常温低基质PN-ANAMMOX耦合工艺脱氮效能及微生物特性研究
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摘要
水体中的氮素过多容易引起富营养化，给水体环境带来危害。与传统硝化反硝化生物脱氮相比，亚硝化（PN）-厌氧氨氧化（ANAMMOX）工艺具有节省曝气能耗、无需外加碳源、污泥产量低等优点，但其主要应用于高温（30-40℃）高氨氮（>500mg/L）污水处理。另外，微生物群落结构是其功能发生的基础，氨氧化菌（AOB）与厌氧氨氧化菌是亚硝化与厌氧氨氧化工艺中的功能微生物，本文通过对亚硝化与厌氧氨氧化反应器内微生物群落特性及功能菌群进行研究，为实现常温低基质PN-ANAMMOX提供理论基础与借鉴。
开展生物膜（SBBR）和活性污泥（SBR）亚硝化试验，并比较两种形式反应器在常温低基质阶段的亚硝化效果。创建了浓度梯度驯化法，利用逐渐降低进水氨氮浓度方式来驯化反应器内AOB，进而实现了常温低基质条件下SBBR和SBR快速启动。SBBR与SBR微生物群落结构均受FA浓度影响而发生变化。在低氨氮阶段，SBR反应器内AOB属于亚硝化单胞均（Nitrosomonas），为反应器内优势菌群，NOB所占比例极少，这是SBR在常温低基质阶段保持稳定亚硝化的原因。
开展低温ANAMMOX试验，以期为厌氧氨氧化工艺在冬季低温下运行提供借鉴。在冬季低温条件下（15-16.5℃），火山岩填料生物滤柱平均总氮去除负荷达1.57kg/(m~3d)，明显高于陶粒填料反应器（0.52kg/(m~3d)）。分析原因发现火山岩填料上ANAMMOX菌所占比例高于陶粒填料，火山岩更适合作为富集ANAMMOX菌的生物滤池填料。对火山岩填料生物滤柱沿层三氮分析，发现大部分氨氮与亚氮在反应器中部成比例去除，分析原因发现ANAMMOX菌在不同滤层菌种相同（Candidatus Kuenenia stuttgartiensis），但其在中部所占比例最高。另外，在反应器不同滤层，存在同一种AOB（Nitrosomonas sp. ENI-11）。
在低温ANAMMOX生物滤柱基础上，通过降低进水氨氮浓度，考察反应器在常温低基质进水下的脱氮效果。试验进水为自然温度（14.5-24.5℃），反应器在低基质阶段Ⅰ（78.7mg/L氨氮）与低基质阶段Ⅱ（46.5mg/L氨氮）都有很好的脱氮效果，其中在低基质阶段Ⅱ总氮去除负荷平均值为2.26kg/(m~3·d)，最高达3.04kg/(m~3d)。反应器中ANAMMOX菌在高、低基质阶段Ⅰ和Ⅱ菌属相同，同为Candidatus Kuenenia stuttgartiensis，但其在三个阶段的相对比例有所差异，分别为41.6%、37.9%和36.3%。在低基质阶段Ⅱ沿层脱氮试验表明，大部分氮素在生物滤柱中部得到去除。Candidatus Kuenenia stuttgartiensis对于温度和基质浓度有着较宽泛的适应性，发现其能在常温低基质环境中保持较高的活性，因而有希望将该ANAMMOX菌种应用于常温低氨氮污水处理。
在前面单体亚硝化（PN）与厌氧氨氧化（ANAMMOX）基础上，进行PN-ANAMMOX串联试验，考察该工艺对常温（18-25℃）低基生活污水的脱氮效果。PN单元启动亚硝化后，推流式连续流运行80天时间内，保持较低的DO浓度（0.1-0.6mg/L），出水NO_2~--N/NH_4~+-N值比较稳定，可以为后续ANAMMOX单元提供适宜的进水。PN-ANAMMOX串联后，ANAMMOX单元添加成熟ANAMMOX污泥25天后启动成功，其最高总氮去除负荷达0.99kg/(m~3d)，最高总氮去除率为85.6%，显示出良好的脱氮潜力。耦合因素分析发现，PN单元出水NO_2~--N/NH_4~+-N为关键因素，比值在1.01-1.45范围内时，ANAMMOX单元出水能够满足一级A标准（GB18918-2002)。
PN-ANAMMOX耦合成功后，亚硝化污泥中以球形菌与短杆菌为主；功能微生物AOB为优势菌群，其菌属为Nitrosomonas sp.；PN单元存在一些异养菌，但并没有影响AOB的优势地位与反应器的亚硝化效果。ANAMMOX单元生物膜上微生物以球形菌为主，ANAMMOX菌为优势菌群；ANAMMOX菌种由接种的Candidatus Kuenenia stuttgartiensis转变为Candidatus Brocadia fulgida，后者更能适应含低基质有机物的生活污水环境，因而成为生物膜上优势ANAMMOX菌种；生物膜上还存在一些AOB与反硝化菌，它们与ANAMMOX菌一起组成功能菌群，以适应进水NO_2~--N/NH_4~+-N比值的微小波动，保持ANAMMOX单元稳定的脱氮效果。
The current wastewater treatment systems are required for removal ofnitrogenous compounds, which could cause eutrophication of surface water bodies.Compared with the conventional nitrification/denitrification process, partialnitrification (PN) coupled with anaerobic ammonium-oxidizing (ANAMMOX)process is an ideal biological nitrogen removal choice. It could save aeration energyconsumption, as well as less biomass production and without organic carbonconsumption. However, most of PN-ANAMMOX processes focused on treatingwarm (>30℃) and concentrated (>500mgN/L) waste streams. On the other hand,the key of promotion process relies on knowing the microbial communityinvolvement. Ammonia oxidizing bacteria (AOB) and ANAMMOX bacteria arefunctional microorganisms in PN-ANAMMOX process. Analysis of microbialcommunity structures and functional microorganisms will contribute to PN andANAMMOX processes optimization. Hence, the aim of the present study was toprovide theoretical guidance for PN-ANAMMOX process treating low strengthwastewater at ambient temperature.
Partial nitrifying experiments were carried out basing on sequencing batchbiofilm reactor (SBBR) and sequencing batch reactor (SBR), which were used forcomparison partial nitrification treating low strength nitrogen wastewater at ambienttemperature. Partial nitrification of SBBR and SBR could be rapidly realized sinceAOB were domesticated in low strength wastewater as influent ammoniumconcentration gradually decreased. Free ammonia (FA) was the key factor affectingmicrobial community of both SBBR and SBR. Nitrosomonas were predominantAOB in SBR with low ammonia influent. The microbial characteristic ofpredominant AOB and less NOB contributed to stable partial nitrification of SBRtreating low strength wastewater at ambient temperature.
Experiments of ANAMMOX were carried out under low temperature, with theaim to provide a reference for ANAMMOX process running and optimization inwinter time. The biofilter filled with volcanic rocks had higher nitrogen removalcapacity than that of ceramic packed biofilter at low temperature (15-16.5℃) inwinter. Total nitrogen removal rate of B2was1.57kg/(m~3d), which was muchhigher than that of B1(0.52kg/(m~3d)). The genus of ANAMMOX bacteria was bothCandidatus Kuenenia stuttgartiensis in two reactors. However, ANAMMOXbacteria had more densely distribution in volcanic rocks biofilter, which indicatedthat volcanic rocks were more suitable for ANAMMOX bacteria enrichment in biofilters. Nitrogen removal rate along volcanic ANAMMOX biofilter wasinvestigated. Results showed that a large proportion of ammonia and nitriteproportional disappeared in the middle part of biofilter. It was owed to theproportion of ANAMMOX bacteria in middle of biofilter was bigger than that inother parts by FISH analysis. There was only one type of ANAMMOX bacteriumand AOB present in different parts of biofilter, which were identified as CandidatusKuenenia stuttgartiensis and Nitrosomonas sp. ENI-11, respectively.
Based on previous volcanic ANAMMOX biofilter, experiments were carriedout to investigate nitrogen removal rate of bioreactor for low strength wastewatertreatment at ambient temperature (14.5-24.5℃). The results showed ANAMMOXbiofilter had the ability to maintain high capacity of nitrogen removal rate underlow ammonia concentrations of periodⅠ(78.7mg/L) and periodⅡ(46.5mg/L). Forexample, average and maximum total nitrogen removal rates were2.26kg/(m~3·d)and3.04kg/(m~3·d) at periodⅡ. The sequence of ANAMMOX16SrRNAidentification revealed that Candidatus Kuenenia stuttgartiensis occurred in all highand low matrix periods. However, it had a little difference of relative proportions inthree periods, which were accounted for41.6%、37.9%and36.3%. Nitrogenremoval rate along biofilter showed that a large proportion of ammonia and nitriteproportional disappeared in the middle part of biofilter. Candidatus Kueneniastuttgartiensis had wide adaptability to variable temperature and substrateconcentrations, which indicated that it had potential to apply such ANAMMOXbacteria for low strength wastewater treatment at ambient temperature.
On the basis of independent running partial nitrification and ANAMMOXreactors, PN-ANAMMOX experiments were developed for sewage treatment atambient temperature. After PN unit start-up, plug-flow continuous stream runningwas carried out with low DO concentration (0.1to0.6mg/L). EffluentNO_2~--N/NH_4~+-N of PN was relatively stable, which was suitable for subsequentANAMMOX unit. Biofilter was successful started up after25days by addingmature ANAMMOX sludge. The bioreactor had a satisfactory nitrogen removalpotential with maximum nitrogen removal rate of0.99kg/(m~3·d) and maximumnitrogen removal efficiency of85.6%. Analysis of PN-ANAMMOX couplingfactors showed that NO_2~--N/NH_4~+-N was the key factor, which could affect nitrogenremoval of ANAMMOX biofilter. The outlet water quality of ANAMMOX unitsatisfied the Standard A of the first class in “Discharge standard of pollutants formunicipal wastewater treatment plant (GB18918-2002)” when the ratio ofNO_2~--N/NH_4~+-N was between1.01to1.45.
After PN-ANAMMOX successfully coupled, spherical and short rod bacteria were predominant in partial nitrifying sludge, in which AOB was the dominantbacteria. The genus of AOB was Nitrosomonas sp., which could be coexisted withother heterotrophic bacteria harmony in PN unit. Combining FISH analysis withSEM observations confirmed the superiority of ANAMMOX bacteria in thecultivated biofilm. The principal anammox bacteria shifted from members related toCandidatus Kuenenia stuttgartiensis present in the initial inoculum to membersrelated to Candidatus Brocadia fulgida in biofilm. Candidatus Brocadia fulgidawere the functional ANAMMOX bacteria in the reactor, which could survivecomfortably in sewage with a limited amount of COD. AOB and denitrifyingbacteria were also found in the bifofilm, which were the members of microbialcommunities living together with ANAMMOX bacteria. The functional microbialpopulations contributed to stable nitrogen removal rate of ANAMMOX unit toaccommodate the slight fluctuation of influent NO_2~--N/NH_4~+-N.
开展生物膜（SBBR）和活性污泥（SBR）亚硝化试验，并比较两种形式反应器在常温低基质阶段的亚硝化效果。创建了浓度梯度驯化法，利用逐渐降低进水氨氮浓度方式来驯化反应器内AOB，进而实现了常温低基质条件下SBBR和SBR快速启动。SBBR与SBR微生物群落结构均受FA浓度影响而发生变化。在低氨氮阶段，SBR反应器内AOB属于亚硝化单胞均（Nitrosomonas），为反应器内优势菌群，NOB所占比例极少，这是SBR在常温低基质阶段保持稳定亚硝化的原因。
开展低温ANAMMOX试验，以期为厌氧氨氧化工艺在冬季低温下运行提供借鉴。在冬季低温条件下（15-16.5℃），火山岩填料生物滤柱平均总氮去除负荷达1.57kg/(m~3d)，明显高于陶粒填料反应器（0.52kg/(m~3d)）。分析原因发现火山岩填料上ANAMMOX菌所占比例高于陶粒填料，火山岩更适合作为富集ANAMMOX菌的生物滤池填料。对火山岩填料生物滤柱沿层三氮分析，发现大部分氨氮与亚氮在反应器中部成比例去除，分析原因发现ANAMMOX菌在不同滤层菌种相同（Candidatus Kuenenia stuttgartiensis），但其在中部所占比例最高。另外，在反应器不同滤层，存在同一种AOB（Nitrosomonas sp. ENI-11）。
在低温ANAMMOX生物滤柱基础上，通过降低进水氨氮浓度，考察反应器在常温低基质进水下的脱氮效果。试验进水为自然温度（14.5-24.5℃），反应器在低基质阶段Ⅰ（78.7mg/L氨氮）与低基质阶段Ⅱ（46.5mg/L氨氮）都有很好的脱氮效果，其中在低基质阶段Ⅱ总氮去除负荷平均值为2.26kg/(m~3·d)，最高达3.04kg/(m~3d)。反应器中ANAMMOX菌在高、低基质阶段Ⅰ和Ⅱ菌属相同，同为Candidatus Kuenenia stuttgartiensis，但其在三个阶段的相对比例有所差异，分别为41.6%、37.9%和36.3%。在低基质阶段Ⅱ沿层脱氮试验表明，大部分氮素在生物滤柱中部得到去除。Candidatus Kuenenia stuttgartiensis对于温度和基质浓度有着较宽泛的适应性，发现其能在常温低基质环境中保持较高的活性，因而有希望将该ANAMMOX菌种应用于常温低氨氮污水处理。
在前面单体亚硝化（PN）与厌氧氨氧化（ANAMMOX）基础上，进行PN-ANAMMOX串联试验，考察该工艺对常温（18-25℃）低基生活污水的脱氮效果。PN单元启动亚硝化后，推流式连续流运行80天时间内，保持较低的DO浓度（0.1-0.6mg/L），出水NO_2~--N/NH_4~+-N值比较稳定，可以为后续ANAMMOX单元提供适宜的进水。PN-ANAMMOX串联后，ANAMMOX单元添加成熟ANAMMOX污泥25天后启动成功，其最高总氮去除负荷达0.99kg/(m~3d)，最高总氮去除率为85.6%，显示出良好的脱氮潜力。耦合因素分析发现，PN单元出水NO_2~--N/NH_4~+-N为关键因素，比值在1.01-1.45范围内时，ANAMMOX单元出水能够满足一级A标准（GB18918-2002)。
PN-ANAMMOX耦合成功后，亚硝化污泥中以球形菌与短杆菌为主；功能微生物AOB为优势菌群，其菌属为Nitrosomonas sp.；PN单元存在一些异养菌，但并没有影响AOB的优势地位与反应器的亚硝化效果。ANAMMOX单元生物膜上微生物以球形菌为主，ANAMMOX菌为优势菌群；ANAMMOX菌种由接种的Candidatus Kuenenia stuttgartiensis转变为Candidatus Brocadia fulgida，后者更能适应含低基质有机物的生活污水环境，因而成为生物膜上优势ANAMMOX菌种；生物膜上还存在一些AOB与反硝化菌，它们与ANAMMOX菌一起组成功能菌群，以适应进水NO_2~--N/NH_4~+-N比值的微小波动，保持ANAMMOX单元稳定的脱氮效果。
The current wastewater treatment systems are required for removal ofnitrogenous compounds, which could cause eutrophication of surface water bodies.Compared with the conventional nitrification/denitrification process, partialnitrification (PN) coupled with anaerobic ammonium-oxidizing (ANAMMOX)process is an ideal biological nitrogen removal choice. It could save aeration energyconsumption, as well as less biomass production and without organic carbonconsumption. However, most of PN-ANAMMOX processes focused on treatingwarm (>30℃) and concentrated (>500mgN/L) waste streams. On the other hand,the key of promotion process relies on knowing the microbial communityinvolvement. Ammonia oxidizing bacteria (AOB) and ANAMMOX bacteria arefunctional microorganisms in PN-ANAMMOX process. Analysis of microbialcommunity structures and functional microorganisms will contribute to PN andANAMMOX processes optimization. Hence, the aim of the present study was toprovide theoretical guidance for PN-ANAMMOX process treating low strengthwastewater at ambient temperature.
Partial nitrifying experiments were carried out basing on sequencing batchbiofilm reactor (SBBR) and sequencing batch reactor (SBR), which were used forcomparison partial nitrification treating low strength nitrogen wastewater at ambienttemperature. Partial nitrification of SBBR and SBR could be rapidly realized sinceAOB were domesticated in low strength wastewater as influent ammoniumconcentration gradually decreased. Free ammonia (FA) was the key factor affectingmicrobial community of both SBBR and SBR. Nitrosomonas were predominantAOB in SBR with low ammonia influent. The microbial characteristic ofpredominant AOB and less NOB contributed to stable partial nitrification of SBRtreating low strength wastewater at ambient temperature.
Experiments of ANAMMOX were carried out under low temperature, with theaim to provide a reference for ANAMMOX process running and optimization inwinter time. The biofilter filled with volcanic rocks had higher nitrogen removalcapacity than that of ceramic packed biofilter at low temperature (15-16.5℃) inwinter. Total nitrogen removal rate of B2was1.57kg/(m~3d), which was muchhigher than that of B1(0.52kg/(m~3d)). The genus of ANAMMOX bacteria was bothCandidatus Kuenenia stuttgartiensis in two reactors. However, ANAMMOXbacteria had more densely distribution in volcanic rocks biofilter, which indicatedthat volcanic rocks were more suitable for ANAMMOX bacteria enrichment in biofilters. Nitrogen removal rate along volcanic ANAMMOX biofilter wasinvestigated. Results showed that a large proportion of ammonia and nitriteproportional disappeared in the middle part of biofilter. It was owed to theproportion of ANAMMOX bacteria in middle of biofilter was bigger than that inother parts by FISH analysis. There was only one type of ANAMMOX bacteriumand AOB present in different parts of biofilter, which were identified as CandidatusKuenenia stuttgartiensis and Nitrosomonas sp. ENI-11, respectively.
Based on previous volcanic ANAMMOX biofilter, experiments were carriedout to investigate nitrogen removal rate of bioreactor for low strength wastewatertreatment at ambient temperature (14.5-24.5℃). The results showed ANAMMOXbiofilter had the ability to maintain high capacity of nitrogen removal rate underlow ammonia concentrations of periodⅠ(78.7mg/L) and periodⅡ(46.5mg/L). Forexample, average and maximum total nitrogen removal rates were2.26kg/(m~3·d)and3.04kg/(m~3·d) at periodⅡ. The sequence of ANAMMOX16SrRNAidentification revealed that Candidatus Kuenenia stuttgartiensis occurred in all highand low matrix periods. However, it had a little difference of relative proportions inthree periods, which were accounted for41.6%、37.9%and36.3%. Nitrogenremoval rate along biofilter showed that a large proportion of ammonia and nitriteproportional disappeared in the middle part of biofilter. Candidatus Kueneniastuttgartiensis had wide adaptability to variable temperature and substrateconcentrations, which indicated that it had potential to apply such ANAMMOXbacteria for low strength wastewater treatment at ambient temperature.
On the basis of independent running partial nitrification and ANAMMOXreactors, PN-ANAMMOX experiments were developed for sewage treatment atambient temperature. After PN unit start-up, plug-flow continuous stream runningwas carried out with low DO concentration (0.1to0.6mg/L). EffluentNO_2~--N/NH_4~+-N of PN was relatively stable, which was suitable for subsequentANAMMOX unit. Biofilter was successful started up after25days by addingmature ANAMMOX sludge. The bioreactor had a satisfactory nitrogen removalpotential with maximum nitrogen removal rate of0.99kg/(m~3·d) and maximumnitrogen removal efficiency of85.6%. Analysis of PN-ANAMMOX couplingfactors showed that NO_2~--N/NH_4~+-N was the key factor, which could affect nitrogenremoval of ANAMMOX biofilter. The outlet water quality of ANAMMOX unitsatisfied the Standard A of the first class in “Discharge standard of pollutants formunicipal wastewater treatment plant (GB18918-2002)” when the ratio ofNO_2~--N/NH_4~+-N was between1.01to1.45.
After PN-ANAMMOX successfully coupled, spherical and short rod bacteria were predominant in partial nitrifying sludge, in which AOB was the dominantbacteria. The genus of AOB was Nitrosomonas sp., which could be coexisted withother heterotrophic bacteria harmony in PN unit. Combining FISH analysis withSEM observations confirmed the superiority of ANAMMOX bacteria in thecultivated biofilm. The principal anammox bacteria shifted from members related toCandidatus Kuenenia stuttgartiensis present in the initial inoculum to membersrelated to Candidatus Brocadia fulgida in biofilm. Candidatus Brocadia fulgidawere the functional ANAMMOX bacteria in the reactor, which could survivecomfortably in sewage with a limited amount of COD. AOB and denitrifyingbacteria were also found in the bifofilm, which were the members of microbialcommunities living together with ANAMMOX bacteria. The functional microbialpopulations contributed to stable nitrogen removal rate of ANAMMOX unit toaccommodate the slight fluctuation of influent NO_2~--N/NH_4~+-N.
引文
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