厌氧氨氧化工艺启动和运行特性及其受抑机理研究
|
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。
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.
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。
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.
引文
[1] Chamchoi N, Nitisoravut S. Anammox enrichment from different conventional sludges[J].Chemosphere,2007,66(11):2225-2232
[2] Dapena-Mora A, Campos J L, Mosquera-Corral A, et al. Stability of the ANAMMOXprocess in a gas-lift reactor and a SBR[J]. J Biotechnol,2004,110(2):159-170
[3] Ruiz G, Jeison D, Chamy R. Nitrification with high nitrite accumulation for the treatmentof wastewater with high ammonia concentration[J]. Water Research,2003,37(6):1371-1377
[4] van Rijn J, Tal Y, Schreier H J. Denitrification in recirculating systems: Theory andapplications[J]. Aquacultural Engineering,2006,34(3):364-376
[5]田智勇.常温低基质浓度下生活污水的厌氧氨氧化生物脱氮研究[D]:[博士学位论文].北京:北京工业大学,2008
[6]刘海春,沈东升,龙焰.城市生活垃圾填埋场渗滤液生物脱氮新技术研究进展[J].中国沼气,2004,22(4):6-9
[7]何岩,赵由才,周恭明.高浓度氨氮废水脱氮技术研究进展[J].工业水处理,2008,28(1):1-4
[8]操卫平,冯玉军,李正山,李自树.高氮低碳废水生物脱氮研究进展[J].化工环保,2004,24(4):266-270
[9] Broda E. Two kinds of lithotrophs missing in nature[J]. Zeitschrift fur Allg.Mikrobiologie,1977,17(6):491-493
[10] van de Graaf A, Mulder A, De Bruijn P, et al. Anaerobic oxidation of ammonium is abiologically mediated process[J]. Applied and Environmental Microbiology,1995,61(4):1246-1251
[11] Jetten M S, Cirpus I, Kartal B, et al.1994-2004:10years of research on the anaerobicoxidation of ammonium[J]. Biochem Soc Trans,2005,33:119-123
[12]操家顺.低氨氮浓度污水亚硝化-厌氧氨氧化生物脱氮途径与机理研究[D]:[博士学位论文].南京:河海大学,2007
[13] Aktan C K, Yapsakli K, Mertoglu B. Inhibitory effects of free ammonia on Anammoxbacteria[J]. Biodegradation,2012,23(5):751-762
[14] van Dongen U, Jetten M S M, van Loosdrecht M C M. The sharon-anammox process fortreatment of ammonium rich wastewate[J]. Water Science and Technology,2001,44(1):153-160
[15] Trimmer M, Nicholls J C, Deflandre B. Anaerobic Ammonium Oxidation Measured inSediments along the Thames Estuary, United Kingdom[J]. Applied and EnvironmentalMicrobiology,2003,69(11):6447-6454
[16] Jin R C, Yang G F, Yu J J, et al. The inhibition of the Anammox process: A review[J].Chemical Engineering Journal,2012,197:67-79
[17] Strous M, Kuenen J G, Jetten M S. Key physiology of anaerobic ammonium oxidation[J].Appl Environ Microbiol,1999,65(7):3248-3250
[18] Schalk J, Oustad H, Kuenen J, et al. The anaerobic oxidation of hydrazine: a novelreaction in microbial nitrogen metabolism[J]. FEMS Microbiology Letters,1998,158:61-67
[19] Fernandez I, Dosta J, Fajardo C, et al. Short-and long-term effects of ammonium andnitrite on the Anammox process[J]. J Environ Manage,2012,95:170-174
[20] Waki M, Tokutomi T, Yokoyama H, et al. Nitrogen removal from animal waste treatmentwater by anammox enrichment[J]. Bioresour Technol,2007,98(14):2775-2780
[21] Tang C J, Zheng P, Mahmood Q, et al. Effect of substrate concentration on stability ofanammox biofilm reactors[J]. Journal of Central South University of Technology,2010,17(1):79-84
[22] Kumar M, Lin J G. Co-existence of anammox and denitrification for simultaneousnitrogen and carbon removal--Strategies and issues[J]. J Hazard Mater,2010,178(1-3):1-9
[23] Lotti T, van der Star W R, Kleerebezem R, et al. The effect of nitrite inhibition on theanammox process[J]. Water Res,2012,46(8):2559-2569
[24] Zhou Y, Oehmen A, Lim M, et al. The role of nitrite and free nitrous acid (FNA) inwastewater treatment plants[J]. Water Res,2011,45(15):4672-4682
[25] Dapena-Mora A, Fernández I, Campos J L, et al. Evaluation of activity and inhibitioneffects on Anammox process by batch tests based on the nitrogen gas production[J].Enzyme and Microbial Technology,2007,40(4):859-865
[26] Furukawa K, Inatomi Y, Qiao S, et al. Innovative treatment system for digester liquorusing anammox process[J]. Bioresour Technol,2009,100(22):5437-5443
[27] Jaroszynski L W, Cicek N, Sparling R, et al. Importance of the operating pH inmaintaining the stability of anoxic ammonium oxidation (anammox) activity in movingbed biofilm reactors[J]. Bioresour Technol,2011,102(14):7051-7056
[28] Cho S, Takahashi Y, Fujii N, et al. Nitrogen removal performance and microbialcommunity analysis of an anaerobic up-flow granular bed anammox reactor[J].Chemosphere,2010,78(9):1129-1135
[29] Kimura Y, Isaka K, Kazama F, et al. Effects of nitrite inhibition on anaerobic ammoniumoxidation[J]. Appl Microbiol Biotechnol,2010,86(1):359-365
[30] Egli K, Fanger U, Alvarez P J J, et al. Enrichment and characterization of an anammoxbacterium from a rotating biological contactor treating ammonium-rich leachate[J].Archives of Microbiology,2001,175(3):198-207
[31] Isaka K, Sumino T, Tsuneda S. High nitrogen removal performance at moderately lowtemperature utilizing anaerobic ammonium oxidation reactions[J]. J Biosci Bioeng,2007,103(5):486-490
[32] Tang C J, Zheng P, Hu B L, et al. Influence of substrates on nitrogen removalperformance and microbiology of anaerobic ammonium oxidation by operating twoUASB reactors fed with different substrate levels[J]. J Hazard Mater,2010,181(1-3):19-26
[33] Oshiki M, Shimokawa M, Fujii N, et al. Physiological characteristics of the anaerobicammonium-oxidizing bacterium 'Candidatus Brocadia sinica'[J]. Microbiology,2011,157:1706-1713
[34] Chen T, Zheng P, Shen L, et al. Kinetic characteristics and microbial community ofAnammox-EGSB reactor[J]. J Hazard Mater,2011,190(1-3):28-35
[35] Jetten M, Strous M, van de Pas-Schoonen K T, et al. The anaerobic oxidation ofammonium[J]. FEMS Microbiology Letters,1999,22:421-437
[36] Tsushima I, Ogasawara Y, Kindaichi T, et al. Development of high-rate anaerobicammonium-oxidizing (anammox) biofilm reactors[J]. Water Res,2007,41(8):1623-1634
[37] Jung J Y, Kang S H, Chung Y C, et al. Factors affecting the activity of anammox bacteriaduring start up in the continuous culture reactor[J]. Water Science&Technology,2007,55(1-2):459-468
[38] Li H, Zhou S, Ma W, et al. Fast start-up of ANAMMOX reactor: Operational strategyand some characteristics as indicators of reactor performance[J]. Desalination,2012,286:436-441
[39] Liao D, Li X, Yang Q, et al. Effect of inorganic carbon on anaerobic ammoniumoxidation enriched in sequencing batch reactor[J]. Journal of Environmental Sciences,2008,20(8):940-944
[40] Tang C J, Zheng P, Zhang L, et al. Enrichment features of anammox consortia frommethanogenic granules loaded with high organic and methanol contents[J].Chemosphere,2010,79(6):613-619
[41] van de Graaf A, de Bruijn P, Robertson L, et al. Autotrophic growth of anaerobicammonium-oxidizing microorganisms in a fluidized bed reactor[J]. Microbiology,1996,142:2187-2196
[42] Chamchoi N, Nitisoravut S, Schmidt J E. Inactivation of ANAMMOX communitiesunder concurrent operation of anaerobic ammonium oxidation (ANAMMOX) anddenitrification[J]. Bioresour Technol,2008,99(9):3331-3336
[43] Shen L D, Hu A H, Jin R C, et al. Enrichment of anammox bacteria from three sludgesources for the startup of monosodium glutamate industrial wastewater treatmentsystem[J]. J Hazard Mater,2012,199-200:193-199
[44] Van Hulle S W H, Vandeweyer H J P, Meesschaert B D, et al. Engineering aspects andpractical application of autotrophic nitrogen removal from nitrogen rich streams[J].Chemical Engineering Journal,2010,162(1):1-20
[45] Guven D, Dapena A, Kartal B, et al. Propionate oxidation by and methanol inhibition ofanaerobic ammonium-oxidizing bacteria[J]. Appl Environ Microbiol,2005,71(2):1066-1071
[46] Ruscalleda M, Lopez H, Ganigue R, et al. Heterotrophic denitrification on granularanammox SBR treating urban landfill leachate[J]. Water Sci Technol,2008,58(9):1749-1755
[47] Tang C J, Zheng P, Wang C H, et al. Suppression of anaerobic ammonium oxidizersunder high organic content in high-rate Anammox UASB reactor[J]. Bioresour Technol,2010,101(6):1762-1768
[48] Molinuevo B, Garcia M C, Karakashev D, et al. Anammox for ammonia removal frompig manure effluents: effect of organic matter content on process performance[J].Bioresour Technol,2009,100(7):2171-2175
[49] Lackner S, Terada A, Smets B F. Heterotrophic activity compromises autotrophicnitrogen removal in membrane-aerated biofilms: results of a modeling study[J]. WaterRes,2008,42(4-5):1102-1112
[50] Kartal B, Rattray J, van Niftrik L A, et al. Candidatus "Anammoxoglobus propionicus" anew propionate oxidizing species of anaerobic ammonium oxidizing bacteria[J]. SystAppl Microbiol,2007,30(1):39-49
[51] Kartal B, van Niftrik L, Rattray J, et al. Candidatus 'Brocadia fulgida': an autofluorescentanaerobic ammonium oxidizing bacterium[J]. FEMS Microbiol Ecol,2008,63(1):46-55
[52] Lan C J, Kumar M, Wang C C, et al. Development of simultaneous partial nitrification,anammox and denitrification (SNAD) process in a sequential batch reactor[J]. BioresourTechnol,2011,102(9):5514-5519
[53] Chen H, Liu S, Yang F, et al. The development of simultaneous partial nitrification,ANAMMOX and denitrification (SNAD) process in a single reactor for nitrogenremoval[J]. Bioresour Technol,2009,100(4):1548-1554
[54] Kuenen J G. Anammox bacteria: from discovery to application[J]. Nat Rev Microbiol,2008,6(4):320-326
[55] Nozhevnikova A N, Simankova M V, Litti Y V. Application of the microbial process ofanaerobic ammonium oxidation (ANAMMOX) in biotechnological wastewatertreatment[J]. Applied Biochemistry and Microbiology,2012,48(8):667-684
[56] Li M, Gu J D. Advances in methods for detection of anaerobic ammonium oxidizing(anammox) bacteria[J]. Appl Microbiol Biotechnol,2011,90(4):1241-1252
[57] Terada A, Zhou S, Hosomi M. Presence and detection of anaerobic ammonium-oxidizing(anammox) bacteria and appraisal of anammox process for high-strength nitrogenouswastewater treatment: a review[J]. Clean Technologies and Environmental Policy,2011,13(6):759-781
[58] Burgin A J, Hamilton S K. Have we overemphasized the role of denitrification in aquaticecosystems? A review of nitrate removal pathways[J]. Frontiers in Ecology and theEnvironment,2007,5(2):89-96
[59] Rysgaard S, Glud R N, Sejr M K, et al. Denitrification activity and oxygen dynamics inArctic sea ice[J]. Polar Biology,2007,31(5):527-537
[60] Yang Q, Jia Z, Liu R, et al. Molecular diversity and anammox activity of novelplanctomycete-like bacteria in the wastewater treatment system of a full-scale alcoholmanufacturing plant[J]. Process Biochemistry,2007,42(2):180-187
[61] Pynaert K, Smets B F, Beheydt D, et al. Start-up of autotrophic nitrogen removal reactorsvia sequential biocatalyst addition[J]. Environ Sci Technol,2004,38(4):1228-1235
[62] Mohamed N M, Saito K, Tal Y, et al. Diversity of aerobic and anaerobicammonia-oxidizing bacteria in marine sponges[J]. ISME J,2010,4(1):38-48
[63] Schubert C J, Durisch-Kaiser E, Wehrli B, et al. Anaerobic ammonium oxidation in atropical freshwater system (Lake Tanganyika)[J]. Environ Microbiol,2006,8(10):1857-1863
[64] Zhang Y, Ruan X H, Op den Camp H J, et al. Diversity and abundance of aerobic andanaerobic ammonium-oxidizing bacteria in freshwater sediments of the Xinyi River(China)[J]. Environ Microbiol,2007,9(9):2375-2382
[65] Humbert S, Tarnawski S, Fromin N, et al. Molecular detection of anammox bacteria interrestrial ecosystems: distribution and diversity[J]. ISME J,2010,4(3):450-454
[66] Kartal B, van Niftrik L, Keltjens J T, et al. Anammox--growth physiology, cell biology,and metabolism[J]. Adv Microb Physiol,2012,60:211-262
[67] Kartal B, Maalcke W J, de Almeida N M, et al. Molecular mechanism of anaerobicammonium oxidation[J]. Nature,2011,479(7371):127-130
[68] Kartal B, Kuenen J G, van Loosdrecht M C. Sewage treatment with anammox[J].Science,2010,328(5979):702-703
[69] Strous M, Pelletier E, Mangenot S, et al. Deciphering the evolution and metabolism of ananammox bacterium from a community genome[J]. Nature,2006,440(7085):790-794
[70] Jetten M, Wagner M, Fuerst J, et al. Microbiology and application of the anaerobicammonium oxidation (anammox) process[J]. Environmental biotechnology,2001,12:283-288
[71] Strous M, van Gerven E, Kuenen J G, et al. Effects of aerobic and microaerobicconditions on anaerobic ammonium-oxidizing (anammox) sludge[J]. Appl EnvironMicrobiol,1997,63(6):2446-2448
[72] Kartal B, Kuypers M M, Lavik G, et al. Anammox bacteria disguised as denitrifiers:nitrate reduction to dinitrogen gas via nitrite and ammonium[J]. Environ Microbiol,2007,9(3):635-642
[73] van de Graaf A, de Bruijn P, Robertson L, et al. Metabolic pathway of anaerobicammonium oxidation on the basis of15N studies in a fluidized bed reactor[J].Microbiology,1997,143:2415-2421
[74] Schalk J, de Vries S, Kuenen J G, et al. Involvement of a novel hydroxylamineoxidoreductase in anaerobic ammonium oxidation[J]. Biochemistry,2000,39(18):5405-5412
[75] Lindsay M, Webb R, Strous M, et al. Cell compartmentalisation in planctomycetes: noveltypes of structural organisation for the bacterial cell[J]. Archives of Microbiology,2001,175(6):413-429
[76] Ma B, Peng Y, Zhang S, et al. Performance of anammox UASB reactor treating lowstrength wastewater under moderate and low temperatures[J]. Bioresour Technol,2013,129:606-611
[77] Isaka K, Date Y, Kimura Y, et al. Nitrogen removal performance using anaerobicammonium oxidation at low temperatures[J]. FEMS Microbiology Letters,2008,282(1):32-38
[78] Dorigo U, Volatier L, Humbert J F. Molecular approaches to the assessment ofbiodiversity in aquatic microbial communities[J]. Water Res,2005,39(11):2207-2218
[79] Bakke I, De Schryver P, Boon N, et al. PCR-based community structure studies ofbacteria associated with eukaryotic organisms: a simple PCR strategy to avoidco-amplification of eukaryotic DNA[J]. J Microbiol Methods,2011,84(2):349-351
[80] Muyzer G, Smalla K. Application of denaturing gradient gel electrophoresis (DGGE) andtemperature gradient gel electrophoresis (TGGE) in microbial ecology[J]. Antonie VanLeeuwenhoek,1998,73(1):127-141
[81] Muyzer G, de Waal E C, Uitterlinden A G. Profiling of complex microbial populations bydenaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplifiedgenes coding for16S rRNA[J]. Appl Environ Microbiol,1993,59(3):695-700
[82] Joss A, Salzgeber D, Eugster J, et al. Full-scale nitrogen removal from digester liquidwith partial nitritation and anammox in one SBR[J]. Environ Sci Technol,2009,43(14):5301-5306
[83] van der Star W R, Abma W R, Blommers D, et al. Startup of reactors for anoxicammonium oxidation: experiences from the first full-scale anammox reactor inRotterdam[J]. Water Res,2007,41(18):4149-4163
[84] Liang Z, Liu J X, Li J. Decomposition and mineralization of aquatic humic substances(AHS) in treating landfill leachate using the Anammox process[J]. Chemosphere,2009,74(10):1315-1320
[85] Toh S K, Ashbolt N J. Adaptation of anaerobic ammonium-oxidising consortium tosynthetic coke-ovens wastewater[J]. Appl Microbiol Biotechnol,2002,59(2-3):344-352
[86] Yamamoto T, Takaki K, Koyama T, et al. Long-term stability of partial nitritation ofswine wastewater digester liquor and its subsequent treatment by Anammox[J].Bioresour Technol,2008,99(14):6419-6425
[87] Tang C J, Zheng P, Chen T T, et al. Enhanced nitrogen removal from pharmaceuticalwastewater using SBA-ANAMMOX process[J]. Water Res,2011,45(1):201-210
[88] Liang Z, Liu J. Landfill leachate treatment with a novel process: anaerobic ammoniumoxidation (Anammox) combined with soil infiltration system[J]. J Hazard Mater,2008,151(1):202-212
[89] Tang C J, Zheng P, Wang C H, et al. Performance of high-loaded ANAMMOX UASBreactors containing granular sludge[J]. Water Res,2011,45(1):135-144
[90] Innerebner G, Insam H, Franke-Whittle I H, et al. Identification of anammox bacteria in afull-scale deammonification plant making use of anaerobic ammonia oxidation[J]. SystAppl Microbiol,2007,30(5):408-412
[91] Wang T, Zhang H, Yang F, et al. Start-up of the Anammox process from the conventionalactivated sludge in a membrane bioreactor[J]. Bioresour Technol,2009,100(9):2501-2506
[92] Zhang J, Zhang Y, Li Y, et al. Enhancement of nitrogen removal in a novel anammoxreactor packed with Fe electrode[J]. Bioresour Technol,2012,114:102-108
[93] Liu S, Yang F, Meng F, et al. Enhanced anammox consortium activity for nitrogenremoval: impacts of static magnetic field[J]. J Biotechnol,2008,138(3-4):96-102
[94] Arrojo B, Mosquera-Corral A, Campos J L, et al. Effects of mechanical stress onAnammox granules in a sequencing batch reactor (SBR)[J]. J Biotechnol,2006,123(4):453-463
[95] Qin L, Tay J H, Liu Y. Selection pressure is a driving force of aerobic granulation insequencing batch reactors[J]. Process Biochemistry,2004,39(5):579-584
[96] Liu Y, Tay J H. The essential role of hydrodynamic shear force in the formation ofbiofilm and granular sludge[J]. Water Res,2002,36(7):1653-1665
[97] Yang G F, Jin R C. Reactivation of effluent granular sludge from a high-rate Anammoxreactor after storage[J]. Biodegradation,2013,24(1):13-32
[98] Bettazzi E, Caffaz S, Vannini C, et al. Nitrite inhibition and intermediates effects onAnammox bacteria: A batch-scale experimental study[J]. Process Biochemistry,2010,45(4):573-580
[99] APHA. Standard Methods for the Examination of Water and Wastewater[J]. USA,1998,20th ed (New York)
[100] Baloch M I, Akunna J C, Kierans M, et al. Structural analysis of anaerobic granules in aphase separated reactor by electron microscopy[J]. Bioresour Technol,2008,99(5):922-929
[101] Shimamura M, Nishiyama T, Shigetomo H, et al. Isolation of a multiheme protein withfeatures of a hydrazine-oxidizing enzyme from an anaerobic ammonium-oxidizingenrichment culture[J]. Appl Environ Microbiol,2007,73(4):1065-1072
[102] Tang C J, Zheng P, Chai L Y, et al. Characterization and quantification of anammoxstart-up in UASB reactors seeded with conventional activated sludge[J]. InternationalBiodeterioration&Biodegradation,2013,82:141-148
[103] Wang T, Zhang H, Gao D, et al. Enrichment of Anammox bacteria in seed sludges fromdifferent wastewater treating processes and start-up of Anammox process[J].Desalination,2011,271(1-3):193-198
[104] Jin R C, Zheng P, Hu A H, et al. Performance comparison of two anammox reactors:SBR and UBF[J]. Chemical Engineering Journal,2008,138(1-3):224-230
[105] Ni S Q, Gao B Y, Wang C C, et al. Fast start-up, performance and microbial communityin a pilot-scale anammox reactor seeded with exotic mature granules[J]. BioresourTechnol,2011,102(3):2448-2454
[106] López H, Puig S, GaniguéR, et al. Start-up and enrichment of a granular anammoxSBR to treat high nitrogen load wastewaters[J]. Journal of Chemical Technology&Biotechnology,2008,83(3):233-241
[107] Tang C J, Zheng P, Mahmood Q, et al. Start-up and inhibition analysis of the Anammoxprocess seeded with anaerobic granular sludge[J]. J Ind Microbiol Biotechnol,2009,36(8):1093-1100
[108] Kieling D D, Reginatto V, Schmidell W, et al. Sludge wash-out as strategy forAnammox process start-up[J]. Process Biochemistry,2007,42(12):1579-1585
[109] Ahn Y H. Sustainable nitrogen elimination biotechnologies: A review[J]. ProcessBiochemistry,2006,41(8):1709-1721
[110] Bagchi S, Biswas R, Nandy T. Start-up and stabilization of an Anammox process from anon-acclimatized sludge in CSTR[J]. J Ind Microbiol Biotechnol,2010,37(9):943-952
[111] Tang C J, Zheng P, Mahmood Q, et al. Start-up and inhibition analysis of the Anammoxprocess seeded with anaerobic granular sludge[J]. Journal of Industrial Microbiology&Biotechnology,2009,36(8):1093-1100
[112] Li X R, Du B, Fu H X, et al. The bacterial diversity in an anaerobicammonium-oxidizing (anammox) reactor community[J]. Syst Appl Microbiol,2009,32(4):278-289
[113] Miura Y, Watanabe Y, Okabe S. Significance of Chloroflexi in performance ofsubmerged membrane bioreactors (MBR) treating municipal wastewater[J]. Environ SciTechnol,2007,41(22):7787-7794
[114] Chen J, Ji Q, Zheng P, et al. Floatation and control of granular sludge in a high-rateanammox reactor[J]. Water Res,2010,44(11):3321-3328
[115] Lu H F, Zheng P, Ji Q X, et al. The structure, density and settlability of anammoxgranular sludge in high-rate reactors[J]. Bioresour Technol,2012,123:312-317
[116] Vazquez-Padin J, Fernadez I, Figueroa M, et al. Applications of Anammox basedprocesses to treat anaerobic digester supernatant at room temperature[J]. BioresourTechnol,2009,100(12):2988-2994
[117] Sabumon P C. Anaerobic ammonia removal in presence of organic matter: a novelroute[J]. J Hazard Mater,2007,149(1):49-59
[118] Kimura Y, Isaka K, Kazama F. Effects of inorganic carbon limitation on anaerobicammonium oxidation (anammox) activity[J]. Bioresour Technol,2011,102(6):4390-4394
[119] Ganigue R, Volcke E I, Puig S, et al. Impact of influent characteristics on a partialnitritation SBR treating high nitrogen loaded wastewater[J]. Bioresour Technol,2012,111:62-69
[120] Veys P, Vandeweyer H, Audenaert W, et al. Performance analysis and optimization ofautotrophic nitrogen removal in different reactor configurations: a modelling study[J].Environ Technol,2010,31(12):1311-1324
[121] Zhang T, Yan Q M, Ye L. Autotrophic biological nitrogen removal from salinewastewater under low DO[J]. Journal of Chemical Technology&Biotechnology,2010,85(10):1340-1345
[122] Ni B J, Zeng R J, Fang F, et al. Evaluation on factors influencing the heterotrophicgrowth on the soluble microbial products of autotrophs[J]. Biotechnology andbioengineering,2011,108(4):804-812
[123] Ni B J, Xie W M, Chen Y P, et al. Heterotrophs grown on the soluble microbial products(SMP) released by autotrophs are responsible for the nitrogen loss in nitrifying granularsludge[J]. Biotechnology and bioengineering,2011,108(12):2844-2852
[124] Ni B J, Ruscalleda M, Smets B F. Evaluation on the microbial interactions of anaerobicammonium oxidizers and heterotrophs in Anammox biofilm[J]. Water Res,2012,46(15):4645-4652
[125] Watt G W, Chrisp J D. A spectrophotometric method for the determination ofhydrazine[J]. Anal. Chem,1952,24(12):2006-2008
[126] Ferear D S, Burrell R C. Spectrophotometric method for determining hydroxylaminereductase activity in higher plants[J]. Anal. Chem,1955,27:1664-1665
[127] Chen J, Zheng P, Yu Y, et al. Promoting sludge quantity and activity results in highloading rates in Anammox UBF[J]. Bioresour Technol,2010,101(8):2700-2705
[128] Ni S Q, Lee P H, Fessehaie A, et al. Enrichment and biofilm formation of Anammoxbacteria in a non-woven membrane reactor[J]. Bioresour Technol,2010,101(6):1792-1799
[129] Liang Z, Li W, Yang S, et al. Extraction and structural characteristics of extracellularpolymeric substances (EPS), pellets in autotrophic nitrifying biofilm and activatedsludge[J]. Chemosphere,2010,81(5):626-632
[130] Adav S S, Lee D J. Extraction of extracellular polymeric substances from aerobicgranule with compact interior structure[J]. J Hazard Mater,2008,154(1-3):1120-1126
[131] Frolund B, Griebe T, Nielsen P H. Enzymatic activity in the activated-sludge flocmatrix[J]. Appl Microbiol Biotechnol,1995,43(4):755-761
[132] Dubois M, Gilles K A, Hamilton J K, et al. Colorimetric method for determination ofsugars and related substances[J]. Analytical Chemistry,1956,28:350-352
[133] Chen M Y, Lee D J, Tay J H. Distribution of extracellular polymeric substances inaerobic granules[J]. Appl Microbiol Biotechnol,2007,73(6):1463-1469
[134] Zhu L, Lv M L, Dai X, et al. Role and significance of extracellular polymericsubstances on the property of aerobic granule[J]. Bioresour Technol,2012,107:46-54
[135] Yu G H, Juang Y C, Lee D J, et al. Enhanced aerobic granulation with extracellularpolymeric substances (EPS)-free pellets[J]. Bioresour Technol,2009,100(20):4611-4615
[136] Wang Z W, Liu Y, Tay J H. Distribution of EPS and cell surface hydrophobicity inaerobic granules[J]. Appl Microbiol Biotechnol,2005,69(4):469-473
[137] Tay J H, Liu Q S, Liu Y. The role of cellular polysaccharides in the formation andstability of aerobic granules[J]. Lett Appl Microbiol,2001,33(3):222-226
[138] Gikas P, Livingston A G. Investigation of biofilm growth and attrition in a three-phaseairlift bioreactor using35SO42as a radiolabelled tracer[J]. Journal of ChemicalTechnology&Biotechnology,2006,81(6):858-865
[139] Gikas P, Livingston A G. Simulation of the cellular anabolic activity within biofilms:Where a new immobilized cell will preferably be born?[J]. Biochemical EngineeringJournal,2007,35(1):29-36
[140] Ye F, Ye Y, Li Y. Effect of C/N ratio on extracellular polymeric substances (EPS) andphysicochemical properties of activated sludge flocs[J]. J Hazard Mater,2011,188(1-3):37-43
[141] Yang S F, Li X Y. Influences of extracellular polymeric substances (EPS) on thecharacteristics of activated sludge under non-steady-state conditions[J]. ProcessBiochemistry,2009,44(1):91-96
[142] Ras M, Lefebvre D, Derlon N, et al. Extracellular Polymeric Substances diversity ofbiofilms grown under contrasted environmental conditions[J]. Water Res,2011,45(4):1529-1538
[143] Zhang Z J, Chen S H, Wang S M, et al. Characterization of extracellular polymericsubstances from biofilm in the process of starting-up a partial nitrification processunder salt stress[J]. Appl Microbiol Biotechnol,2011,89(5):1563-1571
[2] Dapena-Mora A, Campos J L, Mosquera-Corral A, et al. Stability of the ANAMMOXprocess in a gas-lift reactor and a SBR[J]. J Biotechnol,2004,110(2):159-170
[3] Ruiz G, Jeison D, Chamy R. Nitrification with high nitrite accumulation for the treatmentof wastewater with high ammonia concentration[J]. Water Research,2003,37(6):1371-1377
[4] van Rijn J, Tal Y, Schreier H J. Denitrification in recirculating systems: Theory andapplications[J]. Aquacultural Engineering,2006,34(3):364-376
[5]田智勇.常温低基质浓度下生活污水的厌氧氨氧化生物脱氮研究[D]:[博士学位论文].北京:北京工业大学,2008
[6]刘海春,沈东升,龙焰.城市生活垃圾填埋场渗滤液生物脱氮新技术研究进展[J].中国沼气,2004,22(4):6-9
[7]何岩,赵由才,周恭明.高浓度氨氮废水脱氮技术研究进展[J].工业水处理,2008,28(1):1-4
[8]操卫平,冯玉军,李正山,李自树.高氮低碳废水生物脱氮研究进展[J].化工环保,2004,24(4):266-270
[9] Broda E. Two kinds of lithotrophs missing in nature[J]. Zeitschrift fur Allg.Mikrobiologie,1977,17(6):491-493
[10] van de Graaf A, Mulder A, De Bruijn P, et al. Anaerobic oxidation of ammonium is abiologically mediated process[J]. Applied and Environmental Microbiology,1995,61(4):1246-1251
[11] Jetten M S, Cirpus I, Kartal B, et al.1994-2004:10years of research on the anaerobicoxidation of ammonium[J]. Biochem Soc Trans,2005,33:119-123
[12]操家顺.低氨氮浓度污水亚硝化-厌氧氨氧化生物脱氮途径与机理研究[D]:[博士学位论文].南京:河海大学,2007
[13] Aktan C K, Yapsakli K, Mertoglu B. Inhibitory effects of free ammonia on Anammoxbacteria[J]. Biodegradation,2012,23(5):751-762
[14] van Dongen U, Jetten M S M, van Loosdrecht M C M. The sharon-anammox process fortreatment of ammonium rich wastewate[J]. Water Science and Technology,2001,44(1):153-160
[15] Trimmer M, Nicholls J C, Deflandre B. Anaerobic Ammonium Oxidation Measured inSediments along the Thames Estuary, United Kingdom[J]. Applied and EnvironmentalMicrobiology,2003,69(11):6447-6454
[16] Jin R C, Yang G F, Yu J J, et al. The inhibition of the Anammox process: A review[J].Chemical Engineering Journal,2012,197:67-79
[17] Strous M, Kuenen J G, Jetten M S. Key physiology of anaerobic ammonium oxidation[J].Appl Environ Microbiol,1999,65(7):3248-3250
[18] Schalk J, Oustad H, Kuenen J, et al. The anaerobic oxidation of hydrazine: a novelreaction in microbial nitrogen metabolism[J]. FEMS Microbiology Letters,1998,158:61-67
[19] Fernandez I, Dosta J, Fajardo C, et al. Short-and long-term effects of ammonium andnitrite on the Anammox process[J]. J Environ Manage,2012,95:170-174
[20] Waki M, Tokutomi T, Yokoyama H, et al. Nitrogen removal from animal waste treatmentwater by anammox enrichment[J]. Bioresour Technol,2007,98(14):2775-2780
[21] Tang C J, Zheng P, Mahmood Q, et al. Effect of substrate concentration on stability ofanammox biofilm reactors[J]. Journal of Central South University of Technology,2010,17(1):79-84
[22] Kumar M, Lin J G. Co-existence of anammox and denitrification for simultaneousnitrogen and carbon removal--Strategies and issues[J]. J Hazard Mater,2010,178(1-3):1-9
[23] Lotti T, van der Star W R, Kleerebezem R, et al. The effect of nitrite inhibition on theanammox process[J]. Water Res,2012,46(8):2559-2569
[24] Zhou Y, Oehmen A, Lim M, et al. The role of nitrite and free nitrous acid (FNA) inwastewater treatment plants[J]. Water Res,2011,45(15):4672-4682
[25] Dapena-Mora A, Fernández I, Campos J L, et al. Evaluation of activity and inhibitioneffects on Anammox process by batch tests based on the nitrogen gas production[J].Enzyme and Microbial Technology,2007,40(4):859-865
[26] Furukawa K, Inatomi Y, Qiao S, et al. Innovative treatment system for digester liquorusing anammox process[J]. Bioresour Technol,2009,100(22):5437-5443
[27] Jaroszynski L W, Cicek N, Sparling R, et al. Importance of the operating pH inmaintaining the stability of anoxic ammonium oxidation (anammox) activity in movingbed biofilm reactors[J]. Bioresour Technol,2011,102(14):7051-7056
[28] Cho S, Takahashi Y, Fujii N, et al. Nitrogen removal performance and microbialcommunity analysis of an anaerobic up-flow granular bed anammox reactor[J].Chemosphere,2010,78(9):1129-1135
[29] Kimura Y, Isaka K, Kazama F, et al. Effects of nitrite inhibition on anaerobic ammoniumoxidation[J]. Appl Microbiol Biotechnol,2010,86(1):359-365
[30] Egli K, Fanger U, Alvarez P J J, et al. Enrichment and characterization of an anammoxbacterium from a rotating biological contactor treating ammonium-rich leachate[J].Archives of Microbiology,2001,175(3):198-207
[31] Isaka K, Sumino T, Tsuneda S. High nitrogen removal performance at moderately lowtemperature utilizing anaerobic ammonium oxidation reactions[J]. J Biosci Bioeng,2007,103(5):486-490
[32] Tang C J, Zheng P, Hu B L, et al. Influence of substrates on nitrogen removalperformance and microbiology of anaerobic ammonium oxidation by operating twoUASB reactors fed with different substrate levels[J]. J Hazard Mater,2010,181(1-3):19-26
[33] Oshiki M, Shimokawa M, Fujii N, et al. Physiological characteristics of the anaerobicammonium-oxidizing bacterium 'Candidatus Brocadia sinica'[J]. Microbiology,2011,157:1706-1713
[34] Chen T, Zheng P, Shen L, et al. Kinetic characteristics and microbial community ofAnammox-EGSB reactor[J]. J Hazard Mater,2011,190(1-3):28-35
[35] Jetten M, Strous M, van de Pas-Schoonen K T, et al. The anaerobic oxidation ofammonium[J]. FEMS Microbiology Letters,1999,22:421-437
[36] Tsushima I, Ogasawara Y, Kindaichi T, et al. Development of high-rate anaerobicammonium-oxidizing (anammox) biofilm reactors[J]. Water Res,2007,41(8):1623-1634
[37] Jung J Y, Kang S H, Chung Y C, et al. Factors affecting the activity of anammox bacteriaduring start up in the continuous culture reactor[J]. Water Science&Technology,2007,55(1-2):459-468
[38] Li H, Zhou S, Ma W, et al. Fast start-up of ANAMMOX reactor: Operational strategyand some characteristics as indicators of reactor performance[J]. Desalination,2012,286:436-441
[39] Liao D, Li X, Yang Q, et al. Effect of inorganic carbon on anaerobic ammoniumoxidation enriched in sequencing batch reactor[J]. Journal of Environmental Sciences,2008,20(8):940-944
[40] Tang C J, Zheng P, Zhang L, et al. Enrichment features of anammox consortia frommethanogenic granules loaded with high organic and methanol contents[J].Chemosphere,2010,79(6):613-619
[41] van de Graaf A, de Bruijn P, Robertson L, et al. Autotrophic growth of anaerobicammonium-oxidizing microorganisms in a fluidized bed reactor[J]. Microbiology,1996,142:2187-2196
[42] Chamchoi N, Nitisoravut S, Schmidt J E. Inactivation of ANAMMOX communitiesunder concurrent operation of anaerobic ammonium oxidation (ANAMMOX) anddenitrification[J]. Bioresour Technol,2008,99(9):3331-3336
[43] Shen L D, Hu A H, Jin R C, et al. Enrichment of anammox bacteria from three sludgesources for the startup of monosodium glutamate industrial wastewater treatmentsystem[J]. J Hazard Mater,2012,199-200:193-199
[44] Van Hulle S W H, Vandeweyer H J P, Meesschaert B D, et al. Engineering aspects andpractical application of autotrophic nitrogen removal from nitrogen rich streams[J].Chemical Engineering Journal,2010,162(1):1-20
[45] Guven D, Dapena A, Kartal B, et al. Propionate oxidation by and methanol inhibition ofanaerobic ammonium-oxidizing bacteria[J]. Appl Environ Microbiol,2005,71(2):1066-1071
[46] Ruscalleda M, Lopez H, Ganigue R, et al. Heterotrophic denitrification on granularanammox SBR treating urban landfill leachate[J]. Water Sci Technol,2008,58(9):1749-1755
[47] Tang C J, Zheng P, Wang C H, et al. Suppression of anaerobic ammonium oxidizersunder high organic content in high-rate Anammox UASB reactor[J]. Bioresour Technol,2010,101(6):1762-1768
[48] Molinuevo B, Garcia M C, Karakashev D, et al. Anammox for ammonia removal frompig manure effluents: effect of organic matter content on process performance[J].Bioresour Technol,2009,100(7):2171-2175
[49] Lackner S, Terada A, Smets B F. Heterotrophic activity compromises autotrophicnitrogen removal in membrane-aerated biofilms: results of a modeling study[J]. WaterRes,2008,42(4-5):1102-1112
[50] Kartal B, Rattray J, van Niftrik L A, et al. Candidatus "Anammoxoglobus propionicus" anew propionate oxidizing species of anaerobic ammonium oxidizing bacteria[J]. SystAppl Microbiol,2007,30(1):39-49
[51] Kartal B, van Niftrik L, Rattray J, et al. Candidatus 'Brocadia fulgida': an autofluorescentanaerobic ammonium oxidizing bacterium[J]. FEMS Microbiol Ecol,2008,63(1):46-55
[52] Lan C J, Kumar M, Wang C C, et al. Development of simultaneous partial nitrification,anammox and denitrification (SNAD) process in a sequential batch reactor[J]. BioresourTechnol,2011,102(9):5514-5519
[53] Chen H, Liu S, Yang F, et al. The development of simultaneous partial nitrification,ANAMMOX and denitrification (SNAD) process in a single reactor for nitrogenremoval[J]. Bioresour Technol,2009,100(4):1548-1554
[54] Kuenen J G. Anammox bacteria: from discovery to application[J]. Nat Rev Microbiol,2008,6(4):320-326
[55] Nozhevnikova A N, Simankova M V, Litti Y V. Application of the microbial process ofanaerobic ammonium oxidation (ANAMMOX) in biotechnological wastewatertreatment[J]. Applied Biochemistry and Microbiology,2012,48(8):667-684
[56] Li M, Gu J D. Advances in methods for detection of anaerobic ammonium oxidizing(anammox) bacteria[J]. Appl Microbiol Biotechnol,2011,90(4):1241-1252
[57] Terada A, Zhou S, Hosomi M. Presence and detection of anaerobic ammonium-oxidizing(anammox) bacteria and appraisal of anammox process for high-strength nitrogenouswastewater treatment: a review[J]. Clean Technologies and Environmental Policy,2011,13(6):759-781
[58] Burgin A J, Hamilton S K. Have we overemphasized the role of denitrification in aquaticecosystems? A review of nitrate removal pathways[J]. Frontiers in Ecology and theEnvironment,2007,5(2):89-96
[59] Rysgaard S, Glud R N, Sejr M K, et al. Denitrification activity and oxygen dynamics inArctic sea ice[J]. Polar Biology,2007,31(5):527-537
[60] Yang Q, Jia Z, Liu R, et al. Molecular diversity and anammox activity of novelplanctomycete-like bacteria in the wastewater treatment system of a full-scale alcoholmanufacturing plant[J]. Process Biochemistry,2007,42(2):180-187
[61] Pynaert K, Smets B F, Beheydt D, et al. Start-up of autotrophic nitrogen removal reactorsvia sequential biocatalyst addition[J]. Environ Sci Technol,2004,38(4):1228-1235
[62] Mohamed N M, Saito K, Tal Y, et al. Diversity of aerobic and anaerobicammonia-oxidizing bacteria in marine sponges[J]. ISME J,2010,4(1):38-48
[63] Schubert C J, Durisch-Kaiser E, Wehrli B, et al. Anaerobic ammonium oxidation in atropical freshwater system (Lake Tanganyika)[J]. Environ Microbiol,2006,8(10):1857-1863
[64] Zhang Y, Ruan X H, Op den Camp H J, et al. Diversity and abundance of aerobic andanaerobic ammonium-oxidizing bacteria in freshwater sediments of the Xinyi River(China)[J]. Environ Microbiol,2007,9(9):2375-2382
[65] Humbert S, Tarnawski S, Fromin N, et al. Molecular detection of anammox bacteria interrestrial ecosystems: distribution and diversity[J]. ISME J,2010,4(3):450-454
[66] Kartal B, van Niftrik L, Keltjens J T, et al. Anammox--growth physiology, cell biology,and metabolism[J]. Adv Microb Physiol,2012,60:211-262
[67] Kartal B, Maalcke W J, de Almeida N M, et al. Molecular mechanism of anaerobicammonium oxidation[J]. Nature,2011,479(7371):127-130
[68] Kartal B, Kuenen J G, van Loosdrecht M C. Sewage treatment with anammox[J].Science,2010,328(5979):702-703
[69] Strous M, Pelletier E, Mangenot S, et al. Deciphering the evolution and metabolism of ananammox bacterium from a community genome[J]. Nature,2006,440(7085):790-794
[70] Jetten M, Wagner M, Fuerst J, et al. Microbiology and application of the anaerobicammonium oxidation (anammox) process[J]. Environmental biotechnology,2001,12:283-288
[71] Strous M, van Gerven E, Kuenen J G, et al. Effects of aerobic and microaerobicconditions on anaerobic ammonium-oxidizing (anammox) sludge[J]. Appl EnvironMicrobiol,1997,63(6):2446-2448
[72] Kartal B, Kuypers M M, Lavik G, et al. Anammox bacteria disguised as denitrifiers:nitrate reduction to dinitrogen gas via nitrite and ammonium[J]. Environ Microbiol,2007,9(3):635-642
[73] van de Graaf A, de Bruijn P, Robertson L, et al. Metabolic pathway of anaerobicammonium oxidation on the basis of15N studies in a fluidized bed reactor[J].Microbiology,1997,143:2415-2421
[74] Schalk J, de Vries S, Kuenen J G, et al. Involvement of a novel hydroxylamineoxidoreductase in anaerobic ammonium oxidation[J]. Biochemistry,2000,39(18):5405-5412
[75] Lindsay M, Webb R, Strous M, et al. Cell compartmentalisation in planctomycetes: noveltypes of structural organisation for the bacterial cell[J]. Archives of Microbiology,2001,175(6):413-429
[76] Ma B, Peng Y, Zhang S, et al. Performance of anammox UASB reactor treating lowstrength wastewater under moderate and low temperatures[J]. Bioresour Technol,2013,129:606-611
[77] Isaka K, Date Y, Kimura Y, et al. Nitrogen removal performance using anaerobicammonium oxidation at low temperatures[J]. FEMS Microbiology Letters,2008,282(1):32-38
[78] Dorigo U, Volatier L, Humbert J F. Molecular approaches to the assessment ofbiodiversity in aquatic microbial communities[J]. Water Res,2005,39(11):2207-2218
[79] Bakke I, De Schryver P, Boon N, et al. PCR-based community structure studies ofbacteria associated with eukaryotic organisms: a simple PCR strategy to avoidco-amplification of eukaryotic DNA[J]. J Microbiol Methods,2011,84(2):349-351
[80] Muyzer G, Smalla K. Application of denaturing gradient gel electrophoresis (DGGE) andtemperature gradient gel electrophoresis (TGGE) in microbial ecology[J]. Antonie VanLeeuwenhoek,1998,73(1):127-141
[81] Muyzer G, de Waal E C, Uitterlinden A G. Profiling of complex microbial populations bydenaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplifiedgenes coding for16S rRNA[J]. Appl Environ Microbiol,1993,59(3):695-700
[82] Joss A, Salzgeber D, Eugster J, et al. Full-scale nitrogen removal from digester liquidwith partial nitritation and anammox in one SBR[J]. Environ Sci Technol,2009,43(14):5301-5306
[83] van der Star W R, Abma W R, Blommers D, et al. Startup of reactors for anoxicammonium oxidation: experiences from the first full-scale anammox reactor inRotterdam[J]. Water Res,2007,41(18):4149-4163
[84] Liang Z, Liu J X, Li J. Decomposition and mineralization of aquatic humic substances(AHS) in treating landfill leachate using the Anammox process[J]. Chemosphere,2009,74(10):1315-1320
[85] Toh S K, Ashbolt N J. Adaptation of anaerobic ammonium-oxidising consortium tosynthetic coke-ovens wastewater[J]. Appl Microbiol Biotechnol,2002,59(2-3):344-352
[86] Yamamoto T, Takaki K, Koyama T, et al. Long-term stability of partial nitritation ofswine wastewater digester liquor and its subsequent treatment by Anammox[J].Bioresour Technol,2008,99(14):6419-6425
[87] Tang C J, Zheng P, Chen T T, et al. Enhanced nitrogen removal from pharmaceuticalwastewater using SBA-ANAMMOX process[J]. Water Res,2011,45(1):201-210
[88] Liang Z, Liu J. Landfill leachate treatment with a novel process: anaerobic ammoniumoxidation (Anammox) combined with soil infiltration system[J]. J Hazard Mater,2008,151(1):202-212
[89] Tang C J, Zheng P, Wang C H, et al. Performance of high-loaded ANAMMOX UASBreactors containing granular sludge[J]. Water Res,2011,45(1):135-144
[90] Innerebner G, Insam H, Franke-Whittle I H, et al. Identification of anammox bacteria in afull-scale deammonification plant making use of anaerobic ammonia oxidation[J]. SystAppl Microbiol,2007,30(5):408-412
[91] Wang T, Zhang H, Yang F, et al. Start-up of the Anammox process from the conventionalactivated sludge in a membrane bioreactor[J]. Bioresour Technol,2009,100(9):2501-2506
[92] Zhang J, Zhang Y, Li Y, et al. Enhancement of nitrogen removal in a novel anammoxreactor packed with Fe electrode[J]. Bioresour Technol,2012,114:102-108
[93] Liu S, Yang F, Meng F, et al. Enhanced anammox consortium activity for nitrogenremoval: impacts of static magnetic field[J]. J Biotechnol,2008,138(3-4):96-102
[94] Arrojo B, Mosquera-Corral A, Campos J L, et al. Effects of mechanical stress onAnammox granules in a sequencing batch reactor (SBR)[J]. J Biotechnol,2006,123(4):453-463
[95] Qin L, Tay J H, Liu Y. Selection pressure is a driving force of aerobic granulation insequencing batch reactors[J]. Process Biochemistry,2004,39(5):579-584
[96] Liu Y, Tay J H. The essential role of hydrodynamic shear force in the formation ofbiofilm and granular sludge[J]. Water Res,2002,36(7):1653-1665
[97] Yang G F, Jin R C. Reactivation of effluent granular sludge from a high-rate Anammoxreactor after storage[J]. Biodegradation,2013,24(1):13-32
[98] Bettazzi E, Caffaz S, Vannini C, et al. Nitrite inhibition and intermediates effects onAnammox bacteria: A batch-scale experimental study[J]. Process Biochemistry,2010,45(4):573-580
[99] APHA. Standard Methods for the Examination of Water and Wastewater[J]. USA,1998,20th ed (New York)
[100] Baloch M I, Akunna J C, Kierans M, et al. Structural analysis of anaerobic granules in aphase separated reactor by electron microscopy[J]. Bioresour Technol,2008,99(5):922-929
[101] Shimamura M, Nishiyama T, Shigetomo H, et al. Isolation of a multiheme protein withfeatures of a hydrazine-oxidizing enzyme from an anaerobic ammonium-oxidizingenrichment culture[J]. Appl Environ Microbiol,2007,73(4):1065-1072
[102] Tang C J, Zheng P, Chai L Y, et al. Characterization and quantification of anammoxstart-up in UASB reactors seeded with conventional activated sludge[J]. InternationalBiodeterioration&Biodegradation,2013,82:141-148
[103] Wang T, Zhang H, Gao D, et al. Enrichment of Anammox bacteria in seed sludges fromdifferent wastewater treating processes and start-up of Anammox process[J].Desalination,2011,271(1-3):193-198
[104] Jin R C, Zheng P, Hu A H, et al. Performance comparison of two anammox reactors:SBR and UBF[J]. Chemical Engineering Journal,2008,138(1-3):224-230
[105] Ni S Q, Gao B Y, Wang C C, et al. Fast start-up, performance and microbial communityin a pilot-scale anammox reactor seeded with exotic mature granules[J]. BioresourTechnol,2011,102(3):2448-2454
[106] López H, Puig S, GaniguéR, et al. Start-up and enrichment of a granular anammoxSBR to treat high nitrogen load wastewaters[J]. Journal of Chemical Technology&Biotechnology,2008,83(3):233-241
[107] Tang C J, Zheng P, Mahmood Q, et al. Start-up and inhibition analysis of the Anammoxprocess seeded with anaerobic granular sludge[J]. J Ind Microbiol Biotechnol,2009,36(8):1093-1100
[108] Kieling D D, Reginatto V, Schmidell W, et al. Sludge wash-out as strategy forAnammox process start-up[J]. Process Biochemistry,2007,42(12):1579-1585
[109] Ahn Y H. Sustainable nitrogen elimination biotechnologies: A review[J]. ProcessBiochemistry,2006,41(8):1709-1721
[110] Bagchi S, Biswas R, Nandy T. Start-up and stabilization of an Anammox process from anon-acclimatized sludge in CSTR[J]. J Ind Microbiol Biotechnol,2010,37(9):943-952
[111] Tang C J, Zheng P, Mahmood Q, et al. Start-up and inhibition analysis of the Anammoxprocess seeded with anaerobic granular sludge[J]. Journal of Industrial Microbiology&Biotechnology,2009,36(8):1093-1100
[112] Li X R, Du B, Fu H X, et al. The bacterial diversity in an anaerobicammonium-oxidizing (anammox) reactor community[J]. Syst Appl Microbiol,2009,32(4):278-289
[113] Miura Y, Watanabe Y, Okabe S. Significance of Chloroflexi in performance ofsubmerged membrane bioreactors (MBR) treating municipal wastewater[J]. Environ SciTechnol,2007,41(22):7787-7794
[114] Chen J, Ji Q, Zheng P, et al. Floatation and control of granular sludge in a high-rateanammox reactor[J]. Water Res,2010,44(11):3321-3328
[115] Lu H F, Zheng P, Ji Q X, et al. The structure, density and settlability of anammoxgranular sludge in high-rate reactors[J]. Bioresour Technol,2012,123:312-317
[116] Vazquez-Padin J, Fernadez I, Figueroa M, et al. Applications of Anammox basedprocesses to treat anaerobic digester supernatant at room temperature[J]. BioresourTechnol,2009,100(12):2988-2994
[117] Sabumon P C. Anaerobic ammonia removal in presence of organic matter: a novelroute[J]. J Hazard Mater,2007,149(1):49-59
[118] Kimura Y, Isaka K, Kazama F. Effects of inorganic carbon limitation on anaerobicammonium oxidation (anammox) activity[J]. Bioresour Technol,2011,102(6):4390-4394
[119] Ganigue R, Volcke E I, Puig S, et al. Impact of influent characteristics on a partialnitritation SBR treating high nitrogen loaded wastewater[J]. Bioresour Technol,2012,111:62-69
[120] Veys P, Vandeweyer H, Audenaert W, et al. Performance analysis and optimization ofautotrophic nitrogen removal in different reactor configurations: a modelling study[J].Environ Technol,2010,31(12):1311-1324
[121] Zhang T, Yan Q M, Ye L. Autotrophic biological nitrogen removal from salinewastewater under low DO[J]. Journal of Chemical Technology&Biotechnology,2010,85(10):1340-1345
[122] Ni B J, Zeng R J, Fang F, et al. Evaluation on factors influencing the heterotrophicgrowth on the soluble microbial products of autotrophs[J]. Biotechnology andbioengineering,2011,108(4):804-812
[123] Ni B J, Xie W M, Chen Y P, et al. Heterotrophs grown on the soluble microbial products(SMP) released by autotrophs are responsible for the nitrogen loss in nitrifying granularsludge[J]. Biotechnology and bioengineering,2011,108(12):2844-2852
[124] Ni B J, Ruscalleda M, Smets B F. Evaluation on the microbial interactions of anaerobicammonium oxidizers and heterotrophs in Anammox biofilm[J]. Water Res,2012,46(15):4645-4652
[125] Watt G W, Chrisp J D. A spectrophotometric method for the determination ofhydrazine[J]. Anal. Chem,1952,24(12):2006-2008
[126] Ferear D S, Burrell R C. Spectrophotometric method for determining hydroxylaminereductase activity in higher plants[J]. Anal. Chem,1955,27:1664-1665
[127] Chen J, Zheng P, Yu Y, et al. Promoting sludge quantity and activity results in highloading rates in Anammox UBF[J]. Bioresour Technol,2010,101(8):2700-2705
[128] Ni S Q, Lee P H, Fessehaie A, et al. Enrichment and biofilm formation of Anammoxbacteria in a non-woven membrane reactor[J]. Bioresour Technol,2010,101(6):1792-1799
[129] Liang Z, Li W, Yang S, et al. Extraction and structural characteristics of extracellularpolymeric substances (EPS), pellets in autotrophic nitrifying biofilm and activatedsludge[J]. Chemosphere,2010,81(5):626-632
[130] Adav S S, Lee D J. Extraction of extracellular polymeric substances from aerobicgranule with compact interior structure[J]. J Hazard Mater,2008,154(1-3):1120-1126
[131] Frolund B, Griebe T, Nielsen P H. Enzymatic activity in the activated-sludge flocmatrix[J]. Appl Microbiol Biotechnol,1995,43(4):755-761
[132] Dubois M, Gilles K A, Hamilton J K, et al. Colorimetric method for determination ofsugars and related substances[J]. Analytical Chemistry,1956,28:350-352
[133] Chen M Y, Lee D J, Tay J H. Distribution of extracellular polymeric substances inaerobic granules[J]. Appl Microbiol Biotechnol,2007,73(6):1463-1469
[134] Zhu L, Lv M L, Dai X, et al. Role and significance of extracellular polymericsubstances on the property of aerobic granule[J]. Bioresour Technol,2012,107:46-54
[135] Yu G H, Juang Y C, Lee D J, et al. Enhanced aerobic granulation with extracellularpolymeric substances (EPS)-free pellets[J]. Bioresour Technol,2009,100(20):4611-4615
[136] Wang Z W, Liu Y, Tay J H. Distribution of EPS and cell surface hydrophobicity inaerobic granules[J]. Appl Microbiol Biotechnol,2005,69(4):469-473
[137] Tay J H, Liu Q S, Liu Y. The role of cellular polysaccharides in the formation andstability of aerobic granules[J]. Lett Appl Microbiol,2001,33(3):222-226
[138] Gikas P, Livingston A G. Investigation of biofilm growth and attrition in a three-phaseairlift bioreactor using35SO42as a radiolabelled tracer[J]. Journal of ChemicalTechnology&Biotechnology,2006,81(6):858-865
[139] Gikas P, Livingston A G. Simulation of the cellular anabolic activity within biofilms:Where a new immobilized cell will preferably be born?[J]. Biochemical EngineeringJournal,2007,35(1):29-36
[140] Ye F, Ye Y, Li Y. Effect of C/N ratio on extracellular polymeric substances (EPS) andphysicochemical properties of activated sludge flocs[J]. J Hazard Mater,2011,188(1-3):37-43
[141] Yang S F, Li X Y. Influences of extracellular polymeric substances (EPS) on thecharacteristics of activated sludge under non-steady-state conditions[J]. ProcessBiochemistry,2009,44(1):91-96
[142] Ras M, Lefebvre D, Derlon N, et al. Extracellular Polymeric Substances diversity ofbiofilms grown under contrasted environmental conditions[J]. Water Res,2011,45(4):1529-1538
[143] Zhang Z J, Chen S H, Wang S M, et al. Characterization of extracellular polymericsubstances from biofilm in the process of starting-up a partial nitrification processunder salt stress[J]. Appl Microbiol Biotechnol,2011,89(5):1563-1571