应用DGGE监测SBR启动及恶化期微生物群落结构与演替
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摘要
生物除磷系统是一个复杂的微生物群落,探明其中微生物的种类及组成对认识除磷过程,保证反应器的良好运行具有重要意义。纯培养研究很难准确把握反应器内微生物的实时、有效的信息,分子生物学技术如PCR扩增,变性梯度凝胶电泳,荧光原位杂交等得到越来越广泛的应用。本研究利用参数优化后的DGGE技术分析了SBR生物除磷系统启动期和恶化期微生物群落结构及演替,可以为今后的微生物研究和工艺运行提供参考。
本研究监测了SBR除磷反应器启动过程微生物群落结构与演替。启动过程历时30天。前15天磷去除率较低,其原因可能在于Type 0803 filamentous bacterium和Uncultured alpha proteobacterium对聚磷菌的竞争抑制作用,同时功能微生物——聚磷菌数量较少所致。启动过程15天至30天磷去除率增长迅速,此阶段Tetrasphaera elongate , Gemmatimonas aurantiaca和Uncultured gamma proteobacterium这些功能微生物大量存在,同时Candidatus Competibacter phosphatis等起竞争抑制作用的微生物数量较少甚至消失。此时反应器内含量较多的微生物种群为Thauera sp.,Uncultured gamma proteobacterium,Tetrasphaera elongate等类群。
本研究选取处于恶化期的第81天上清液,排泥和混合液的污泥样品进行DGGE分析。结果表明,上清液与排泥的微生物类群基本相同,而各种微生物的相对数量有所差别,排泥与混合液的DGGE图谱基本相同。恶化期含量较多的微生物种群为Uncultured bacterium clone SBRQ157 ,Uncultured actinobacterium clone GCP18 , Tetrasphaera elongata和Candidatus Competibacter phosphatis clone SBRQ22。其中Candidatus Competibacter phosphatis clone SBRQ22含量最多,为造成除磷效果恶化的重要原因。
本研究比较了反应器启动末期和恶化期的微生物群落结构,发现其群落结构差别很大,但是其中的功能微生物基本相同,都为Tetrasphaera elongate和Gemmatimonas aurantiaca。同时,反应器启动末期Candidatus Competibacter phosphatis clone SBRQ22很少,对聚磷菌的抑制作用有限,而恶化期其数量在群落中最多。
Biological phosphorus removal system is a complex microbial community. It is significant to notice the microbes’species and composition for understand phosphorus removal process and keep a good operation. Pure culture presents the localization to catch the real-time and efficient information. Molecular biological methods such as PCR, DGGE, FISH etc. gets its application more and more extensively on this field. Parameter optimized DGGE protocol is used to analysis the microbial community structure and succession in start up and deteriorate period of a SBR phosphorus removal system.
The period of start up lasted 30 days. In the former 15 days, it hadn’t reached a good phosphorus removal effort. The reason was that Type 0803 filamentous bacterium and Uncultured alpha proteobacterium had competition on Polyphosphate Accumulating Organisms (PAOs). The quanatity of PAOs was relatively less. In the following time, phosphorus removal rate increased rapidly. Tetrasphaera elongate, Gemmatimonas aurantiaca and Uncultured gamma proteobacterium existed in a large amount as founctional microbes and Candidatus Competibacter phosphatis clone SBRQ22 are rather less, which led to rapid increase of phosphorus removal efficiency. After the achievement of stable phosphorus removal, Thauera sp., Uncultured gamma proteobacterium and Tetrasphaera elongate were dominant in reactor.
Sludge samples of supernatant, discharge sludge and mixed liquid in the 81th day (deteriorate period) were chosen for DGGE analysis. The results showed that microbial groups of supernatant and discharge sludge were basically the same with the different quantity separately. The DGGE profiles of discharge sludge and mixed liquid were rather similar. Tetrasphaera elongate and Gemmatimonas aurantiaca were still the function microbes which can not lead to a higher phosphorus removal because of their less quantity. Uncultured bacterium clone SBRQ157,Uncultured actinobacterium clone GCP18,Tetrasphaera elongate and Candidatus Competibacter phosphatis clone SBRQ22 were dominant in deteriorate period. Candidatus Competibacter phosphatis clone SBRQ22 was in the largest quantity and responsible for worse operation effort.
There was a larger difference between start up and deteriorate period of microbe community structure, but the functional microbes were the same (Tetrasphaera elongate and Gemmatimonas aurantiaca). At the end of the start up period, the amount of Candidatus Competibacter phosphatis clone SBRQ22 was less and caused less restraint to PAOs, while it accounted for the largest amount in deteriorate period.
本研究监测了SBR除磷反应器启动过程微生物群落结构与演替。启动过程历时30天。前15天磷去除率较低,其原因可能在于Type 0803 filamentous bacterium和Uncultured alpha proteobacterium对聚磷菌的竞争抑制作用,同时功能微生物——聚磷菌数量较少所致。启动过程15天至30天磷去除率增长迅速,此阶段Tetrasphaera elongate , Gemmatimonas aurantiaca和Uncultured gamma proteobacterium这些功能微生物大量存在,同时Candidatus Competibacter phosphatis等起竞争抑制作用的微生物数量较少甚至消失。此时反应器内含量较多的微生物种群为Thauera sp.,Uncultured gamma proteobacterium,Tetrasphaera elongate等类群。
本研究选取处于恶化期的第81天上清液,排泥和混合液的污泥样品进行DGGE分析。结果表明,上清液与排泥的微生物类群基本相同,而各种微生物的相对数量有所差别,排泥与混合液的DGGE图谱基本相同。恶化期含量较多的微生物种群为Uncultured bacterium clone SBRQ157 ,Uncultured actinobacterium clone GCP18 , Tetrasphaera elongata和Candidatus Competibacter phosphatis clone SBRQ22。其中Candidatus Competibacter phosphatis clone SBRQ22含量最多,为造成除磷效果恶化的重要原因。
本研究比较了反应器启动末期和恶化期的微生物群落结构,发现其群落结构差别很大,但是其中的功能微生物基本相同,都为Tetrasphaera elongate和Gemmatimonas aurantiaca。同时,反应器启动末期Candidatus Competibacter phosphatis clone SBRQ22很少,对聚磷菌的抑制作用有限,而恶化期其数量在群落中最多。
Biological phosphorus removal system is a complex microbial community. It is significant to notice the microbes’species and composition for understand phosphorus removal process and keep a good operation. Pure culture presents the localization to catch the real-time and efficient information. Molecular biological methods such as PCR, DGGE, FISH etc. gets its application more and more extensively on this field. Parameter optimized DGGE protocol is used to analysis the microbial community structure and succession in start up and deteriorate period of a SBR phosphorus removal system.
The period of start up lasted 30 days. In the former 15 days, it hadn’t reached a good phosphorus removal effort. The reason was that Type 0803 filamentous bacterium and Uncultured alpha proteobacterium had competition on Polyphosphate Accumulating Organisms (PAOs). The quanatity of PAOs was relatively less. In the following time, phosphorus removal rate increased rapidly. Tetrasphaera elongate, Gemmatimonas aurantiaca and Uncultured gamma proteobacterium existed in a large amount as founctional microbes and Candidatus Competibacter phosphatis clone SBRQ22 are rather less, which led to rapid increase of phosphorus removal efficiency. After the achievement of stable phosphorus removal, Thauera sp., Uncultured gamma proteobacterium and Tetrasphaera elongate were dominant in reactor.
Sludge samples of supernatant, discharge sludge and mixed liquid in the 81th day (deteriorate period) were chosen for DGGE analysis. The results showed that microbial groups of supernatant and discharge sludge were basically the same with the different quantity separately. The DGGE profiles of discharge sludge and mixed liquid were rather similar. Tetrasphaera elongate and Gemmatimonas aurantiaca were still the function microbes which can not lead to a higher phosphorus removal because of their less quantity. Uncultured bacterium clone SBRQ157,Uncultured actinobacterium clone GCP18,Tetrasphaera elongate and Candidatus Competibacter phosphatis clone SBRQ22 were dominant in deteriorate period. Candidatus Competibacter phosphatis clone SBRQ22 was in the largest quantity and responsible for worse operation effort.
There was a larger difference between start up and deteriorate period of microbe community structure, but the functional microbes were the same (Tetrasphaera elongate and Gemmatimonas aurantiaca). At the end of the start up period, the amount of Candidatus Competibacter phosphatis clone SBRQ22 was less and caused less restraint to PAOs, while it accounted for the largest amount in deteriorate period.
引文
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2邵林广.控磷除磷在水体富营养化控制中的作用.环境与开发. 1999,14(2):19~20.
3郑兴灿,李亚新.污水除磷脱氮技术.中国建筑工业出版社, 1998:33~34
4徐亚同.废水的生物除磷.环境科学研究. 1994, 7(5):1~6.
5王宝贞,王琳.水污染治理新技术.科学出版社, 2003:33~34
6李红,周友兵,陈炳耀等.分子生物学技术在微生物多样性研究中的应用.河北大学学报(自然科学版). 2003, 23(4):450~454.
7郑金伟,冉炜,钟增涛等.增强型生物除磷过程中聚磷酸盐积累微生物的研究进展.应用生态学报. 2004, 15(8):1487~1490
8 M. C. M. van Loosdrecht, G. J. Smolders, T. Kuba, et al. Metabolism of Micro-organisms Responsible for Enhanced Biological Phosphorus Removal from Wastewater. Antonie van Leeuwenhoek. 1997, 71: 109–116.
9 R. J. Seviour, T. Mino, M. Onuki. The Microbiology of Biological Phosphorus Removal in Activated Sludge Systems. FEMS Microbiology Reviews. 2003, 27:99-127.
10 M. C. Wentzel, L. H. Loetter, R. E. Loewenthal, et al. Metabolic Behaviour of Acinetobacter spp. in Enhanced Biological Phosphorus Removal - A Biochemical Model. Water SA. 1986, 12:209-224.
11 T. Mino, Y. Tsuzuki, T. Matsuo. Effect of Phosphorus Accumulation on Acetate Metabolism in the Biological Phosphorus Removal Process. In: Biological Phosphate Removal from Wastewaters (Ramadori, R., Ed.). 1987, 27-38.
12 M. C. Wentzel, L. H. Loetter, G. A. Ekama, et al. Evaluation of Biochemical Models for Biological Excess Phosphorus Removal. Water Sci Technol. 1991, 23:567-576.
13 J. S. Cech, P. Hartman. Glucose Induced Breakdown of Enhanced Biological Phosphate Removal. Environ. Technol. 1990, 11:651-656.
14 J. S. Cech, P. Hartman. Competition between Polyphosphate andPolysaccharide Accumulating Bacteria in Enhanced Biological Phosphate Removal Systems. Water Res. 1993, 27:1219-1225.
15 A. Carucci, M. Majone, R. Ramadori, et al. Dynamics of Phosphorus and Organic Substrates in Anaerobic and Aerobic Phases of A Sequencing Batch Reactor. Water Sci. Technol. 1994, 30:237-246.
16 H. Imai, K. Endoh, T. Kozuka. Magnesium Requirement for Biological Removal of Phosphate by Activated Sludge. J. Ferment. Technol. 1988, 66:657-666.
17 O. J. Che, M.P. Jong. Enhanced Biological Phosphorus Removal in A Sequencing Batch Reactor Supplied with Glucose as A Sole Carbon Source. Water Research. 2000, 34:2160~2170
18 R. H. Cathy, A. A. Randall. A Biochemical Hypothesis Explaining the Response of Enhanced Biological Phosphorus Removal Biomass to Organic Substrates. Water Research. 2001, 35:2758-2766
19 H. Satoh, T. Mino, T. Matsuo. Anaerobic Uptake of Glutamate and Aspartate By Enhanced Biological Phosphorus Removal Activated Sludge. Wat Science and Technology. 1998, 37:579-582
20 T. Kuba, G. Smolders, M. C. M. van Loosdrecht, et al. Biological phosphorus removal from wastewater by anaerobic-anoxic sequencing batch reactor. Water Sci. Technol. 1993, 27:241-252.
21 S. Chuang, C. Ouyang, Y. Wang, Kinetic Competition between Phosphorus Release and Denitrification on Sludge under Anoxic Condition. Water Research, 1996, 30:2961~2968
22 G. W. Fuhs, M. Chen. Microbiological Basis of Phosphate Removal in the Activated Sludge Process for the Treatment of Wastewater Microb. Ecol. 1975, 2:119~138
23 T. Mino, M. C. M. Van Loosdrecht, J. J. Heijnen. Microbiology and Biochemistry of the Enhanced Biological Phosphate Removal Process. Wat Res. 1998, 32:3193-3207
24 K. Nakamura, A. Hiraishi, Y. Yoshimi, et al. Microlunatus phosphovorus gen. nov. sp. nov., A New Gram-positive Polyphosphate Accumulating Bacterium Isolated from Activated Sludge. Int. J. Syst. Bacteriol. 1995, 45:17-22.
25 L. Stante, C. M. Cellamare, F. Malaspina, et al. Biological PhosphorusRemoval by Pure Culture of Lampropedia spp. Water Res. 1997, 31:1317-1324.
26 J. S. Cech, P. Hartman. Competition between Polyphosphate and Polysaccharide Accumulating Bacteria in Enhanced Biological Phosphate Removal by Systems. Wat. Res. 1993, 27:1219-1225
27 T. Panswad, A. Doungchai, J. Anotai. Temperature Effect on Microbial Community of Enhanved Biological Phosphorus Removal System. Water Research. 2003, 37:409-415
28 A. Oehmen, Z. Yuan, L.L. Blackall, et al. Comparison of Acetate and Propionate Uptake by PolyphosphateAccumulating Organisms and Glycogen Accumulating Organisms. Biotechnology and Bioengineering. 2005, 91:162-168.
29 A. Oehmen, M. T. Vives, H. Lu. The Effect of pH on the Competition between Polyphosphate Accumulating Organisms and Glycogen-Accumulating Organisms. Water Research. 2005, 39:3727-3737.
30刘志培,杨惠芳.微生物分子生态学进展.应用与环境生物学报. 1999, 5(Suppl):43～48
31 S. G. Fischer, L.S. Lerman. Length-independent Separation of DNA Restriction Fragments inTwo Dimensional Gel Electrophoresis. Cell. 1979, 16:191-200.
32 G. Muyzer, E. C. Waal, A. G. Uitterlinden. Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction Amplicied Genes Encoding for 16S rRNA. Applied Environ Microbial, 1993, 59:695-700
33付琳林,李海星,曹郁生.利用变性梯度凝胶电泳分析微生物的多样性.生物技术通报. 2004, 2:38~41.
34赵翔,安志东.现代生物技术在环境微生物学中的应用:Ⅲ.限制性片段长度多态性分析、变性P温度梯度凝胶电泳和报道基因.氨基酸和生物资源. 2003, 25(2):48～51.
35宫曼丽,任南琪,邢德峰. DGGE/TGGE技术及其在微生物分子生态学中的应用.微生物学报. 2004, 44(6):845-848
36邢德峰,任南琪.应用DGGE研究微生物群落时的常见问题分析.微生物学报. 2006, 46(2):331-335
37马悦欣, Carola Holmstrm, Jeremy Webb et al.变性梯度凝胶电泳(DGGE)在微生物生态学中的应用.生态学报. 2003, 23(8):1561-1569
38张洪勋,王晓谊,齐鸿雁.微生物生态学研究方法进展.生态学报. 2003, 23(5):988-996
39 M. Onuki, H. Satoh, T. Mino Analysis of Microbial Community that Performs Enhanced Biological Phosphorus Removal in Activated Sludge Fed with Acetate. Water Science and Technology. 46:145-154
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42 T. Shoji T. Nittami, M. Onuki, et al. Microbial Community of Biological Phosphorus Removal Process Fed with Municipal Wastewater under Different Electron Acceptor Conditions
43 A. J. Schuler, M. Onuki, H. Satoh, et al. Density Separation and Molecular Methods to Characterize Enhanced Biological Phosphorus Removal System Populations. Water Science & Technology 46:195-198
44 A. T. Nielsen, W.T Liu, C. Filipe, et al. Identification of a Novel Group of Bacteria in Sludge from a Deteriorated Biological Phosphorus Removal Reactor. Appled and Environmental Microbiology. 1999,1251–1258
45 S. L. Yu, Y. N. Liu, G. L. Jing, et al. Analysis of Phosphate - accumulating Organisms Cultivated under Different Carbon Sources with Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis Assay. Journal of Environmental Sciences. 2005, 17:611~614
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49 W. V. Siglera, C. Miniaci, J. Zeyer. Electrophoresis Time Impacts the Denaturing Gradient Gel Electrophoresis-based Assessment of Bacterial Community Structure. Journal of Microbiological Methods. 2004, 57:17~22.
50 M. Beer, Y. H. Kong, R. J. Seviour. Are Some Putative Glycogen Accumulating Organisms (GAO) in Anaerobic: Aerobic Activated Sludge Systems Members of the a-Proteobacteria? Microbiology. 2004, 150:2267–2275
51 H. Zhang, Y. Sekiguchi, S. Hanada, et al. Gemmatimonas aurantiaca gen. nov., sp. nov., a Gram-negative, Aerobic, Polyphosphate Accumulating Micro-organism, the First Cultured Representative of the New Bacterial Phylum Gemmatimonadetes phyl. nov. International Journal of Systematic and Evolutionary Microbiology. 2003, 53:1155–1163
52 S. Hanada, W. T. Liu, T. Shintani, et al. Tetrasphaera elongata sp. nov., a Polyphosphate -Accumulating Bacterium Isolated from Activated Sludge. International Journal of Systematic and Evolutionary Microbiology. 2002, 52:883–887
2邵林广.控磷除磷在水体富营养化控制中的作用.环境与开发. 1999,14(2):19~20.
3郑兴灿,李亚新.污水除磷脱氮技术.中国建筑工业出版社, 1998:33~34
4徐亚同.废水的生物除磷.环境科学研究. 1994, 7(5):1~6.
5王宝贞,王琳.水污染治理新技术.科学出版社, 2003:33~34
6李红,周友兵,陈炳耀等.分子生物学技术在微生物多样性研究中的应用.河北大学学报(自然科学版). 2003, 23(4):450~454.
7郑金伟,冉炜,钟增涛等.增强型生物除磷过程中聚磷酸盐积累微生物的研究进展.应用生态学报. 2004, 15(8):1487~1490
8 M. C. M. van Loosdrecht, G. J. Smolders, T. Kuba, et al. Metabolism of Micro-organisms Responsible for Enhanced Biological Phosphorus Removal from Wastewater. Antonie van Leeuwenhoek. 1997, 71: 109–116.
9 R. J. Seviour, T. Mino, M. Onuki. The Microbiology of Biological Phosphorus Removal in Activated Sludge Systems. FEMS Microbiology Reviews. 2003, 27:99-127.
10 M. C. Wentzel, L. H. Loetter, R. E. Loewenthal, et al. Metabolic Behaviour of Acinetobacter spp. in Enhanced Biological Phosphorus Removal - A Biochemical Model. Water SA. 1986, 12:209-224.
11 T. Mino, Y. Tsuzuki, T. Matsuo. Effect of Phosphorus Accumulation on Acetate Metabolism in the Biological Phosphorus Removal Process. In: Biological Phosphate Removal from Wastewaters (Ramadori, R., Ed.). 1987, 27-38.
12 M. C. Wentzel, L. H. Loetter, G. A. Ekama, et al. Evaluation of Biochemical Models for Biological Excess Phosphorus Removal. Water Sci Technol. 1991, 23:567-576.
13 J. S. Cech, P. Hartman. Glucose Induced Breakdown of Enhanced Biological Phosphate Removal. Environ. Technol. 1990, 11:651-656.
14 J. S. Cech, P. Hartman. Competition between Polyphosphate andPolysaccharide Accumulating Bacteria in Enhanced Biological Phosphate Removal Systems. Water Res. 1993, 27:1219-1225.
15 A. Carucci, M. Majone, R. Ramadori, et al. Dynamics of Phosphorus and Organic Substrates in Anaerobic and Aerobic Phases of A Sequencing Batch Reactor. Water Sci. Technol. 1994, 30:237-246.
16 H. Imai, K. Endoh, T. Kozuka. Magnesium Requirement for Biological Removal of Phosphate by Activated Sludge. J. Ferment. Technol. 1988, 66:657-666.
17 O. J. Che, M.P. Jong. Enhanced Biological Phosphorus Removal in A Sequencing Batch Reactor Supplied with Glucose as A Sole Carbon Source. Water Research. 2000, 34:2160~2170
18 R. H. Cathy, A. A. Randall. A Biochemical Hypothesis Explaining the Response of Enhanced Biological Phosphorus Removal Biomass to Organic Substrates. Water Research. 2001, 35:2758-2766
19 H. Satoh, T. Mino, T. Matsuo. Anaerobic Uptake of Glutamate and Aspartate By Enhanced Biological Phosphorus Removal Activated Sludge. Wat Science and Technology. 1998, 37:579-582
20 T. Kuba, G. Smolders, M. C. M. van Loosdrecht, et al. Biological phosphorus removal from wastewater by anaerobic-anoxic sequencing batch reactor. Water Sci. Technol. 1993, 27:241-252.
21 S. Chuang, C. Ouyang, Y. Wang, Kinetic Competition between Phosphorus Release and Denitrification on Sludge under Anoxic Condition. Water Research, 1996, 30:2961~2968
22 G. W. Fuhs, M. Chen. Microbiological Basis of Phosphate Removal in the Activated Sludge Process for the Treatment of Wastewater Microb. Ecol. 1975, 2:119~138
23 T. Mino, M. C. M. Van Loosdrecht, J. J. Heijnen. Microbiology and Biochemistry of the Enhanced Biological Phosphate Removal Process. Wat Res. 1998, 32:3193-3207
24 K. Nakamura, A. Hiraishi, Y. Yoshimi, et al. Microlunatus phosphovorus gen. nov. sp. nov., A New Gram-positive Polyphosphate Accumulating Bacterium Isolated from Activated Sludge. Int. J. Syst. Bacteriol. 1995, 45:17-22.
25 L. Stante, C. M. Cellamare, F. Malaspina, et al. Biological PhosphorusRemoval by Pure Culture of Lampropedia spp. Water Res. 1997, 31:1317-1324.
26 J. S. Cech, P. Hartman. Competition between Polyphosphate and Polysaccharide Accumulating Bacteria in Enhanced Biological Phosphate Removal by Systems. Wat. Res. 1993, 27:1219-1225
27 T. Panswad, A. Doungchai, J. Anotai. Temperature Effect on Microbial Community of Enhanved Biological Phosphorus Removal System. Water Research. 2003, 37:409-415
28 A. Oehmen, Z. Yuan, L.L. Blackall, et al. Comparison of Acetate and Propionate Uptake by PolyphosphateAccumulating Organisms and Glycogen Accumulating Organisms. Biotechnology and Bioengineering. 2005, 91:162-168.
29 A. Oehmen, M. T. Vives, H. Lu. The Effect of pH on the Competition between Polyphosphate Accumulating Organisms and Glycogen-Accumulating Organisms. Water Research. 2005, 39:3727-3737.
30刘志培,杨惠芳.微生物分子生态学进展.应用与环境生物学报. 1999, 5(Suppl):43～48
31 S. G. Fischer, L.S. Lerman. Length-independent Separation of DNA Restriction Fragments inTwo Dimensional Gel Electrophoresis. Cell. 1979, 16:191-200.
32 G. Muyzer, E. C. Waal, A. G. Uitterlinden. Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction Amplicied Genes Encoding for 16S rRNA. Applied Environ Microbial, 1993, 59:695-700
33付琳林,李海星,曹郁生.利用变性梯度凝胶电泳分析微生物的多样性.生物技术通报. 2004, 2:38~41.
34赵翔,安志东.现代生物技术在环境微生物学中的应用:Ⅲ.限制性片段长度多态性分析、变性P温度梯度凝胶电泳和报道基因.氨基酸和生物资源. 2003, 25(2):48～51.
35宫曼丽,任南琪,邢德峰. DGGE/TGGE技术及其在微生物分子生态学中的应用.微生物学报. 2004, 44(6):845-848
36邢德峰,任南琪.应用DGGE研究微生物群落时的常见问题分析.微生物学报. 2006, 46(2):331-335
37马悦欣, Carola Holmstrm, Jeremy Webb et al.变性梯度凝胶电泳(DGGE)在微生物生态学中的应用.生态学报. 2003, 23(8):1561-1569
38张洪勋,王晓谊,齐鸿雁.微生物生态学研究方法进展.生态学报. 2003, 23(5):988-996
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