城市污水处理过程硝化菌群功能与群落特征研究
摘要
本研究中首先设计和优化了针对污水处理系统氨氧化细菌(Ammoni Odxidizing Bacteria，AOB)和亚硝酸盐氧化细菌(Nitrite Oxidizing Bacteria，NOB)分析的DGGE和Real-Time PCR技术，可分别用于AOB和NOB菌群(包括Nitrospira和Nitrobacter两类)菌群结构和群落密度的分析。实验中采取嵌套式扩增的方法，利用Nitrospira和Nitrobacter菌群16SrDNA保守区域设计和筛选了引物序列，用于DGGE可分离片段的扩增实验；还设计了Nitrobacter菌群Real-Time PCR定量测定方案，此外还对文献报道的硝化细菌分析方法进行了优化。结果证实，通过以上方法可以有效地对污水处理样品中硝化细菌的特定核酸片段进行扩增和密度测定，能够获取污泥或生物膜样品中特定功能细菌群落的组成特征和密度信息。
利用建立的方法对几种新型的污水处理系统的硝化过程和硝化细菌群落进行了分析，包括好氧颗粒污泥系统、两级生物滤池系统、AmOn一体化处理系统和化学生物絮凝处理工艺。研究中发现，前三种处理系统均具有一定的同步硝化反硝化(Simultaneous Nitrification and Denitrification，SND)效果。
在好氧颗粒污泥实验中，利用厌氧颗粒污泥作为接种污泥在40～60天内可成功培养出好氧颗粒污泥，平均颗粒尺寸在1.9～2.3mm左右。利用人工合成废水和实际污水作为研究对象，先后试验了合成废水启动、碳氮比、运行周期、运行温度、曝气强度、原污水溶解氧(Dissolved Oxygen，DO)控制启动等运行条件，此外还与活性污泥工艺进行了对比研究。结果表明：(1)主要的运行参数对于好氧颗粒污泥的SND性能均有影响。优化后的碳氮比条件为5／1～7／1；运行周期为4小时；DO浓度约为1.2～0.5mg／L。优化条件下，好氧颗粒污泥对COD_(Cr)、氨氮和总氮的去除率分别可达75％，76％，和58％左右；(2)与传统活性污泥相比，好氧颗粒污泥的结构特征使其具有更好的SND效果。(3)在启动过程中通过高氨氮浓度和DO控制可以达到亚硝酸盐的稳定积累并促使反硝化过程通过亚硝酸盐的途径进行。(4)同步进行的微生物群落分析表明，主要运行参数的调节会对硝化菌群的结构组成以及密度水平产生影响，且变化规律较为复杂。AOB菌群中优势菌株主要属于Nitrosomonas属；NOB中，Nitrospira为主要的类别，Nitrobacter类的密度比Nitrospira类低一个数量级左右，并且在各种运行条件变化过程中，Nitrobacter类的变化程度较小，AOB与Nitrospira的动念变化更加明显。
在两级生物滤池研究中，通过在首级生物滤池中的DO限制，可以达到一定水平的SND作用。在人工合成污水的条件下，试验了碳氮比和曝气强度的影响，试验表明：(1)较好的碳氮比和DO浓度分别为5／1和1.6～0.8mg/L，在优化的条件下，两级生物滤池对COD_(Cr)、氨氮和总氮的去除率分别可达79％，91％和76％。(2)一级和二级生物滤池中会形成各具特征的硝化菌群结构，当DO超过1.6mg/L，硝化菌群的结构变化显著；在一级生物滤池中，AOB和NOB菌群在特定的碳氮比范围内会保持一定的稳定，Nitrobacter菌群的稳定性比Nitrospira菌群高。
在AmOn处理工艺实验中，重点考察了悬浮填料填充比对于污染物去除效果和硝化菌群的影响。实验表明，AmOn一体化的运行方式能够形成特定的硝化细菌群落结构和优势菌株，Nitrosomonas和Nitrospira属分别为主要的AOB和NOB菌属。悬浮填料填充比的改变能够明显影响硝化菌群的结构，且相对于AOB菌群和Nitrobacter菌属，Nitrospira菌群结构的改变更为明显。
在化学生物絮凝处理工艺中，发现硝化悬浮填料塔中AOB菌群多样性较为丰富，在不同的气水比和水力停留条件下，悬浮填料塔中的AOB菌群显示出较好的稳定性。
研究的中讨论了城市污水处理厂污泥中三种常见的金属离子Mn~(2+)，Cu~(2+)和Cr~(6+)对活性污泥、颗粒污泥和生物膜三种不同特征的生物固体硝化代谢过程的影响。实验发现：(1)对于三种金属离子的影响，不同生物固体类型对金属离子抑制敏感性顺序为：活性污泥＞好氧颗粒污泥＞填料生物膜，表明生物固体的结构特征对于金属离子的抑制具有一定的减弱作用；在受到冲击后的恢复速度上，填料生物膜和颗粒污泥也具有优于活性污泥的表现。不同金属离子的抑制水平大小顺序为：Cr~(6+)＞Cu~(2+)＞Mn~(2+)；在长期Mn~(2+)离子培养后，抑制作用普遍减弱，而在Cu~(2+)和Cr~(6+)长期培养后则会出现强化的趋势。(2)硝化细菌群落多样性会在金属离子高浓度培养下逐渐下降，其细菌密度也会呈下降趋势，总体上而言，活性污泥中AOB菌群密度下降程度最高，生物膜和颗粒污泥中略低。
研究中还将经典的Monod模型和硝化菌群结构特征分析结合起来，选取具有代表性的城市污水处理系统的不同生物固体样品，测定氨氮氧化和亚硝酸盐氧化模型中的最大比增长速率常数、半速率常数和自由铵(Feee Ammonia，FA)抑制常数。微生物群落特征研究中分析了AOB和NOB菌群的组成特征和菌群密度水平。在以上三种常数与硝化菌群特征相关性分析中主要发现：(1)在环境样
品复杂菌群作用下，氨氧化过程和亚硝酸盐氧化过程的最大比反应常数与功能
菌群密度之间仍然具有良好的相关性。(2)AOB菌群的组成特征与半速率常数和
FA抑制常数间具有一定的相关性，表明AOB菌群结构与这两种常数间存在一
定内在联系。
In this paper, DGGE and Real-Time PCR methods are designed and optimized for study of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) communities in wastewater treatment process. Two kinds of methods can be used for analysis of communities' structure and population size for AOB and NOB, respectively. The nested PCR method and the primers based on 16SrDNA of Nitrospira and Nitrobacter groups are applied in DGGE experiment. Quantitative determination for cells density of Nitrobacter group is also developed in this paper. The results prove the availability of the upper methods in analysis of samples from wastewater treatment system. Certain region of special bacterial 16SrDNA can be successfully amplified for biodiversity and quantitative study in sludge and biofilm samples.
The upper methods are utilized in study of several newly developed wastewater treatment process including aerobic granular sludge reactor, two-stage biofilter and integrated AmOn reactor, in which simultaneous nitrification and denitrification (SND) are all found.
In the study of aerobic granular sludge, it can be successfully cultured in 40-60 days using the anaerobic granular sludge as seed sludge. The cultured aerobic granular sludge has the mean size of 1.9-2.3mm. Under the influent condition of synthetic and real wastewater, we study the influence of start-up process, carbon/nitrogen ratio, cycle length, operational temperature and air flow level on contaminations removal performance. Besides these, we also study the dissolved oxygen (DO) restriction in start-up process and compare the removal performance of aerobic granular sludge to that of activated sludge. The results show that SND performance is affected by upper operational conditions in different levels. The optimal conditions in this study including C/N=5/1-7/1, cycle length=4h, DO level=1.2-0.5mg/L, the higher(35℃ in this study) temperature also increase the
SND performance. Under the optimal operational conditions, the removal rates of COD_(Cr), NH_4~+-N and TN are 75%, 76%, and 58%, respectively. Compared with traditional sludge, granular sludge has better SND performance because of the special structure and characters. A certainly high ammonia or DO restriction in start-up process can lead to stable nitrite accumulation, and denitrification can be achieved via nitrite. The adjustment of primary operational conditions should have relatively complex impact on nitrifying bacterial community structure and population size. Nitrosomonas genus is the dominant AOB in aerobic granular sludge, and Nitrospira group takes the dominant position in NOB community. Population size of Nitrobacter is an order less than that of Nitrospira. And in the adjustment of operational conditions, AOB and Nitrospira groups show obvious change, while Nitrobacter group can keep relatively stable.
In the study of two-stage biofilter, SND can be achieved by DO restriction in first stage biofilter. The C/N ratio and air flow are studied in experiment with the influent of synthetic wastewater. The removal rates of CODEr, NH_4~+-N and TN are 79%, 91%, and 76% under optimal C/N=5/1 and DO=1.6-0.8mg/L(in the first stage), respectively. Special nitrifying bacteria communities are revealed in two stages and obvious change can be found when DO level is higher than 1.6mg/L. In the first stage, AOB and NOB communities can keep stable within certain C/N region. The Nitrobacter group also shows higher stability than Nitrospira.
In the study of AmOn process, we focus on the impact of packing ratio on primary contaminations removal and nitrifying bacteria community. The result shows that Nitrosomonas and Nitrospira groups are dominant bacteria AOB and NOB, respaectively. The change of packing ratio obviously affect dominant species in nitrifying bacteria community, and the Nitrospira seem to be more sensitive for the variety of packing ratio than AOB and Nitrobacter.
We investigate the short-term and long-term impact of three metal ions Mn~(2+),Cu~(2+),Cr~(6+) on nitrification and nitrifying bacteria community in activated sludge, granular sludge and biofilm, respectively. Results reveal that different bio-solid has different sensitivity for short-term and long-term impact of upper three metal ions, it is found that activated sludge＞granular sludge＞biofilm, which means biofilm can reduce inhibition of metal ions in certain level. The biofilm and granular sludge have faster recovery than activated sludge after 24-hour inhibition of three metal ions. Different metal ions also show inhibition at different level, and it is found Cr~(6+)＞Cu~(2+)＞Mn~(2+) in this study. After long-term culture of Mn~(2+), inhibition level became to be weak. while heavier inhibition is found after long-term culture of Cu~(2+) and Cr~(6+). The biodiversity and population size of nitrifying bacteria community gradually decrease in the culture of high concentration of metal ions, and it is more obvious in activated sludge than in biofilm and granular sludge.
To study the correlation between classical Monod model and characters of nitrifying bacteria, different kinds of samples collected from sewage treatment processes are investigated. Three constants of ammonia-oxidizing and nitrite-oxidizing processes are measured, including maxium specific substrate utilization rate, half-velocity constant and inhibition conatant of FA (Free Ammonia). In molecular microbial analysis, the dominant species and population size of AOB and NOB are investigated. Results prove the high positive correlation between maxium specific substrate utilization rate and cells density of functional bacteria for ammonia and nitrite oxidizing process even in complex environmental samples such as sludge and biofilm. It is revealed that dominant species affect the half-velocity constant and inhibition conatant of FA too, which indicates the certain inner relation between nitrifying bacteria community structure and two kinds of constants.
利用建立的方法对几种新型的污水处理系统的硝化过程和硝化细菌群落进行了分析，包括好氧颗粒污泥系统、两级生物滤池系统、AmOn一体化处理系统和化学生物絮凝处理工艺。研究中发现，前三种处理系统均具有一定的同步硝化反硝化(Simultaneous Nitrification and Denitrification，SND)效果。
在好氧颗粒污泥实验中，利用厌氧颗粒污泥作为接种污泥在40～60天内可成功培养出好氧颗粒污泥，平均颗粒尺寸在1.9～2.3mm左右。利用人工合成废水和实际污水作为研究对象，先后试验了合成废水启动、碳氮比、运行周期、运行温度、曝气强度、原污水溶解氧(Dissolved Oxygen，DO)控制启动等运行条件，此外还与活性污泥工艺进行了对比研究。结果表明：(1)主要的运行参数对于好氧颗粒污泥的SND性能均有影响。优化后的碳氮比条件为5／1～7／1；运行周期为4小时；DO浓度约为1.2～0.5mg／L。优化条件下，好氧颗粒污泥对COD_(Cr)、氨氮和总氮的去除率分别可达75％，76％，和58％左右；(2)与传统活性污泥相比，好氧颗粒污泥的结构特征使其具有更好的SND效果。(3)在启动过程中通过高氨氮浓度和DO控制可以达到亚硝酸盐的稳定积累并促使反硝化过程通过亚硝酸盐的途径进行。(4)同步进行的微生物群落分析表明，主要运行参数的调节会对硝化菌群的结构组成以及密度水平产生影响，且变化规律较为复杂。AOB菌群中优势菌株主要属于Nitrosomonas属；NOB中，Nitrospira为主要的类别，Nitrobacter类的密度比Nitrospira类低一个数量级左右，并且在各种运行条件变化过程中，Nitrobacter类的变化程度较小，AOB与Nitrospira的动念变化更加明显。
在两级生物滤池研究中，通过在首级生物滤池中的DO限制，可以达到一定水平的SND作用。在人工合成污水的条件下，试验了碳氮比和曝气强度的影响，试验表明：(1)较好的碳氮比和DO浓度分别为5／1和1.6～0.8mg/L，在优化的条件下，两级生物滤池对COD_(Cr)、氨氮和总氮的去除率分别可达79％，91％和76％。(2)一级和二级生物滤池中会形成各具特征的硝化菌群结构，当DO超过1.6mg/L，硝化菌群的结构变化显著；在一级生物滤池中，AOB和NOB菌群在特定的碳氮比范围内会保持一定的稳定，Nitrobacter菌群的稳定性比Nitrospira菌群高。
在AmOn处理工艺实验中，重点考察了悬浮填料填充比对于污染物去除效果和硝化菌群的影响。实验表明，AmOn一体化的运行方式能够形成特定的硝化细菌群落结构和优势菌株，Nitrosomonas和Nitrospira属分别为主要的AOB和NOB菌属。悬浮填料填充比的改变能够明显影响硝化菌群的结构，且相对于AOB菌群和Nitrobacter菌属，Nitrospira菌群结构的改变更为明显。
在化学生物絮凝处理工艺中，发现硝化悬浮填料塔中AOB菌群多样性较为丰富，在不同的气水比和水力停留条件下，悬浮填料塔中的AOB菌群显示出较好的稳定性。
研究的中讨论了城市污水处理厂污泥中三种常见的金属离子Mn~(2+)，Cu~(2+)和Cr~(6+)对活性污泥、颗粒污泥和生物膜三种不同特征的生物固体硝化代谢过程的影响。实验发现：(1)对于三种金属离子的影响，不同生物固体类型对金属离子抑制敏感性顺序为：活性污泥＞好氧颗粒污泥＞填料生物膜，表明生物固体的结构特征对于金属离子的抑制具有一定的减弱作用；在受到冲击后的恢复速度上，填料生物膜和颗粒污泥也具有优于活性污泥的表现。不同金属离子的抑制水平大小顺序为：Cr~(6+)＞Cu~(2+)＞Mn~(2+)；在长期Mn~(2+)离子培养后，抑制作用普遍减弱，而在Cu~(2+)和Cr~(6+)长期培养后则会出现强化的趋势。(2)硝化细菌群落多样性会在金属离子高浓度培养下逐渐下降，其细菌密度也会呈下降趋势，总体上而言，活性污泥中AOB菌群密度下降程度最高，生物膜和颗粒污泥中略低。
研究中还将经典的Monod模型和硝化菌群结构特征分析结合起来，选取具有代表性的城市污水处理系统的不同生物固体样品，测定氨氮氧化和亚硝酸盐氧化模型中的最大比增长速率常数、半速率常数和自由铵(Feee Ammonia，FA)抑制常数。微生物群落特征研究中分析了AOB和NOB菌群的组成特征和菌群密度水平。在以上三种常数与硝化菌群特征相关性分析中主要发现：(1)在环境样
品复杂菌群作用下，氨氧化过程和亚硝酸盐氧化过程的最大比反应常数与功能
菌群密度之间仍然具有良好的相关性。(2)AOB菌群的组成特征与半速率常数和
FA抑制常数间具有一定的相关性，表明AOB菌群结构与这两种常数间存在一
定内在联系。
In this paper, DGGE and Real-Time PCR methods are designed and optimized for study of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) communities in wastewater treatment process. Two kinds of methods can be used for analysis of communities' structure and population size for AOB and NOB, respectively. The nested PCR method and the primers based on 16SrDNA of Nitrospira and Nitrobacter groups are applied in DGGE experiment. Quantitative determination for cells density of Nitrobacter group is also developed in this paper. The results prove the availability of the upper methods in analysis of samples from wastewater treatment system. Certain region of special bacterial 16SrDNA can be successfully amplified for biodiversity and quantitative study in sludge and biofilm samples.
The upper methods are utilized in study of several newly developed wastewater treatment process including aerobic granular sludge reactor, two-stage biofilter and integrated AmOn reactor, in which simultaneous nitrification and denitrification (SND) are all found.
In the study of aerobic granular sludge, it can be successfully cultured in 40-60 days using the anaerobic granular sludge as seed sludge. The cultured aerobic granular sludge has the mean size of 1.9-2.3mm. Under the influent condition of synthetic and real wastewater, we study the influence of start-up process, carbon/nitrogen ratio, cycle length, operational temperature and air flow level on contaminations removal performance. Besides these, we also study the dissolved oxygen (DO) restriction in start-up process and compare the removal performance of aerobic granular sludge to that of activated sludge. The results show that SND performance is affected by upper operational conditions in different levels. The optimal conditions in this study including C/N=5/1-7/1, cycle length=4h, DO level=1.2-0.5mg/L, the higher(35℃ in this study) temperature also increase the
SND performance. Under the optimal operational conditions, the removal rates of COD_(Cr), NH_4~+-N and TN are 75%, 76%, and 58%, respectively. Compared with traditional sludge, granular sludge has better SND performance because of the special structure and characters. A certainly high ammonia or DO restriction in start-up process can lead to stable nitrite accumulation, and denitrification can be achieved via nitrite. The adjustment of primary operational conditions should have relatively complex impact on nitrifying bacterial community structure and population size. Nitrosomonas genus is the dominant AOB in aerobic granular sludge, and Nitrospira group takes the dominant position in NOB community. Population size of Nitrobacter is an order less than that of Nitrospira. And in the adjustment of operational conditions, AOB and Nitrospira groups show obvious change, while Nitrobacter group can keep relatively stable.
In the study of two-stage biofilter, SND can be achieved by DO restriction in first stage biofilter. The C/N ratio and air flow are studied in experiment with the influent of synthetic wastewater. The removal rates of CODEr, NH_4~+-N and TN are 79%, 91%, and 76% under optimal C/N=5/1 and DO=1.6-0.8mg/L(in the first stage), respectively. Special nitrifying bacteria communities are revealed in two stages and obvious change can be found when DO level is higher than 1.6mg/L. In the first stage, AOB and NOB communities can keep stable within certain C/N region. The Nitrobacter group also shows higher stability than Nitrospira.
In the study of AmOn process, we focus on the impact of packing ratio on primary contaminations removal and nitrifying bacteria community. The result shows that Nitrosomonas and Nitrospira groups are dominant bacteria AOB and NOB, respaectively. The change of packing ratio obviously affect dominant species in nitrifying bacteria community, and the Nitrospira seem to be more sensitive for the variety of packing ratio than AOB and Nitrobacter.
We investigate the short-term and long-term impact of three metal ions Mn~(2+),Cu~(2+),Cr~(6+) on nitrification and nitrifying bacteria community in activated sludge, granular sludge and biofilm, respectively. Results reveal that different bio-solid has different sensitivity for short-term and long-term impact of upper three metal ions, it is found that activated sludge＞granular sludge＞biofilm, which means biofilm can reduce inhibition of metal ions in certain level. The biofilm and granular sludge have faster recovery than activated sludge after 24-hour inhibition of three metal ions. Different metal ions also show inhibition at different level, and it is found Cr~(6+)＞Cu~(2+)＞Mn~(2+) in this study. After long-term culture of Mn~(2+), inhibition level became to be weak. while heavier inhibition is found after long-term culture of Cu~(2+) and Cr~(6+). The biodiversity and population size of nitrifying bacteria community gradually decrease in the culture of high concentration of metal ions, and it is more obvious in activated sludge than in biofilm and granular sludge.
To study the correlation between classical Monod model and characters of nitrifying bacteria, different kinds of samples collected from sewage treatment processes are investigated. Three constants of ammonia-oxidizing and nitrite-oxidizing processes are measured, including maxium specific substrate utilization rate, half-velocity constant and inhibition conatant of FA (Free Ammonia). In molecular microbial analysis, the dominant species and population size of AOB and NOB are investigated. Results prove the high positive correlation between maxium specific substrate utilization rate and cells density of functional bacteria for ammonia and nitrite oxidizing process even in complex environmental samples such as sludge and biofilm. It is revealed that dominant species affect the half-velocity constant and inhibition conatant of FA too, which indicates the certain inner relation between nitrifying bacteria community structure and two kinds of constants.
引文
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