反硝化聚磷菌快速富集、培养及其荧光原位杂交技术鉴别
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摘要
以污水处理厂A2/O厌氧段污泥为种泥,采用膜生物反应器(membrane bio-reactor,MBR)对反硝化聚磷菌(denitrifyingphosphate-removal bacteria,DPB)进行快速富集及培养,并提供一种鉴别方法.试验中以乙酸钠为碳源,并在缺氧段投加一定浓度的硝酸盐,结果表明,在膜组件的高效截留作用下,经过厌氧-好氧和厌氧-缺氧2个阶段的富集培养,35 d内反硝化聚磷菌占全部聚磷菌(phosphate-accumulating organisms)的比例从24%上升到93%.此时系统的脱氮、除磷效率均可保持在90%以上.通过荧光原位杂交技术(fluorescence in situ hybridization,FISH)并结合常规测定手段对活性污泥进行鉴别,确定Pseudomonas sp.和Rhodocyclus sp.为主要的优势菌.
The present work focused on a rapid enrichment and cultivation of denitrifying phosphate-removal bacteria(DPB) in a membrane bio-reactor(MBR) by using A2/ O anaerobic sludge from a wastewater treatment plant as seed,as well as providing an identification method.In the experiments,sodium acetate was used as the carbon source and a certain amount of nitrate was added to the MBR in the anoxic stage.Results showed that,with the efficient trap of the hollow-fiber membrane module,the proportion of DPB in all the phosphate-accumulating organisms(PAOs) increased from 24% to 93% within 35 days after two-stage's cultivation including anaerobic / aerobic and anaerobic / anoxic,during which the removal efficiency of nitrogen and phosphorus reached more than 90%.The activated sludge was identified by combining a regular method and the fluorescence in situ hybridization(FISH) technique,which demonstrated that Pseudomonas sp.and Rhodocyclus sp.were the dominant bacteria in the used bioreactor.
The present work focused on a rapid enrichment and cultivation of denitrifying phosphate-removal bacteria(DPB) in a membrane bio-reactor(MBR) by using A2/ O anaerobic sludge from a wastewater treatment plant as seed,as well as providing an identification method.In the experiments,sodium acetate was used as the carbon source and a certain amount of nitrate was added to the MBR in the anoxic stage.Results showed that,with the efficient trap of the hollow-fiber membrane module,the proportion of DPB in all the phosphate-accumulating organisms(PAOs) increased from 24% to 93% within 35 days after two-stage's cultivation including anaerobic / aerobic and anaerobic / anoxic,during which the removal efficiency of nitrogen and phosphorus reached more than 90%.The activated sludge was identified by combining a regular method and the fluorescence in situ hybridization(FISH) technique,which demonstrated that Pseudomonas sp.and Rhodocyclus sp.were the dominant bacteria in the used bioreactor.
引文
[1]Kuba T,Smolders G,van Loosdrecht M C M,et al.Biologicalphosphorus removal from wastewater by anaerobic-anoxicsequencing batch reactor[J].Water Science&Technology,1993,27(5-6):241-252.
[2]Kuba T,van Loosdrecht M C M,Heijnen J J.Phosphorus andnitrogen removal with minimal COD requirement by integration ofdenitrifying dephosphatation and nitrification in a two sludgesystem[J].Water Research,1996,30(7):1702-1710.
[3]李勇智,王淑滢,吴凡松,等.强化生物除磷体系中反硝化聚磷菌的选择与富集[J].环境科学学报,2004,24(1):45-49.
[4]Zeng R J,Saunders A M,Yuan Z G,et al.Identification andcomparison of aerobic and denitrifying polyphosphate-accumulating organisms[J].Biotechnology and Bioengineering,2003,83(2):140-148.
[5]Lee D S,Jeon C O,Park J M.Biological nitrogen removal withenhanced phosphate uptake in a sequencing batch reactor usingsingle sludge system[J].Water Research,2001,35(16):3968-3976.
[6]杨文婷,沈耀良.SBR中反硝化聚磷菌的培养驯化研究[J].环境科学与技术,2009,32(8):6-8,12.
[7]刘娇娇,刘涛.反硝化除磷菌富集试验研究[J].化工文摘,2009,(2):33-35.
[8]Ekama G A,Wentzel M C.Difficulties and developments inbiological nutrient removal technology and modelling[J].WaterScience&Technology,1999,39(6):1-11.
[9]代文臣,张捍民,肖景霓,等.序批式膜生物反应器中反硝化聚磷菌的富集[J].环境科学,2007,28(3):517-521.
[10]颜强,谢有奎.FISH技术在污水生物除磷脱氮研究中的应用[J].重庆建筑大学学报,2004,26(6):71-73.
[11]李冰冰,肖波,李蓓.FISH技术及其在环境微生物监测中的应用[J].生物技术,2007,17(5):94-97.
[12]Adham S,Gagliardo P,Boulos L,et al.Feasibility of themembrane bioreactor process for water reclamation[J].WaterScience&Technology,2001,43(10):203-209.
[13]Soejima K,Oki K,Terada A,et al.Effects of acetate and nitriteaddition on fraction of denitrifying phosphate-accumulatingorganisms and nutrient removal efficiency in anaerobic/aerobic/anoxic process[J].Bioprocess and Biosystems engineering,2006,29(5-6):305-313.
[14]Zillers J L,Peccia J,Kim M W,et al.Involvement ofRhodocyclus-related organisms in phosphorus removal in full-scale wastewater treatment plants[J].Applied andEnvironmental Microbiology,2002,68(6):2763-2769.
[15]徐微,吕锡武,蒋彬.反硝化聚磷污泥的培养驯化及关键参数研究[J].中国给水排水,2009,25(5):5-8.
[16]国家环保局编委会.水和废水检测分析方法[M].北京:环境科学出版社,1994.
[17]Wachtmeister A,Kuba T,van Loosdrecht M C M,et al.Asludge Characterization assay for aerobic and denitrifyingphosphorus removing sludge[J].Water Research,1997,31(3):471-478.
[18]Meinhold J,Filipe C D M,Dagger G T,et al.Characterizationof the denitrifying fraction of phosphate accumulating organisms inbiological phosphate removal[J].Water Science&Technology,1999,39(1):31-42.
[19]Kerrnr Jespersen J P,Henze M.Biological phosphorus uptakeunder anoxic and aerobic condition[J].Water Research,1993,27(4):617-624.
[20]Hu J Y,Ong S L,Ng W J,et al.A new method forcharacterizing denitrifying phosphorus removal bacteria by usingthree different types of electron acceptors[J].Water Research,2003,37(14):3463-3471.
[21]朱琳,尹立红,浦跃朴,等.荧光原位杂交法检测环境硝化细菌实验条件优化及应用[J].东南大学学报(自然科学版),2005,35(2):266-270.
[22]张勇,宋吟玲.荧光原位杂交法检测反应器中聚磷菌实验条件优化及应用[J].环境科学与管理,2008,33(2):126-129.
[23]Schleifer K H,Amann R I,Ludwig W,et al.Nucleic acidprobes for the identification and in situ detection of Pseudomonasspp[A].In:Galii E,Silver S,Witholt B(Eds.).Pseudomonas:Molecular Biology and Biotechnology[C].Washington D.C.:American Society for Microbiology,1992.127-134.
[24]Amann R I.In situ identification of micro-organisms by wholecell hybridization with rRNA-targeted nucleic acid probes[A].In:Akkermans A D L,Elsas J D,de Bruij F J(Eds.).Molecular Microbial Ecology Manual[C].London:Kluwer,1995.1-5.
[25]张洁,田立江,张雁秋.反硝化聚磷菌快速驯化的对比研究[J].华中师范大学学报(自然科学版),2007,41(3):411-414.
[26]曹桂萍.反硝化聚磷菌富集条件的研究[J].中国给水排水,2010,26(13):60-63.
[27]周康群,刘晖,孙彦富,等.反硝化聚磷菌的富集及富集污泥活性研究[J].环境科学与技术,2008,31(4):110-115.
[2]Kuba T,van Loosdrecht M C M,Heijnen J J.Phosphorus andnitrogen removal with minimal COD requirement by integration ofdenitrifying dephosphatation and nitrification in a two sludgesystem[J].Water Research,1996,30(7):1702-1710.
[3]李勇智,王淑滢,吴凡松,等.强化生物除磷体系中反硝化聚磷菌的选择与富集[J].环境科学学报,2004,24(1):45-49.
[4]Zeng R J,Saunders A M,Yuan Z G,et al.Identification andcomparison of aerobic and denitrifying polyphosphate-accumulating organisms[J].Biotechnology and Bioengineering,2003,83(2):140-148.
[5]Lee D S,Jeon C O,Park J M.Biological nitrogen removal withenhanced phosphate uptake in a sequencing batch reactor usingsingle sludge system[J].Water Research,2001,35(16):3968-3976.
[6]杨文婷,沈耀良.SBR中反硝化聚磷菌的培养驯化研究[J].环境科学与技术,2009,32(8):6-8,12.
[7]刘娇娇,刘涛.反硝化除磷菌富集试验研究[J].化工文摘,2009,(2):33-35.
[8]Ekama G A,Wentzel M C.Difficulties and developments inbiological nutrient removal technology and modelling[J].WaterScience&Technology,1999,39(6):1-11.
[9]代文臣,张捍民,肖景霓,等.序批式膜生物反应器中反硝化聚磷菌的富集[J].环境科学,2007,28(3):517-521.
[10]颜强,谢有奎.FISH技术在污水生物除磷脱氮研究中的应用[J].重庆建筑大学学报,2004,26(6):71-73.
[11]李冰冰,肖波,李蓓.FISH技术及其在环境微生物监测中的应用[J].生物技术,2007,17(5):94-97.
[12]Adham S,Gagliardo P,Boulos L,et al.Feasibility of themembrane bioreactor process for water reclamation[J].WaterScience&Technology,2001,43(10):203-209.
[13]Soejima K,Oki K,Terada A,et al.Effects of acetate and nitriteaddition on fraction of denitrifying phosphate-accumulatingorganisms and nutrient removal efficiency in anaerobic/aerobic/anoxic process[J].Bioprocess and Biosystems engineering,2006,29(5-6):305-313.
[14]Zillers J L,Peccia J,Kim M W,et al.Involvement ofRhodocyclus-related organisms in phosphorus removal in full-scale wastewater treatment plants[J].Applied andEnvironmental Microbiology,2002,68(6):2763-2769.
[15]徐微,吕锡武,蒋彬.反硝化聚磷污泥的培养驯化及关键参数研究[J].中国给水排水,2009,25(5):5-8.
[16]国家环保局编委会.水和废水检测分析方法[M].北京:环境科学出版社,1994.
[17]Wachtmeister A,Kuba T,van Loosdrecht M C M,et al.Asludge Characterization assay for aerobic and denitrifyingphosphorus removing sludge[J].Water Research,1997,31(3):471-478.
[18]Meinhold J,Filipe C D M,Dagger G T,et al.Characterizationof the denitrifying fraction of phosphate accumulating organisms inbiological phosphate removal[J].Water Science&Technology,1999,39(1):31-42.
[19]Kerrnr Jespersen J P,Henze M.Biological phosphorus uptakeunder anoxic and aerobic condition[J].Water Research,1993,27(4):617-624.
[20]Hu J Y,Ong S L,Ng W J,et al.A new method forcharacterizing denitrifying phosphorus removal bacteria by usingthree different types of electron acceptors[J].Water Research,2003,37(14):3463-3471.
[21]朱琳,尹立红,浦跃朴,等.荧光原位杂交法检测环境硝化细菌实验条件优化及应用[J].东南大学学报(自然科学版),2005,35(2):266-270.
[22]张勇,宋吟玲.荧光原位杂交法检测反应器中聚磷菌实验条件优化及应用[J].环境科学与管理,2008,33(2):126-129.
[23]Schleifer K H,Amann R I,Ludwig W,et al.Nucleic acidprobes for the identification and in situ detection of Pseudomonasspp[A].In:Galii E,Silver S,Witholt B(Eds.).Pseudomonas:Molecular Biology and Biotechnology[C].Washington D.C.:American Society for Microbiology,1992.127-134.
[24]Amann R I.In situ identification of micro-organisms by wholecell hybridization with rRNA-targeted nucleic acid probes[A].In:Akkermans A D L,Elsas J D,de Bruij F J(Eds.).Molecular Microbial Ecology Manual[C].London:Kluwer,1995.1-5.
[25]张洁,田立江,张雁秋.反硝化聚磷菌快速驯化的对比研究[J].华中师范大学学报(自然科学版),2007,41(3):411-414.
[26]曹桂萍.反硝化聚磷菌富集条件的研究[J].中国给水排水,2010,26(13):60-63.
[27]周康群,刘晖,孙彦富,等.反硝化聚磷菌的富集及富集污泥活性研究[J].环境科学与技术,2008,31(4):110-115.
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