EBPR系统中聚磷菌与聚糖菌的竞争和调控的基础研究
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摘要
随着环境水体问题的日益突出,“水体富营养化”问题已得到国内外学术专家、环保工作者及政府部门的高度重视,污水排放标准不断严格化,污水处理技术逐渐从以单一去除有机物为目的逐渐转变为既要去除有机物,又要脱氮除磷的深度处理,以控制富营养化为目的的脱氮除磷已成为当今污水处理领域的研究热点之一。强化生物除磷(EBPR)是污水除磷的有效而经济的方法,但是近年来很多学者发现由于系统中存在大量的聚糖菌(glycogen accumulating organisms,GAO),它与具有除磷功能的聚磷菌(phosphorus accumulating organisms,PAO)竞争有限基质和环境因素等条件,常常导致强化生物除磷系统恶化甚至崩溃。为促进强化生物除磷工艺的发展,尤其是促进聚磷菌与聚糖菌竞争原理及调控方法方面的探讨,本课题以SBR反应器为试验装置,分别研究了以实际生活污水和人工合成配水为基质的强化生物除磷系统,这对于人们认识EBPR,寻求调节菌群之间的竞争手段具有比较重要的意义。
首先,本研究以P/C和碳源种类这2个关键因素控制强化生物除磷系统种群富集的方向,试图通过富集获得近似于纯培养的大量的PAO,GAO和α-GAO,高P/C下以乙酸丙酸交替富集PAO,在低P/C下可以以乙酸富集GAO,以丙酸富集α-GAO。富集结果三个反应器中PAO、GAO和α-GAO各占80%,90%,80%以上。
鉴于纯培养产物对于研究聚磷菌的重要性,本研究采用传统的倾注法和涂布法分离,用连续划线法纯化,以具有强化生物除磷功能的活性污泥作为菌种来源,对聚磷菌进行了分离,纯化和筛选。分离纯化得到纯菌87株。经过蓝白斑法初筛,有4株菌在限磷培养基和高磷培养基上都显篮斑,经过吸放磷试验复筛,得到一株具有厌氧放磷好氧吸磷能力的菌株P4,经考察,它同时具有在厌氧和好氧交替的条件下转换PHA,糖原以及聚磷的功能。结合P4的个体和菌落形态特征、生理生化试验指标,以16sRNA技术鉴定P4为不动杆菌Acinetobacter sp.DG880[AY258108]。
在环境条件对于EBPR系统的影响以及对于PAO和GAO竞争的影响试验中,本研究主要探讨了pH、温度、碳源、C/P以及亚硝态氮的影响。
厌氧段pH影响小试试验表明,较高的pH对系统放磷有利,在6.0到8.5范围内pH越高,系统放磷量就越大,厌氧段pH的差异对系统去除COD的能力基本没有影响,却对PHA的合成影响显著,在厌氧段pH为6.0和8.5时,系统合成PHA的能力都受到了明显的抑制。好氧段pH影响小试试验表明,6.0明显是一个非常不利于聚磷菌吸磷的pH条件,在6.5到8.5的范围内,高pH对好氧吸磷有利。厌氧段pH长期影响试验表明,8.0是比较适合聚磷菌放磷的pH条件,在pH为7.0-8.0时除磷率可以达到80%以上,在pH为6.5时放磷量和吸磷量都有显著下降,在pH为6.0和8.5时,系统几乎完全丧失除磷功能,但是聚磷菌只是被不良条件抑制,一旦恢复正常的不调节pH的操作模式,除磷率迅速提升。因此,以实际生活污水驯化的污泥具有一定的适应不良pH影响的能力。
在以实际生活污水驯化污泥的强化生物除磷系统不能在低温(低于15℃)下长期运行。低温会破坏EBPR的生态系统平衡。
在碳源影响的正交试验中,与投加碳源量和投加磷量相比,碳源种类是影响EBPR系统磷去除率的最关键因素,葡萄糖的均值最大(96.357),说明葡萄糖是对以生活污水驯化的污泥中的PAO来说最好的碳源,以葡萄糖为碳源,系统去除磷的平均效率为96.357%;此外,投加碳源量是影响系统放磷和吸磷的最关键因素,投加碳源越多,系统放磷/吸磷量就越大,对于系统放磷/吸磷来说,丙酸钠是最好的碳源。
C/P比影响试验结果显示,C/P对COD去除率的影响微乎其微,在C/P小于或等于30时,磷去除率随着C/P的增加而增加,但是当C/P大于30时,磷去除率的变化就不再显著;随着C/P的增加,系统合成PHA的量没有明显差异,但系统降解COD/释放P的比例增加,两者之间呈线性关系。
亚硝态氮影响试验表明,随着系统好氧结束时亚硝态氮积累越来越多,SBR系统除磷能力不断的下降,当亚硝态氮积累超过10mg/L时,磷去除率从原来的60%左右下降为20%,所以,10mg/L的亚硝态氮对于强化生物除磷系统来说是一个可以容忍的上限值。
以丙酸为底物,普通絮状活性污泥为接种污泥,在SBR中通过一系列手段可以实现富集PAO的好氧活性污泥的颗粒化。培养的富集PAO的颗粒污泥对COD去除率可达94%,磷去除率可达100%。成熟的富集PAO的好氧颗粒污泥平均直径为2mm,白色,表面致密,外层有毛状物,同时具有低含水率和低比重等优良性质。与普通的富集PAO的活性污泥相比,富集PAO的颗粒污泥具有生物量大,沉淀速度快,抗COD冲击负荷能力强等众多优点,具有一定的理论和实际意义。
In nowadays, people, including the enrironmental experts, the enriomental protection postulants and the government, pay more and more attention on the eutrophication questions because of the degeneration of the waterbody. The standard of the wastewater discharge was controlled more strictly to achieve not only COD removal but also simultaneous COD, nitrogen and phosphorus removal. So the nitrogen and phosphorus removal technology aimed for eutrophication control has been the study focus in the wastewater treatment area. Enhanced biological phosphorus removal system is accepted in many country for its effective and low cost, while it often faced to deteriotated or collapse because of the existence or enrichment of glycogen accumulating organisms(GAO) in the system. GAO can competed with the phosphorus removal sponsor: phosphorus accumulating organisms(PAO) for the limited carbon souce and other substance. In order to accelerate the investigation development of phosphorus removal, especially on the competition principle and control method between PAO and GAO, this research use lab SBR fed with raw water or synthetic wastewater to study the enhanced biological phosphorus removal system(EBPR). We hope this kind of work can benefit for the people who dedicated in the biological phosphoros removal.
Because of the absent of PAO or GAO’s pure culture in the world, this research aimed to obtain large quantity of PAO、GAO orα-GAO by enrichment works. This kind of enrichment cultures is very important for people to know the physiological and biochemical characterists and the metabolic pathway of these three groups. Other than some suitable conditions just like pH, tempreture and load, P/C and carben source were two important selective factors for this enrichment works. As results, PAO reached 80%of the total bacteria,the efficiency of P removal of the system was over than 98%. GAO could reach 90%of the total bacteria, and also,α-GAO was a absolutely predominant group in its reactor.
Although people haven’t isolated PAO successfully till now, but this research tried to isolate and purify the phosphorus accumulating bacterial because of the importance of the pure culture for the understanding of the EBPR system.
87 strains were isolated from the activated sludge with phosphorus removal ability. After pre-screening by blue-white blaze test, re-screening by phosphorus release/uptake test, 1 strain, P4, with high phosphorus release ability in anaerobic phase and high phosphorus uptake ability in aerobic phase was achieved, and also, it could convert PHA, glycogen and poly-P in the anaerobic-aerobic condititions. Through observing the morphological feature of YP4, analyzing the physiology and biochemistry index, combining the 16SrRNA technique, YP4 was identified as acinetovacter sp.DG880.
Among many factors that can affect the competition between PAO and GAO, this research focused on pH, tempreture, carbon source, C/P and nitrite.
The short term effect operated by batch test showed that the amount of phosphorus released was related to the anaerobic pH, PAO prefered higher pH for phosphorus release, the higher pH was, the more phosphorus was released into the water. But on the other hand, the varision of pH did nothing on COD removal but it could affect the PHA synthetic significantly. The system fed with raw water showed steady state when anaerobic pH ranged from 6.5 to 8.0. A slight change of pH from 6.5 to 6.0 or from 8.0 to 8.5 led to a complete loss of phosphate-removing capability. Compared to PAO, GAO fed with raw water had a wider anaerobic pH survival range. When the anaerobic pH changed from 6.0 to 8.5, there was no significant effect of pH on GAO. The activated sludge fed by raw water had ability to be suit for the undesired pH conditions. Aerobic pH affect test showed that 6.0 was not fit for PAO to uptake phosphorus, while other pH conditions(6.5-8.0) had nothing to do with phosphorus uptake.
The enhanced biological phosphorus removal system, using activated sludge fed by raw water couldn’t be operated in low(<15℃)temperature for a long period, for it might destroy the other bacteria’s metablic leading to destroy the balance of PAO and the other bacteria.
In the orthogonal tests, all the three factors, including the kind of carbon source, quantity of added carbon source and quantity of added phosphate, could affect the phosphorus removal rate. The kind of carbon source was the crucial factor to affect the phosphorus removal rate, and the phosphorus removal rate increased with the addition quantity of carbon source (from 100mg/L to 300mg/L) and the decrease of the phosphate source (from 21mg/L to 5mg/L). The experiment showed that glucose could be utilized by phosphorus accumulating organisms as the most available carbon source compared with both sodium acetate and sodium propionate in actual WWTP operation. A different result from the traditional concept obtained in this study is that the system could release and uptake more phosphorus when it was fed by propionate than when it was fed by acetate, since the system we used is enriched and cultured by raw water, so it showed different performance from the one cultured by synthetic wastewater.
Batch experiment showed that phosphorus removal rate varied remarkably with the C/P ratio at the value of 30 or less, while the COD removal rate did not. And also, C/P ratio also effected the COD reduction/ P release greatly.
The accumulated nitrite in the aerobic phase would affect the phosphorus removal ability of the system. When the concentration of nitrite was over 10mg/L and increased with a rapid speed, the phosphorus removal rate decreased from 60% to 20% darmatically. So 10mg/L was the endurable highest nitrite concentration for the EBPR system. Besides, nitrite could affect phosphorus uptake more than phosphorus release.
After inoculated with floc activated sludge, the culturing of PAO enriched granular sludge could be achieved by using propionate as substrate, alternating the concentration of COD load, shortening settling time, providing hydrodynamic shear stress and high DO concentration. GAO was washed out of the system and PAO became the dominant population when acetate was used as substrate. The PAO enriched granular sludge had perfect COD removal rate of 94% and phosphorus removal rate of 100%. The mature PAO enriched granules had compact structure, with mean diameter of 2mm and white flagellum on the surface. And also, the mature granules had high OUR, density, integrality rate and low aquiferous rate, leading to the reactor could contain high biomass concentration with good settling properties.
首先,本研究以P/C和碳源种类这2个关键因素控制强化生物除磷系统种群富集的方向,试图通过富集获得近似于纯培养的大量的PAO,GAO和α-GAO,高P/C下以乙酸丙酸交替富集PAO,在低P/C下可以以乙酸富集GAO,以丙酸富集α-GAO。富集结果三个反应器中PAO、GAO和α-GAO各占80%,90%,80%以上。
鉴于纯培养产物对于研究聚磷菌的重要性,本研究采用传统的倾注法和涂布法分离,用连续划线法纯化,以具有强化生物除磷功能的活性污泥作为菌种来源,对聚磷菌进行了分离,纯化和筛选。分离纯化得到纯菌87株。经过蓝白斑法初筛,有4株菌在限磷培养基和高磷培养基上都显篮斑,经过吸放磷试验复筛,得到一株具有厌氧放磷好氧吸磷能力的菌株P4,经考察,它同时具有在厌氧和好氧交替的条件下转换PHA,糖原以及聚磷的功能。结合P4的个体和菌落形态特征、生理生化试验指标,以16sRNA技术鉴定P4为不动杆菌Acinetobacter sp.DG880[AY258108]。
在环境条件对于EBPR系统的影响以及对于PAO和GAO竞争的影响试验中,本研究主要探讨了pH、温度、碳源、C/P以及亚硝态氮的影响。
厌氧段pH影响小试试验表明,较高的pH对系统放磷有利,在6.0到8.5范围内pH越高,系统放磷量就越大,厌氧段pH的差异对系统去除COD的能力基本没有影响,却对PHA的合成影响显著,在厌氧段pH为6.0和8.5时,系统合成PHA的能力都受到了明显的抑制。好氧段pH影响小试试验表明,6.0明显是一个非常不利于聚磷菌吸磷的pH条件,在6.5到8.5的范围内,高pH对好氧吸磷有利。厌氧段pH长期影响试验表明,8.0是比较适合聚磷菌放磷的pH条件,在pH为7.0-8.0时除磷率可以达到80%以上,在pH为6.5时放磷量和吸磷量都有显著下降,在pH为6.0和8.5时,系统几乎完全丧失除磷功能,但是聚磷菌只是被不良条件抑制,一旦恢复正常的不调节pH的操作模式,除磷率迅速提升。因此,以实际生活污水驯化的污泥具有一定的适应不良pH影响的能力。
在以实际生活污水驯化污泥的强化生物除磷系统不能在低温(低于15℃)下长期运行。低温会破坏EBPR的生态系统平衡。
在碳源影响的正交试验中,与投加碳源量和投加磷量相比,碳源种类是影响EBPR系统磷去除率的最关键因素,葡萄糖的均值最大(96.357),说明葡萄糖是对以生活污水驯化的污泥中的PAO来说最好的碳源,以葡萄糖为碳源,系统去除磷的平均效率为96.357%;此外,投加碳源量是影响系统放磷和吸磷的最关键因素,投加碳源越多,系统放磷/吸磷量就越大,对于系统放磷/吸磷来说,丙酸钠是最好的碳源。
C/P比影响试验结果显示,C/P对COD去除率的影响微乎其微,在C/P小于或等于30时,磷去除率随着C/P的增加而增加,但是当C/P大于30时,磷去除率的变化就不再显著;随着C/P的增加,系统合成PHA的量没有明显差异,但系统降解COD/释放P的比例增加,两者之间呈线性关系。
亚硝态氮影响试验表明,随着系统好氧结束时亚硝态氮积累越来越多,SBR系统除磷能力不断的下降,当亚硝态氮积累超过10mg/L时,磷去除率从原来的60%左右下降为20%,所以,10mg/L的亚硝态氮对于强化生物除磷系统来说是一个可以容忍的上限值。
以丙酸为底物,普通絮状活性污泥为接种污泥,在SBR中通过一系列手段可以实现富集PAO的好氧活性污泥的颗粒化。培养的富集PAO的颗粒污泥对COD去除率可达94%,磷去除率可达100%。成熟的富集PAO的好氧颗粒污泥平均直径为2mm,白色,表面致密,外层有毛状物,同时具有低含水率和低比重等优良性质。与普通的富集PAO的活性污泥相比,富集PAO的颗粒污泥具有生物量大,沉淀速度快,抗COD冲击负荷能力强等众多优点,具有一定的理论和实际意义。
In nowadays, people, including the enrironmental experts, the enriomental protection postulants and the government, pay more and more attention on the eutrophication questions because of the degeneration of the waterbody. The standard of the wastewater discharge was controlled more strictly to achieve not only COD removal but also simultaneous COD, nitrogen and phosphorus removal. So the nitrogen and phosphorus removal technology aimed for eutrophication control has been the study focus in the wastewater treatment area. Enhanced biological phosphorus removal system is accepted in many country for its effective and low cost, while it often faced to deteriotated or collapse because of the existence or enrichment of glycogen accumulating organisms(GAO) in the system. GAO can competed with the phosphorus removal sponsor: phosphorus accumulating organisms(PAO) for the limited carbon souce and other substance. In order to accelerate the investigation development of phosphorus removal, especially on the competition principle and control method between PAO and GAO, this research use lab SBR fed with raw water or synthetic wastewater to study the enhanced biological phosphorus removal system(EBPR). We hope this kind of work can benefit for the people who dedicated in the biological phosphoros removal.
Because of the absent of PAO or GAO’s pure culture in the world, this research aimed to obtain large quantity of PAO、GAO orα-GAO by enrichment works. This kind of enrichment cultures is very important for people to know the physiological and biochemical characterists and the metabolic pathway of these three groups. Other than some suitable conditions just like pH, tempreture and load, P/C and carben source were two important selective factors for this enrichment works. As results, PAO reached 80%of the total bacteria,the efficiency of P removal of the system was over than 98%. GAO could reach 90%of the total bacteria, and also,α-GAO was a absolutely predominant group in its reactor.
Although people haven’t isolated PAO successfully till now, but this research tried to isolate and purify the phosphorus accumulating bacterial because of the importance of the pure culture for the understanding of the EBPR system.
87 strains were isolated from the activated sludge with phosphorus removal ability. After pre-screening by blue-white blaze test, re-screening by phosphorus release/uptake test, 1 strain, P4, with high phosphorus release ability in anaerobic phase and high phosphorus uptake ability in aerobic phase was achieved, and also, it could convert PHA, glycogen and poly-P in the anaerobic-aerobic condititions. Through observing the morphological feature of YP4, analyzing the physiology and biochemistry index, combining the 16SrRNA technique, YP4 was identified as acinetovacter sp.DG880.
Among many factors that can affect the competition between PAO and GAO, this research focused on pH, tempreture, carbon source, C/P and nitrite.
The short term effect operated by batch test showed that the amount of phosphorus released was related to the anaerobic pH, PAO prefered higher pH for phosphorus release, the higher pH was, the more phosphorus was released into the water. But on the other hand, the varision of pH did nothing on COD removal but it could affect the PHA synthetic significantly. The system fed with raw water showed steady state when anaerobic pH ranged from 6.5 to 8.0. A slight change of pH from 6.5 to 6.0 or from 8.0 to 8.5 led to a complete loss of phosphate-removing capability. Compared to PAO, GAO fed with raw water had a wider anaerobic pH survival range. When the anaerobic pH changed from 6.0 to 8.5, there was no significant effect of pH on GAO. The activated sludge fed by raw water had ability to be suit for the undesired pH conditions. Aerobic pH affect test showed that 6.0 was not fit for PAO to uptake phosphorus, while other pH conditions(6.5-8.0) had nothing to do with phosphorus uptake.
The enhanced biological phosphorus removal system, using activated sludge fed by raw water couldn’t be operated in low(<15℃)temperature for a long period, for it might destroy the other bacteria’s metablic leading to destroy the balance of PAO and the other bacteria.
In the orthogonal tests, all the three factors, including the kind of carbon source, quantity of added carbon source and quantity of added phosphate, could affect the phosphorus removal rate. The kind of carbon source was the crucial factor to affect the phosphorus removal rate, and the phosphorus removal rate increased with the addition quantity of carbon source (from 100mg/L to 300mg/L) and the decrease of the phosphate source (from 21mg/L to 5mg/L). The experiment showed that glucose could be utilized by phosphorus accumulating organisms as the most available carbon source compared with both sodium acetate and sodium propionate in actual WWTP operation. A different result from the traditional concept obtained in this study is that the system could release and uptake more phosphorus when it was fed by propionate than when it was fed by acetate, since the system we used is enriched and cultured by raw water, so it showed different performance from the one cultured by synthetic wastewater.
Batch experiment showed that phosphorus removal rate varied remarkably with the C/P ratio at the value of 30 or less, while the COD removal rate did not. And also, C/P ratio also effected the COD reduction/ P release greatly.
The accumulated nitrite in the aerobic phase would affect the phosphorus removal ability of the system. When the concentration of nitrite was over 10mg/L and increased with a rapid speed, the phosphorus removal rate decreased from 60% to 20% darmatically. So 10mg/L was the endurable highest nitrite concentration for the EBPR system. Besides, nitrite could affect phosphorus uptake more than phosphorus release.
After inoculated with floc activated sludge, the culturing of PAO enriched granular sludge could be achieved by using propionate as substrate, alternating the concentration of COD load, shortening settling time, providing hydrodynamic shear stress and high DO concentration. GAO was washed out of the system and PAO became the dominant population when acetate was used as substrate. The PAO enriched granular sludge had perfect COD removal rate of 94% and phosphorus removal rate of 100%. The mature PAO enriched granules had compact structure, with mean diameter of 2mm and white flagellum on the surface. And also, the mature granules had high OUR, density, integrality rate and low aquiferous rate, leading to the reactor could contain high biomass concentration with good settling properties.
引文
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