【作者】 李媛；

【导师】 阮文权；

【作者基本信息】 江南大学 ， 环境工程， 2014， 博士

【摘要】 厌氧氨氧化(ANaerobic AMMonium Oxidation，ANAMMOX)是一种新型的高效生物脱氮工艺，其脱氮效率优于传统生物脱氮技术，具有较好的工业化应用前景。但是厌氧氨氧化菌倍增时间长，对反应器运行条件较为敏感，从而一定程度上限制了ANAMMOX工艺的稳定性和实际应用潜力。因此进一步了解ANAMMOX工艺的运行特性及其对环境条件变化的响应，有助于推动ANAMMOX的理论发展和工业应用。有鉴于此，本论文以闲置两年的厌氧氨氧化污泥为研究对象，采用UASB反应器研究ANAMMOX工艺的启动过程和运行特性。在此基础上，从微生物群落结构、关键中间代谢产物、胞外多聚物(EPS)等多维水平解析ANAMMOX系统对反应器控制条件变化的响应及其受抑机理。主要研究结果如下：(1)将常温下闲置两年的厌氧氨氧化污泥接种至UASB反应器，以人工配水为原水，通过逐步提高水力压力，在运行68天后成功地实现了ANAMMOX工艺的启动。反应器运行稳定，在水力停留时间(HRT)为4h，进水总氮负荷为1.2kg N·m-3·d-1的条件下，氨氮、亚硝酸盐氮和总氮的去除负荷分别达到了0.52kg N·m-3·d-1、0.59kg N·m-3·d-1和1.01kg N·m-3·d-1。稳定运行的ANAMMOX反应器内的氨氮、亚硝酸盐氮消耗量与硝酸盐氮生成量之比约为1：1.08：0.26。污泥表观特性可以直观地描述厌氧氨氧化菌群的快速富集过程。随着水力压力的增加，颗粒污泥颜色逐渐由黑色变为红棕色。反应器启动成功后的厌氧氨氧化污泥平均粒径为3-4mm，沉降速度在20-78m·h-1之间，具备较好的污泥沉降性能。酶学分析结果表明，厌氧氨氧化比活性、联氨氧化酶比活性与氨氮去除负荷之间呈现显著的正相关关系，两者能够较好地表征ANAMMOX的启动过程。在HRT为4h时厌氧氨氧化比活性和联氨氧化酶比活性分别达到了125.38±3.01mg N·gVSS-1·d-1和339.42±6.83μmol·g VSS-1·min-1。高通量测序结果显示，随着ANAMMOX的逐渐恢复，种群丰富度和多样性下降，微生物群落结构发生明显演替。启动成功的ANAMMOX反应器中，优势菌群主要隶属于变形菌门(Proteobacteria)、绿菌门(Chlorobi)，未分类细菌(Unclassified Bacteria)和放线菌门(Actinobacteria)。(2)在不同进水氮浓度下，研究了水力压力对ANAMMOX工艺运行效能的影响。在进水总氮浓度较低的情况下(200mg·L-1)，水力压力对反应器氮去除能力影响不大，总氮去除率都在90%以上。当进水总氮浓度为400mg·L-1，HRT为6h时，反应器达到最佳的处理能力。此时总氮、氨氮和亚硝酸盐氮的去除负荷分别为1.32kg N·m-3·d-1、0.76kg N·m-3·d-1和0.75kg N·m-3·d-1，氨氮和亚硝酸盐氮的去除率分别达到95%和94%；进一步将HRT缩短至4h，虽然总氮去除负荷可以达到最大值1.39kg N·m-3·d-1，但是氨氮和亚硝酸盐氮的去除率却分别下降至58%和71%。在进水总氮浓度较高的情况下(600mg·L-1)，当HRT为6h时，氨氮和亚硝酸氮的去除率分别为58%和40%；而HRT降低至4h时，氨氮和亚硝酸氮去除率仅为34%和18%，处理效能明显降低。以上结果表明，相较于水力压力的变化，进水氮浓度变化对ANAMMOX效能的影响更为显著。基于聚合酶链反应-变形梯度凝胶电泳(PCR-DGGE)指纹图谱的聚类分析结果表明，当进水总氮浓度为200mg·L-1和400mg·L-1时，随着HRT不断缩短，微生物群落结构的差异度较小，菌群稳定性较好；多样性指数计算表明，微生物种群Shannon多样性指数、均匀度指数与优势度指数均升高，而丰富度指数则有所下降。当进水总氮浓度为600mg·L-1时，随着HRT的变化，微生物群落结构差异明显，菌群稳定性减弱；同时微生物种群Shannon多样性指数、优势度指数，均匀度指数均有所下降。以上结果表明，在一定的进水氮浓度下，通过控制反应器水力压力和氮负荷，可以实现厌氧氨氧化菌群的富集；但是过高的氮浓度会影响群落结构的稳定性和作用效能。随着进水负荷的提高，反应器中优势微生物菌群主要隶属于蓝藻门(Cyanobacteria)、浮霉菌门(Planctomycetes)和变形菌门。(3)在不同进水氮负荷下，研究了COD对反应器运行效能的影响。结果表明，在合适的COD浓度下，厌氧氨氧化菌的活性和丰度没有受到显著影响，反应器的脱氮效能均优于对应的空白对照试验(无COD添加)，且ANAMMOX对氮去除的贡献率在80%以上。此时的优势菌群主要隶属于绿菌门、绿弯菌门(Chloroflexi)、浮霉菌门、放线菌门和厚壁菌门。但是当进水COD浓度过高时，厌氧氨氧化菌的活性和丰度受到明显抑制，反硝化作用贡献率显著上升。此时反应器运行主要表现为氨氮去除率急剧下降，而亚硝酸盐和硝酸盐氮几近完全去除。系统中优势微生物菌群主要隶属于绿菌门、变形菌门，厚壁菌门(Firmicutes)和放线菌门。基于PCR-DGGE指纹图谱的聚类分析研究表明，在一定的进水氮负荷下，随着COD浓度的逐渐增加，微生物群落结构差异越发显著。而在COD浓度一定的情况下，逐渐提高进水氨氮和亚硝酸盐氮浓度，微生物种群结构则更趋于稳定。以上结果表明，进水COD浓度变化会显著影响微生物群落结构和脱氮效能。此外一定的进水COD浓度可以在维持较高的ANAMMOX贡献率的同时，进一步强化反应器的脱氮能力。(4)将反应器HRT固定为6h，通过逐渐提高进水氨氮和亚硝酸盐氮浓度，考察基质浓度变化对ANAMMOX脱氮效能的影响。结果表明，随着进水总氮浓度的逐渐升高，总氮去除负荷逐渐增加。当进水总氮浓度达到400mg·L-1时，氮去除负荷达到1.43kgN·m-3·d-1，总氮去除率达到90%左右。当总氮浓度继续增加时，氮去除负荷没有明显变化，但是总氮去除率则显著降低。当进水总氮浓度为800mg·L-1时，氨氮和亚硝态氮的去除率仅为51%和34%。以上结果表明，本研究中反应器的最大处理负荷在1.43kgN·m-3·d-1左右，而过高的进水氮浓度反而会对ANAMMOX系统产生一定的抑制作用。关键中间代谢产物的分析表明，无论进水总氮浓度的高低，羟氨浓度都会维持在较低的水平。但是较高的总氮浓度会造成一定程度的联氨积累。通过研究中间代谢产物的转化过程，证实了本研究中ANAMMOX菌群的代谢多样性，其存在着羟氨歧化反应、联氨歧化反应以及氨氮和羟氨的归中反应。研究关键代谢产物浓度对ANAMMOX菌群代谢的影响，结果表明高浓度的羟氨不会导致联氨的累积，但是会产生一定量的氨氮；而随着联氨添加浓度的升高，联氨转化率逐渐降低，且氨氮累积更加明显。由此可见，联氨积累的主要途径是氨氮和羟氨的归中反应而不是羟氨的歧化反应；而当联氨合成量增加时，联氨还原酶催化的还原反应并不能及时将联氨转化成N2，成为主要的限速步骤。联氨积累则可能是抑制ANAMMOX效能的主要因素之一。论文进一步研究了COD干扰下中间代谢产物的转化过程。结果表明，添加一定浓度COD没有对ANAMMOX代谢途径产生显著影响，因此可以认为COD对ANAMMOX菌群的影响机制主要发生在微生物群落结构水平而并非厌氧氨氧化菌的代谢水平。考察ANAMMOX菌群在不同受抑情况后的恢复过程，结果表明高基质浓度引起的ANAMMOX活性抑制可通过降低进水氮浓度来快速恢复，而高浓度COD(900mg·L-1)引起的ANAMMOX活性抑制现象则很难通过降低进水COD浓度来有效恢复。(5)论文研究了COD浓度对ANAMMOX污泥颗粒化的影响。污泥特性研究表明，在无COD添加的反应器中，颗粒污泥粒径以0.4-0.6mm为主，当COD浓度为100mg·L-1和200mg·L-1时，颗粒污泥粒径范围主要在1.39-1.65mm。通过研究污泥EPS的分布和组成揭示COD对ANAMMOX污泥颗粒化的影响机制。结果表明，添加COD可以增加EPS合成以促进颗粒化，其中蛋白质的增加更明显；此外COD还可以增加紧密型EPS的比例，结合荧光共聚焦显微镜的观察，可以发现此时颗粒污泥结构更为紧密。而紧密的污泥结构可以强化ANAMMOX菌的固定，从而有利于维持其在反应器整体脱氮过程中的优势地位和贡献率。研究还发现合适的COD浓度在促进颗粒化的同时，并不影响ANAMMOX的效能。但是过高的COD浓度会破坏菌群的生物相结构，在强化异养反硝化菌作用的同时，显著降低和弱化了ANAMMOX菌的丰度和贡献，因此反应器中氨氮和总氮去除率明显下降。由此可见，合适的COD浓度不仅能够促进ANAMMOX污泥的颗粒化，而且更有利于总氮的去除。更多还原

【Abstract】 ANaerobic AMMonium Oxidation (ANAMMOX) is a promising biological nitrogenremoval process with a good application potential. However, the stability and application ofthe process was usually limited due to the low growth rate of ANAMMOX bacteria and itssensitivity to environment variation. In order to enhance ANAMMOX process, it is necessaryto further investigate its operational performance and metabolic characteristics. In thisdissertation, an UASB (Upflow Anaerobic Sludge Bed) reactor inoculated with ANAMMOXsludge idled for2years as inoculum was used to investigate the start-up and operationalperformance of ANAMMOX process. Furthermore, microbial responses and repressedmechanisms to operational condition variation were multidimensionally revealed based on theanalysis of microbial community structure, key intermediate metabolites and EPS(Extracellular Polymers Substances).The main conclusions are as follows:(1) The UASB reactor inoculated with ANAMMOX consortium idled for2years wasadopted to investigate the start-up of ANAMMOX process via hydrodynamic stress control.Results showed that the ANAMMOX process was successfully start-up within68days operation. Moreover, at a4h HRT (Hydraulic Retention Time) with an influent TN (TotalNitrogen) loading rate of1.2kg TN·m-3·d-1, the reactor maintained high nitrogen removalperformance with ammonium removal rate of0.52kg N·m-3·d-1, a nitrite removal rate of0.59kg N·m-3·d-1and total nitrogen removal rate of1.01kg N·m-3·d-1. The stoichiometriccoefficients of removal NH4+-N to removal NO2--N to generated NO3--N were1:1.08:0.26.The rapid enrichment of ANAMMOX bacteria can be described by the visually sludgecharacteristics. The color of granular sludge gradually turned from black to red-brown withincreasing of hydrodynamic stress. The average diameter of granular sludge was3-4mm. Thecultivated ANAMMOX granular had excellent settling characteristics with sedimentation rateof20-78m·h-1. Enzymatic analysis showed that the SAA (Specific ANAMMOX Activity) andHZO (Hydrazine Oxidoreductase) activity presented a positive correlation with ammoniumremoval rate, and both of them can represent the start-up process of ANAMMOX. Themaximum SAA and HZO activity were125.38±3.01mg N·g VSS-1·d-1and339.42±6.83μmol·min-1·g VSS-1at4h HRT, respectively. High-throughput sequencing results showed thatthe microbial community was obviously shifted with recovery of ANAMMOX bacteria, andthe species richness and diversity of ANAMMOX bacteria declined. The dominant bacteria inthe cultivated ANAMMOX reactor were: Proteobacteria, Chlorobi, Unclassified Bacteria andActinobacteria.(2) The influence of hydraulic stress on ANAMMOX reactor performance underdifferent influent nitrogen concentration was carried out. Resluts showed that the hydraulicstress did not have significant effect on nitrogen removal when the influent TN concentrationwas200mg·L-1, the removal efficiency of TN was higher than90%. The optimal operationalperformance was obtained when the influent TN concentration was400mg·L-1with HRT of6h. The removal rates of TN, ammonium and nitrite were1.32kg N·m-3·d-1,0.76kg N·m-3·d-1 and0.75kg N·m-3·d-1, respectively, and the removal efficiencies of ammonium and nitritewere95%and94%. However, the removal efficiencies of ammonium and nitrite weredecreased to58%and71%when HRT was decreased to4h, although the removal rate of TNreached up to1.39kg N·m-3·d-1. When the influent TN concentration was600mg·L-1andHRT was6h, the removal efficiencies of ammonium and nitrite were58%and40%.However, the operational performance declined when HRT decreased to4h, and the removalefficiencies of ammonium and nitrite were only34%and18%. As a result, influent nitrogenconcentration, compared with hydraulic stress, played a more positive effect on operationalperformance of ANAMMOX process. Results of clusting analysis based on fingerprint ofPCR-DGGE showed that smaller diversity factors and good stability of microorganismcommunity structure were obtained with decreasing of HRT when the influent TNconcentrations were200mg·L-1and400mg·L-1. The Shannon diversity index, evennessindex and dominance index increased, while the richness index decreased. However, obviouschange of species diversity and poor stability of community structure were found when theinfluent TN concentration was600mg·L-1, and the Shannon diversity index, evenness indexand dominance index decreased. The above results showed that ANAMMOX bacteria can beenriched by controlling the hydraulic stress and nitrogen loading rate under certain influentnitrogen concentration, while the stability of microbial community structure and operationalperformance was affected by higher nitrogen concentration. The dominant bacteria wereenriched with increasing of influent nitrogen loading rate, and they were Cyanobacteria,Planctomycetes and Proteobacteria.(3) The influence of COD (Chemical Oxygen Demand) on ANAMMOX reactorperformance under different influent nitrogen loading rate showed that ANAMMOX activityand richness index were scarcely affected under appropriate COD concentration.ANAMMOX performance of nitrogen removal was better than the control experiment(without COD addition) and ANAMMOX contribution to nitrogen removal was higher than80%. The dominant bacteria were Chlorobi, Proteobacteria, Planctomycetes, Firmicutes andActinobacteria. However, ANAMMOX acitivity and richness index were significantlyaffected under higher COD concentration, and denitrification contribution increasedobviously. At the same time, removal efficiency of ammonium decreased sharply, while nitriteand nitrate were almost totally removed. The dominant bacteria were Chlorobi,Proteobacteria, Firmicutes and Actinobacteria. Results of clusting analysis based onfingerprint of PCR-DGGE showed that significant differences of microbial communitystructural were investigated with increasing of COD concentration under certain nitrogenloading rate. However, more stable microbial community structural were obtained wheninfluent ammonium and nitrite concentrations increased under certain COD concentration. Asa result, microbial community structural and nitrogen removal performance were significantaffected by influent COD concentration. Furthermore, certain COD concentration not onlymaintained higher ANAMMOX contribution, but also strengthened the nitrogen removalcapacity.(4) The influence of influent nitrogen concentration on ANAMMOX nitrogen removalperformance was further investigated by increasing the influent ammonium and nitrite concentration under HRT of6h. Results showed that total nitrogen removal rate increasedwith increasing of influent TN concentration. When the influent TN concentration was400mg·L-1, the TN removal rate and efficiency were1.43kg N·m-3·d-1and90%. However, TNremoval efficiency decreased when influent TN concentration increased, although no obviouschange in nitrogen removal rate was obtained. The removal efficiencies of ammonium andnitrite were only51%and34%when the influent TN concentration was800mg·L-1. Thus, thehighest nitrogen removal rate achieved in this study was around1.43kg N·m-3·d-1, andANAMMOX activity could be somewhat inhibited by higher influent nitrogen concentration.Conversions of key intermediate products showed that hydroxylamine concentrationmaintained at a lower level regardless of the influent TN concentration; however, hydrazineaccumulation was investigated at higher influent TN concentration. Metabolic diversity ofANAMMOX bacteria was confirmed through studying on conversion of intermediatemetabolite, and hydroxylamine disproportionation, hydrazine disproportionation andsymproportionation of ammonium and hydroxylamine were found exist in ANAMMOXsystem. The influence of intermediate products on metabolism of ANAMMOX bacteriashowed that a certain amount of ammonium would be produced without hydrazineaccumulation at higher hydroxylamine concentration, while apparent ammoniumaccumulation was obtained and conversion rate of hydrazine reduced with increasing ofhydrazine concentration. Hence, the hydrazine accumulation could be attributed tosymproportionation of ammonium and hydroxylamine instead of hydroxylaminedisproportionation. The reduction reaction step of converting hydrazine to nitrogen catalyzedby hydrazine reductase was the limited step when the production of hydrazine increased.Therefore, hydrazine accumulation may be one of the main factors inhibiting ANAMMOXperformance. Furthermore, effect of COD disturbance on conversions of intermediateproduction was investigated. Results showed that the level of microbial community structurewas changed instead of metabolism level of ANAMMOX bacteria under COD disturbance.ANAMMOX metabolic pathways were hardly influenced by COD addition. The inhibitedphenomenon caused by higher nitrogen concentration could be recovered by decreasing theinfluent nitrogen concentration; however, it was difficult to recover by decreasing CODconcentration when the inhibited phenomenon was caused by higher COD disturbance withCOD concentration of900mg·L-1.(5) ANAMMOX granulation influenced by COD concentration was qualitativelydiscussed by CLSM (Confocal Laser Scanning Microscopy) analysis and quantitativelyanalyzed by EPS determination. The granular diameter was mainly between0.4-0.6mmwithout COD addition, while the granular diameter enlarged to1.39-1.65mm when influentCOD concentration were100mg·L-1and200mg·L-1. To reveal the mechanism of theenhanced ANAMMOX granulation, EPS composition and distribution were studied. Resultsshowed that EPS formation was promoted by certain COD stimulation, especially for proteincontent. Furthermore, the ratio of TB (Tightly Bound)-EPS was increased by certain CODaddition. The increase could strengthen the granular compactness, which were shown by theCLSM observation. The compact granular structure was able to enhance ANAMMOXbacteria immobilization, which could contribute to maintain the dominant position of ANAMMOX bacteria and its contribution to nitrogen removal. Besides promotinggranulation, an appropriate COD concentration also enhanced the total nitrogen removalduring ANAMMOX process by enabling a stable synergism between ANAMMOX bacteriaand heterotrophic denitrificans. However, excessive COD disturbance disrupted the highlyefficient biofacies structure, which then decreased the ANAMMOX competitiveness andrichness. Therefore, the removal efficiencies of ammonium and total nitrogen decreased. As aresult, an appropriate COD concentration could not only accelerate ANAMMOX granulation,but also promote total nitrogen removal.更多还原