate reduction. Furthermore, the methodology for determining the most important parameter in anoxic storage, i.e., anoxic reduction factor, was established based on an analysis of anoxic storage mechanisms. The performance of storage process in a granule-based denitrification system was accurately modeled.6. EPS produced by mixed microbial community were characterized using gel-permeating chromatography (GPC) and 3-dimensional excitation emission matrix (EEM) fluorescence spectroscopy measurement. The production of EPS, as well as its molecular weight (MW), depended on the external substrate utilization. The quantity of produced EPS increases significantly in the substrate utilization process. With the parallel factor analysis (PARAFAC) approach, two components of the polymer matrix are identified by the EEM analysis, one as proteins at Ex/Em 280/340 nm and one matrix associated as fulvic-acid-like substances at 320/400 nm.7. A novel and convenient approach to evaluate the EPS production kinetics was developed. The weighted least-squares analysis was employed to calculate approximate differences in EPS concentration between model predictions and experimental results. An iterative search routine in Monte Carlo simulation was utilized for optimizing the objective function by minimizing the sum of squared weighted errors. Parameters estimation results indicate that the kinetic coefficients of EPS production by activated sludge and their practical identifiability information could be obtained accurately and convenient ly with this approach. Electrons from the external substrate were distributed in the following order: new biomass synthesis of 61%, oxygen for respiration of 21%, and EPS of 18%.8. The sub-fractions of the SMP, i.e., utilization-associated products (UAP) and biomass-associated products (BAP), excreted by activated sludge were characterized in terms of formation sequence, MW and chemical natures, using MW and dissolved organic carbon (DOC) measurements, coupled with oxygen utilization rate determination, polysaccharide and protein measurement, 3-dimensional EEM fluorescence spectroscopy and Fourier transform infrared spectroscopy (FTIR) analysis. The UAP, produced in the substrate utilization process, were found to be carbonaceous compounds with an MW lower than 290 kDa. The BAP were mainly cellularmacromolecules with an MW in a range of 290-5000 kDa, and were further classified into the growth-associated BAP (GBAP) and the endogeny-associated BAP (EBAP).9. A new approach for determinating SMP, UAP and BAP and their production kinetics was established. A mathematical model was developed to further quantitatively and qualitatively describe the production kinetics and sub-fractionation of SMP. The objective functions of UAP and BAP were constructed based on the integrated substrate utilization equation. A spreadsheet program was utilized to optimize the objective function by minimizing the sum of squared weighted errors. In this way, the best estimations of the rate coefficients μH, K S and k BAP and the two yield coefficients for active bacteria (Y H) and UAP (k UAP) were determined. Furthermore, the relationships among the formation of the three sub-fractions of the SMP and the substrate utilization, as well as the SMP formation mechanisms, were elucidated.10. The formation of EPS, SMP and internal storage products (X STO) in aerobic granular sludge was investigated using experimental and modeling approaches. An expanded unified model describing the production and the consumption of EPS, SMP, and X STO was formulated after integrating the electron flows from the external substrate to EPS, SMP, and X STO. The effect of the sludge retention time (SRT) of the reactor on the formation of EPS, SMP, and X STO in the aerobic granular sludge was analyzed. The new model could be used for process control and thus for the optimization of aerobic-granule-based reactors.11. The formation mechanism, component characterization, and mathematical modeling of the microbial products of autotrophs in the aerobic granular sludge were investigated systemically. The formation mechanism of EPS and SMP by autotrophs was clarified. Information on the MW and chemical natures of microbial products in the nitrifying sludge was explored using EEM fluorescence spectroscopy and GPC measurement. The heterotrophic growth on the microbial products in the nitrifying sludge feeding with non-organic carbon source was also evaluated. The aerobic growth of the NOB occurred at the expense of nitrite as an electron donor and resulted in the production of new biomass, UAP, EPS, and nitrate. These autotrophic microbial products could be utilized as the sole electrons and carbon source for the heterotrophic growth in autotrophic systems.12. For the first time, aerobic granules with an excellent settling ability were cultivated in a pilot-scale SBR for the treatment of low-strength municipal wastewater. The key factors in thegranulation of activated sludge grown on the low-strength municipal wastewater were identifed. Mathematical modeling of this pilot-scale granule-based reactor was successfully achieved. The granules had a diameter ranging from 0.2 to 0.8 mm and had good settling ability with a settling velocity of 18-40 m h-1. Three bacteria with rod, coccus and filament morphologies coexisted in the granules. The volume exchange ratio and settling time were found to be two key factors in the granulation of activated sludge grown on such a low-concentration wastewater in an SBR.13. The accelerating startup of anaerobic ammonium oxidation (ANAMMOX) process in an upflow anaerobic sludge blanket (UASB) reactor was achieved by seeding aerobic granules. The structure and physicochemical properties of the ANAMMOX granules and the reactor performance were characterized. Microbial community analysis was performed to reveal the composition and dynamics of the microbial consortium based on denaturing gradient gel electrophoresisanalysis and 16S rRNA genes amplified from the granular sludge. The obtained results demonstrate that aerobic granular sludge could be effectively used to inoculate ANAMMOX reactors.Key words: Aerobic granular sludge; mathematical modeling; microbial products; sequencing batch reactor (SBR); pilot-scale granular sludge reactor。