基于亚硝化的全程自养脱氮工艺（CANON）效能及微生物特征研究
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摘要
基于亚硝化的全程自养脱氮工艺（CANON）相比于传统的硝化/反硝化脱氮工艺具有诸多明显的优势，然而，目前对该工艺的研究成果和实践经验主要集中于低负荷、高氨氮浓度、高温废水的处理上；针对常温、低氨氮基质浓度的城市生活污水的处理方面存在若干亟待解决的问题，其中最为关键的问题在于如何在常温、低氨氮基质环境中快速启动并获取稳定、高效的脱氮性能。因此，本课题以温度和进水基质浓度作为两个重点考察因素，以实验室规模的数个CANON反应装置为研究对象，首次将宏观工艺运行性能和微观生物学特征两个方面相结合，研究了CANON反应器的启动策略和脱氮性能，并对CANON系统内的功能微生物在生理生化、形态特征、空间分布、种群数量、群落特征及遗传学等方面的特征进行了研究，以期加强对CANON工艺的机理及微生物动态变化规律的认知，并基于微生物研究结果指导并优化工艺运行，从而为CANON工艺应用于城市生活污水的脱氮处理提供技术支持。
CANON工艺中的功能微生物为AOB和anammox菌，通过二者的协同作用实现氮素的去除。本文对不同工艺运行条件下的若干CANON反应装置内的微生物特征进行研究，结果表明不同的CANON系统在生物膜形态、功能微生物的空间分布、种属特征等方面具有共性：首先，火山岩填料表面的微生物以直径为0.2~1.0μm的球形及椭球形菌为主，易成簇生长。然而，生物膜在较强的水力剪切作用下容易受损，从而影响CANON的脱氮效能。其次，CANON内部的功能微生物彼此共生，并未呈现出分布在生物膜外层是AOB而内部是anammox菌的特征；第三，参与亚硝化作用的主要功能菌为亚硝化单胞菌属（Nitrosomonas），参与厌氧氨氧化作用的主要功能菌为待定布罗卡地菌属（Candidatus Brocadia），且AOB的生物多样性明显高于anammox菌，这种生物多样性保证了CANON系统一定的抗冲击能力；第四，CANON系统中还存在希瓦氏菌属（Shewanella）、假单胞菌属（Pseudomonas）、依格纳季氏菌属（Ignatzschineria）、脱氯单胞菌属（Dechloromonas）等，它们与AOB和anammox菌共同构成CANON系统内的微生物群落；最后，利用特定的AOB选择性培养基，从CANON反应器中筛选出4个AOB菌株。AOB的成功筛选为今后的微生物富集培养、固定化及基因工程的应用提供了理论基础。
本文考察了不同温度对CANON的影响，研究表明：高温（30℃）和室温（16~23℃）条件下系统的总氮去除负荷分别为2.21kg N/(m3·d)和1.00kgN/(m3·d)。微生物试验表明：在常温下AOB和anammox菌的数量明显下降，而NOB数量有所增加，总细菌和AOB的群落多样性在常温条件下也略有降低，菌胶团的体积和间距变大，这些可能是造成反应器在常温条件下脱氮能力下降的主要原因。而温度对功能微生物的空间分布以及anammox菌的群落结构无显著影响。基于微生物学研究结果，笔者提出了可以通过补充CANON污泥、设置污泥截留装置、间歇曝气等方式提高常温下CANON的脱氮性能。
除温度外，进水氨氮浓度也是影响CANON运行效果的另一重要因素。研究表明：常温下CANON在较低氨氮浓度时（NH_4~+-N>200mg/L）能够实现稳定的运行；当氨氮浓度降至100mg/L时，系统仍具有一定的脱氮性能，但此时生物膜被破坏，生物量流失严重，微生物菌团的间距增大，结构松散，且多以单细胞方式存在。在整个降基质运行过程中，AOB群落结构变化明显而anammox菌群落结构保持稳定。此外，系统内部总细菌和AOB数量随基质浓度的降低而减少，Nitrospira数量显著增加，anammox菌数量略有减少，而Nitrobacter很少检测到。基于微生物试验结果，本文提出了在常温、低基质浓度条件下，可以通过改用软性或者半软性填料、增设反冲洗、降低曝气、投加适量的亚硝态氮等途径来提高系统的脱氮性能。
基于上述试验结果，本文在常温低氨氮条件下，通过接种污水处理厂（A~2/O工艺）曝气池回流污泥，经过好氧/间歇曝气/限氧三个阶段，在很短的时期内（180天）首次成功启动了CANON工艺，总氮去除负荷可达1.10kg N/(m~3·d)。相比于完全限氧方式，采用间歇曝气有利于维持系统内AOB和anammox的平衡关系，从而大大缩短了CANON的启动周期。在启动的全过程中Nitrobacter没有完全被淘汰，其群落结构变化明显，而AOB和anammox菌的群落结构相对稳定。
为了使得CANON工艺发挥更好的脱氮效能，本文又进行了微生物优化控制方面的研究，结果表明：CANON滤层不同高度处微生物分布差异明显，在滤层下方微生物数量较多，易成簇生长，AOB的群落多样性很高，具有更强的脱氮能力和抗冲击负荷。为了使得功能微生物的沿程分布更为合理，建议定期改变进水方向，对不同滤层处进行均匀曝气或者对滤料进行重新排布；其次，常温条件下的生物膜厚度远未达到其理论最佳厚度值，因此，增加常温下的生物膜厚度或者适当降低系统内的DO浓度能够有效提高系统的脱氮效能。
Compared with traditional nitrification/denitrification nitrogen-removalprocess, completely autotrophic nitrogen-removal over nitrite (CANON) process hasmany distinct advantages. However, most of the research achievements and practicalexperiences are concerning wastewater that with low loading, high ammoniaconcentration and high temperature; and there are still some limitaions andchallenges of CANON to treat domestic sewage with low ammonia concentrationand ambient temperature, one of which is how to start up CANON quickly andobtain stable and effective nitrogen-removal performance at room temperature andlow ammonia concentration. Therefore, concerning of the two most importantfactors of temperature and influent ammonia concentration, this study combined themacroscopical process performance and microscopical biological characteristics forthe first time and investigated the physiology and biochemistry, morphologicalcharacteristics, spatial distribution, population, community structure and geneticsfeature of the functional microorganisms in CANON system based on a number oflab-scale CANON reactors. Except for the microbial characteristics, the start-upstrategy and nitrogen-removal performance were also analyzed, both of which mightstrengthen the awareness of the mechanism of CANON process and microbialcharacteristics, and provide theoretical guidance and technical support forCANON’s application to treat domestic sewage.
The function microorganisms in CANON system are AOB and anammoxbacteria, and the removal of nitrogen relies on the harmonious balance between thetwo kinds of function microorganisms. The microbiological characteristics havebeen investigated in a number of CANON reactors operated in different conditions.The results showed some common features in the biofilm morphology, spatialdistribution of function microorganisms and species identification in differentCANON reactors. First, most microorganisms on the volcanic filter surface werespherical or ellipsoidal with a diameter of0.2~1.0μm, which showed clusteredappearance. However, the biofilm might be broken as a result of the strong hydraulicshearing when the hydraulic load increased, which might affect nitrogen removalcapacity. Second, AOB and anammox bacteria were side by side with each other,which was not consistant with previous reports that AOB distributed in the outerlayer while anaerobic anammox bacteria was in the inner part of the biofilm. Third,the main functional bacteria contributing to short-cut nitrification process wasNitrosomonas while Candidatus Brocadia contributed to anammox process. Thebiodiversity of AOB was better than that of anammox bacteria, which enhanced the anti-impact capactiy of CANON. Fourth, except for AOB and anammox bacteria,some other kinds of bacteria like Shewanella, Pseudomonas, Ignatzschineria andDechloromonas existed in CANON system. Finally, four AOB strains were screenedsuccessfully from the CANON reactor by using specific selective medium for AOB,which provided theoretical guides for future microbial enrichment, immobilizationand application of genetic engineering.
The study of the effect of different temperature on CANON indicated theaverage TN removal loading was2.21kg N/(m3·d) and1.00kg N/(m3·d) respectivelyat high temperature (30℃) and ambient temperature (16~23℃). Microbiologicalexperiments showed that the population of AOB and anammox bacteria decreasedwhile NOB increased at ambient temperature. Besides, the community diversity oftotal bacteria and AOB also lowered slightly and the volume and spacing ofzoogloea increased. All the changes of microbial features mentioned above might bethe main reason for the decrease of nitrogen-removal capacity of CANON atambient temperature. It was also worth mentioning that the change of temperaturehad little effect on the spatial distribution of the functional microorganisms or thecommunity structure of anammox bacteria. Based on the microbial experimentalresults, such strategies should be in consideration so as to improve nitrogen-removalcapacity at ambient temperature: to add some CANON sludge, to set sludgeintercept instrument or to aerating intermittently.
Except for temperature, influent ammonia concentration is the other mostimportant factor to effect CANON. The study indicated that CANON showed abetter performance when the influent ammonia concentration was higher than200mg/L. When influent ammonia concentration reduced to100mg/L, thenitrogen-removal capacity still existed but the biofilm destroyed and some biomasslost, and the SEM pictures showed the increased zoogloea spacing and loosezoogloea structure. During the whole decreasing of influent ammonia concentration,the community structure of AOB changed obviously, which was not the same withanammox bacteria. Besides, the population of total bacteria and AOB decreasedwhile Nitrospira increased. The population of anammox bacteria decreased slightlywhiel little Nitrobacter could be detected. Based on the microbial experimentsresults, using soft or half-soft filter, adding backwash, reducing aeration or adding acertain amount of nitrite might be some feasible approaches to improve theperformance of CANON at ambient temperature and low substrate concentration.
In this study, a lab-scale CANON reactor was started up successfully within avery few period (180days) by inoculating with activated sludge obtained fromaeration tank of WWTP (A2/O process) at ambient temperature and low substrateconcentration for the first time based on the results above. After three phases ofaeration/intermittent aeration/limited aeration, stable CANON process was achieved with the TN removal loading of1.10kg N/(m3·d). Compared to completely stopaerating, intermittent aeration was conducive to maintaining the balance betweenAOB and anammox bacteria, leading to a much shorter start-up period. Nitrobacterwas hard to eliminated completely through the whole process and the communitystructure of it changed sharply, which was not the same wtih AOB and anammoxbacteria.
In order to improve nitrogen removal capacity of CANON, optimal microbialcontrol was investigated and the results showed that the population was much biggerin the lower part of the filter, which trended to cluster. While in the above layer, itshowed less the number of microorganisms, more loosely, larger spacing and lessdiversity of AOB. It was suggested to change the inflow direction, to aerate equallyalong the filter or to rearrange filter every once in a while so as to optimizemicrobial distribution along the filter. Besides, the biofilm thickness at ambienttemperature was far from the optimal thickness value. Therefore, it was suggested toincrease the biofilm thickness or reduce DO concentration to some degree so as toimprove nitrogen removal capacity of CANON at ambient temperature.
CANON工艺中的功能微生物为AOB和anammox菌，通过二者的协同作用实现氮素的去除。本文对不同工艺运行条件下的若干CANON反应装置内的微生物特征进行研究，结果表明不同的CANON系统在生物膜形态、功能微生物的空间分布、种属特征等方面具有共性：首先，火山岩填料表面的微生物以直径为0.2~1.0μm的球形及椭球形菌为主，易成簇生长。然而，生物膜在较强的水力剪切作用下容易受损，从而影响CANON的脱氮效能。其次，CANON内部的功能微生物彼此共生，并未呈现出分布在生物膜外层是AOB而内部是anammox菌的特征；第三，参与亚硝化作用的主要功能菌为亚硝化单胞菌属（Nitrosomonas），参与厌氧氨氧化作用的主要功能菌为待定布罗卡地菌属（Candidatus Brocadia），且AOB的生物多样性明显高于anammox菌，这种生物多样性保证了CANON系统一定的抗冲击能力；第四，CANON系统中还存在希瓦氏菌属（Shewanella）、假单胞菌属（Pseudomonas）、依格纳季氏菌属（Ignatzschineria）、脱氯单胞菌属（Dechloromonas）等，它们与AOB和anammox菌共同构成CANON系统内的微生物群落；最后，利用特定的AOB选择性培养基，从CANON反应器中筛选出4个AOB菌株。AOB的成功筛选为今后的微生物富集培养、固定化及基因工程的应用提供了理论基础。
本文考察了不同温度对CANON的影响，研究表明：高温（30℃）和室温（16~23℃）条件下系统的总氮去除负荷分别为2.21kg N/(m3·d)和1.00kgN/(m3·d)。微生物试验表明：在常温下AOB和anammox菌的数量明显下降，而NOB数量有所增加，总细菌和AOB的群落多样性在常温条件下也略有降低，菌胶团的体积和间距变大，这些可能是造成反应器在常温条件下脱氮能力下降的主要原因。而温度对功能微生物的空间分布以及anammox菌的群落结构无显著影响。基于微生物学研究结果，笔者提出了可以通过补充CANON污泥、设置污泥截留装置、间歇曝气等方式提高常温下CANON的脱氮性能。
除温度外，进水氨氮浓度也是影响CANON运行效果的另一重要因素。研究表明：常温下CANON在较低氨氮浓度时（NH_4~+-N>200mg/L）能够实现稳定的运行；当氨氮浓度降至100mg/L时，系统仍具有一定的脱氮性能，但此时生物膜被破坏，生物量流失严重，微生物菌团的间距增大，结构松散，且多以单细胞方式存在。在整个降基质运行过程中，AOB群落结构变化明显而anammox菌群落结构保持稳定。此外，系统内部总细菌和AOB数量随基质浓度的降低而减少，Nitrospira数量显著增加，anammox菌数量略有减少，而Nitrobacter很少检测到。基于微生物试验结果，本文提出了在常温、低基质浓度条件下，可以通过改用软性或者半软性填料、增设反冲洗、降低曝气、投加适量的亚硝态氮等途径来提高系统的脱氮性能。
基于上述试验结果，本文在常温低氨氮条件下，通过接种污水处理厂（A~2/O工艺）曝气池回流污泥，经过好氧/间歇曝气/限氧三个阶段，在很短的时期内（180天）首次成功启动了CANON工艺，总氮去除负荷可达1.10kg N/(m~3·d)。相比于完全限氧方式，采用间歇曝气有利于维持系统内AOB和anammox的平衡关系，从而大大缩短了CANON的启动周期。在启动的全过程中Nitrobacter没有完全被淘汰，其群落结构变化明显，而AOB和anammox菌的群落结构相对稳定。
为了使得CANON工艺发挥更好的脱氮效能，本文又进行了微生物优化控制方面的研究，结果表明：CANON滤层不同高度处微生物分布差异明显，在滤层下方微生物数量较多，易成簇生长，AOB的群落多样性很高，具有更强的脱氮能力和抗冲击负荷。为了使得功能微生物的沿程分布更为合理，建议定期改变进水方向，对不同滤层处进行均匀曝气或者对滤料进行重新排布；其次，常温条件下的生物膜厚度远未达到其理论最佳厚度值，因此，增加常温下的生物膜厚度或者适当降低系统内的DO浓度能够有效提高系统的脱氮效能。
Compared with traditional nitrification/denitrification nitrogen-removalprocess, completely autotrophic nitrogen-removal over nitrite (CANON) process hasmany distinct advantages. However, most of the research achievements and practicalexperiences are concerning wastewater that with low loading, high ammoniaconcentration and high temperature; and there are still some limitaions andchallenges of CANON to treat domestic sewage with low ammonia concentrationand ambient temperature, one of which is how to start up CANON quickly andobtain stable and effective nitrogen-removal performance at room temperature andlow ammonia concentration. Therefore, concerning of the two most importantfactors of temperature and influent ammonia concentration, this study combined themacroscopical process performance and microscopical biological characteristics forthe first time and investigated the physiology and biochemistry, morphologicalcharacteristics, spatial distribution, population, community structure and geneticsfeature of the functional microorganisms in CANON system based on a number oflab-scale CANON reactors. Except for the microbial characteristics, the start-upstrategy and nitrogen-removal performance were also analyzed, both of which mightstrengthen the awareness of the mechanism of CANON process and microbialcharacteristics, and provide theoretical guidance and technical support forCANON’s application to treat domestic sewage.
The function microorganisms in CANON system are AOB and anammoxbacteria, and the removal of nitrogen relies on the harmonious balance between thetwo kinds of function microorganisms. The microbiological characteristics havebeen investigated in a number of CANON reactors operated in different conditions.The results showed some common features in the biofilm morphology, spatialdistribution of function microorganisms and species identification in differentCANON reactors. First, most microorganisms on the volcanic filter surface werespherical or ellipsoidal with a diameter of0.2~1.0μm, which showed clusteredappearance. However, the biofilm might be broken as a result of the strong hydraulicshearing when the hydraulic load increased, which might affect nitrogen removalcapacity. Second, AOB and anammox bacteria were side by side with each other,which was not consistant with previous reports that AOB distributed in the outerlayer while anaerobic anammox bacteria was in the inner part of the biofilm. Third,the main functional bacteria contributing to short-cut nitrification process wasNitrosomonas while Candidatus Brocadia contributed to anammox process. Thebiodiversity of AOB was better than that of anammox bacteria, which enhanced the anti-impact capactiy of CANON. Fourth, except for AOB and anammox bacteria,some other kinds of bacteria like Shewanella, Pseudomonas, Ignatzschineria andDechloromonas existed in CANON system. Finally, four AOB strains were screenedsuccessfully from the CANON reactor by using specific selective medium for AOB,which provided theoretical guides for future microbial enrichment, immobilizationand application of genetic engineering.
The study of the effect of different temperature on CANON indicated theaverage TN removal loading was2.21kg N/(m3·d) and1.00kg N/(m3·d) respectivelyat high temperature (30℃) and ambient temperature (16~23℃). Microbiologicalexperiments showed that the population of AOB and anammox bacteria decreasedwhile NOB increased at ambient temperature. Besides, the community diversity oftotal bacteria and AOB also lowered slightly and the volume and spacing ofzoogloea increased. All the changes of microbial features mentioned above might bethe main reason for the decrease of nitrogen-removal capacity of CANON atambient temperature. It was also worth mentioning that the change of temperaturehad little effect on the spatial distribution of the functional microorganisms or thecommunity structure of anammox bacteria. Based on the microbial experimentalresults, such strategies should be in consideration so as to improve nitrogen-removalcapacity at ambient temperature: to add some CANON sludge, to set sludgeintercept instrument or to aerating intermittently.
Except for temperature, influent ammonia concentration is the other mostimportant factor to effect CANON. The study indicated that CANON showed abetter performance when the influent ammonia concentration was higher than200mg/L. When influent ammonia concentration reduced to100mg/L, thenitrogen-removal capacity still existed but the biofilm destroyed and some biomasslost, and the SEM pictures showed the increased zoogloea spacing and loosezoogloea structure. During the whole decreasing of influent ammonia concentration,the community structure of AOB changed obviously, which was not the same withanammox bacteria. Besides, the population of total bacteria and AOB decreasedwhile Nitrospira increased. The population of anammox bacteria decreased slightlywhiel little Nitrobacter could be detected. Based on the microbial experimentsresults, using soft or half-soft filter, adding backwash, reducing aeration or adding acertain amount of nitrite might be some feasible approaches to improve theperformance of CANON at ambient temperature and low substrate concentration.
In this study, a lab-scale CANON reactor was started up successfully within avery few period (180days) by inoculating with activated sludge obtained fromaeration tank of WWTP (A2/O process) at ambient temperature and low substrateconcentration for the first time based on the results above. After three phases ofaeration/intermittent aeration/limited aeration, stable CANON process was achieved with the TN removal loading of1.10kg N/(m3·d). Compared to completely stopaerating, intermittent aeration was conducive to maintaining the balance betweenAOB and anammox bacteria, leading to a much shorter start-up period. Nitrobacterwas hard to eliminated completely through the whole process and the communitystructure of it changed sharply, which was not the same wtih AOB and anammoxbacteria.
In order to improve nitrogen removal capacity of CANON, optimal microbialcontrol was investigated and the results showed that the population was much biggerin the lower part of the filter, which trended to cluster. While in the above layer, itshowed less the number of microorganisms, more loosely, larger spacing and lessdiversity of AOB. It was suggested to change the inflow direction, to aerate equallyalong the filter or to rearrange filter every once in a while so as to optimizemicrobial distribution along the filter. Besides, the biofilm thickness at ambienttemperature was far from the optimal thickness value. Therefore, it was suggested toincrease the biofilm thickness or reduce DO concentration to some degree so as toimprove nitrogen removal capacity of CANON at ambient temperature.
引文
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