影响剩余污泥减量细菌的实时定量PCR研究
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摘要
剩余污泥如何达到零排放,一直是废水处理的一大难题,虽然一些新工艺的出现,已经减少了剩余污泥的排放,但在运行过程中,并不能很好的控制污水中的细菌生长环境。污泥中的菌群直接影响污水的处理效果,只有了解微生物群落多样性和动态性等信息,才能提高对处理系统的控制能力。污水生物处理系统可视为一个适应了极端环境的人工生态系统,如自然生态系统一样,它有自身的结构,生物的群落结构也有时空的变化。
分子生物学技术的出现,使得在分子级别上进行生命现象研究得到了快速的发展。应用实时定量PCR法,建立污泥中部分功能性细菌及总细菌的标准曲线,从而得到它们的数量。应用该方法来对剩余污泥减量起作用的一些特定细菌做动态分析。
本实验以生物砾间接触氧化工艺为基础,以分子生物技术为手段,以污泥中的细菌为研究对象,利用实时定量PCR技术来研究对剩余污泥减量起作用的一些功能性细菌的定量分析。在实际工程中,通过适当的人为方式来改变反应器中一些细菌的相对数量,改善反应器中内环境条件,最终达到污泥零排放的目的。
本实验选择生物砾间接触氧化反应器和普通活性污泥反应器做平行对比实验,通过对比两种反应器中的细菌数量,来反映细菌在污泥减量中是如何起作用的。首先进行两个反应器的启动、挂膜、正常运行,经过长时间的稳定运行,使出水水质稳定并达标(污水综合排放标准一级,GB8987-1996)。在运行期间,提取活性污泥和生物膜中细菌的DNA进行分子生物实验,并对其做动态定量分析。
How to not discharging the excess sludge, which is always a major problem of sewage treatment, although there are some new crafts, we could not control the bacterial habitat well during the course of operation. The bacteria in the sludge impact on the treatment effect of the wastewater, we could improve the control capacity of the treatment system by understanding the bacterial diversity and dynamic information. The biological system of wastewater treatment system can be considered as an artificial ecosystem, which adapted to the extreme environment, just like the natural ecosystems, it has its own structure, its biological community structure also changes in time and space.
Molecular biology technology makes the research on the phenomenon of life developed quickly on the molecule-level. Application of real-time quantitative PCR method, we could establish the standard curve of some functional bacteria and the total bacteria in the sludge, and get their quantity. Using this method, we could make a dynamic analysis to some specifically bacteria, which make the excess sludge demineralization.
This experiment based on the gravel contact oxidation technology, taking molecular biotechnology as a method, taking the bacteria in the sludge as the object of study, and applying the real-time quantitative PCR technology to make a quantitative analysis on some functional bacteria which make the excess sludge demineralization. Applying some appropriate human approaches during the operation of the project to change the relative number of some bacteria in the reactor to improve the habit of the reactor, ultimately achieve the purpose of not discharging the remaining sludge.
This experiment choose the conventional activated sludge reactor as contrast object, in order to reflect the way of bacterial working in the sludge by contrasting bacterial quantity of two actors.
First of all, two reactors start-up,linked to film, operated normally ,then after a long period of stable operation, the water became stability and water quality standards (integrated wastewater discharge standard, GB 8987-1996)can be achieved. During the operation, extracting the DNA of the bacterial in the activated sludge and biofilm to do the molecular biology experiments, and making a dynamic analysis to the bacterial in the sludge.
分子生物学技术的出现,使得在分子级别上进行生命现象研究得到了快速的发展。应用实时定量PCR法,建立污泥中部分功能性细菌及总细菌的标准曲线,从而得到它们的数量。应用该方法来对剩余污泥减量起作用的一些特定细菌做动态分析。
本实验以生物砾间接触氧化工艺为基础,以分子生物技术为手段,以污泥中的细菌为研究对象,利用实时定量PCR技术来研究对剩余污泥减量起作用的一些功能性细菌的定量分析。在实际工程中,通过适当的人为方式来改变反应器中一些细菌的相对数量,改善反应器中内环境条件,最终达到污泥零排放的目的。
本实验选择生物砾间接触氧化反应器和普通活性污泥反应器做平行对比实验,通过对比两种反应器中的细菌数量,来反映细菌在污泥减量中是如何起作用的。首先进行两个反应器的启动、挂膜、正常运行,经过长时间的稳定运行,使出水水质稳定并达标(污水综合排放标准一级,GB8987-1996)。在运行期间,提取活性污泥和生物膜中细菌的DNA进行分子生物实验,并对其做动态定量分析。
How to not discharging the excess sludge, which is always a major problem of sewage treatment, although there are some new crafts, we could not control the bacterial habitat well during the course of operation. The bacteria in the sludge impact on the treatment effect of the wastewater, we could improve the control capacity of the treatment system by understanding the bacterial diversity and dynamic information. The biological system of wastewater treatment system can be considered as an artificial ecosystem, which adapted to the extreme environment, just like the natural ecosystems, it has its own structure, its biological community structure also changes in time and space.
Molecular biology technology makes the research on the phenomenon of life developed quickly on the molecule-level. Application of real-time quantitative PCR method, we could establish the standard curve of some functional bacteria and the total bacteria in the sludge, and get their quantity. Using this method, we could make a dynamic analysis to some specifically bacteria, which make the excess sludge demineralization.
This experiment based on the gravel contact oxidation technology, taking molecular biotechnology as a method, taking the bacteria in the sludge as the object of study, and applying the real-time quantitative PCR technology to make a quantitative analysis on some functional bacteria which make the excess sludge demineralization. Applying some appropriate human approaches during the operation of the project to change the relative number of some bacteria in the reactor to improve the habit of the reactor, ultimately achieve the purpose of not discharging the remaining sludge.
This experiment choose the conventional activated sludge reactor as contrast object, in order to reflect the way of bacterial working in the sludge by contrasting bacterial quantity of two actors.
First of all, two reactors start-up,linked to film, operated normally ,then after a long period of stable operation, the water became stability and water quality standards (integrated wastewater discharge standard, GB 8987-1996)can be achieved. During the operation, extracting the DNA of the bacterial in the activated sludge and biofilm to do the molecular biology experiments, and making a dynamic analysis to the bacterial in the sludge.
引文
[1] 黄培堂等译.Joseph Sambrook,David W. Russell[M]. 分子克隆实验指南(第三版).北京,科学出版社, 2002,8 月.
[2] 严兴,徐正茂,冯晓西等.焦化废水工业处理装置和实验室装置硝化菌群的分子比较[N].微生物学报,47(2):301-30.
[3] 熊开容,张智明等.活性污泥总 DNA 提取方法的研究[J].生物技术,2005,15(4):43-45.
[4] 国家环境保护局.水和废水监测分析方法. 北京:中国环境科学出版社,1989.
[5]Yoshikazu Koizumi,Hisaya Kojima, et al. Characterization of depth-related microbial community structure in lake sediment by denaturing gradient gel electrophoresis of amplified 16rDNA and reversely transcribed 16S r RNA fragments. FEMS Microbiology Ecology,2003:147-157.
[6]Ruben Araya,Katsuji Tani, et al. Bacterial activity and community composition in stream water an iofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis. FEMS Microbiology Ecology,2003:111-119.
[7]Hans Henrik Larsen,Joseph A,Kovacs, et a .Development of a rapid real-time PCR assay for quantitation of Pneumocystis carinii f.sp.carinii. Journal of Clinical Microbiology,2002,40(8):2989-2993.
[8]K.E.Shannon,D.-Y.Lee, et al .Applicant of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment. Science of the Total Environment,2007,382:121-129.
[9]A.M.Ibekwe,C.m.Grieve.Changes in developing plant microbial community structure as affected by contaminated water.FEMS Microbiology Ecology,2004,48:239-248.
[10] Harms G, Layton AC, Dionisi HM, et al . Real2time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant .Environ Sci Technol,2003,37:343 -351.
[11] Markku J. Lehtola, Eila Torvinen, et al .Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis inPotable-Water Biofilms. Applied And Environmental Microbiology, Jan. 2006,72(1):848–853.
[12] 王爱杰,任南琪.环境中的分子生物学诊断技术[M].北京:化学工业出社,2004:1-14.
[13]朱彤,张洪军等.应用FISH 法对污水生物处理系统中细菌数量的计数[J].环境保护科学,2007,33(1):22-25.
[14] 孙寓姣,左剑恶等,厌氧颗粒污泥中微生物种群变化的分子生物学解析[J]. 中国环境科学,2006,26(2):183-187.
[15]孙淑斌,李宝珍等.水稻低丰度表达基因 OsAMT1;3 实时荧光定量 PCR 方法 的建立及其应用[J].中国水稻科学,2006,20(1):8-12.
[16] 邢德峰,任南琪等.不同16S rDNA靶序列对DGGE分析活性污泥群落的影响[J].环境科学,2006,27(7):1424-1428.
[17] 梁鹏,黄霞,钱易,丁国际.污泥减量化技术的研究进展[J].环境污染治理技术与设备,2003-1,4(1).
[18] 乐毅全,顾国维. 16SrRNA基因技术在环境科学领域中的应用[J].四川环境,2003,Vo(l22),No(6).
[19] ]Godon J J, Zumstein E, Dabert P, et al. Moecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis[J]. Appl. Environ. Microbiol.,1997,63(7):2802-2813.
[20] Pace N R, Stahl D A, Lane D J, Olsen GJ ,The analysis of natural microbial populations by ribosomal RNA sequence[J]. Advanced in Microbial Ecology,1986,(9):1 55.
[21] Skeiguchi Y, Kamagata Y, Syutsubo K, et al. Phylogenetic diversity of mesophilic and the rmophilic granular sludges determined by 16SrRNA gene analysis [J]. Microbiol.,1998,144(9):2655-2665.
[22] Lemarchand K, Berthiaume F, Maynard C et al. Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays[J].J Microbiological Methods, 2005, 63, 115-126.
[23] 叶芬霞,立波 活性污泥工艺中剩余污泥减量化技术 中国给水排水,2003.
[24] Gulnur Coskuner, Stuart J. Ballinger,et al. Agreement between Theory and Measurement in Quantification of Ammonia-Oxidizing Bacteria[J]. Applied And Environmental Microbiology,2005,71(10):6325-6334.
[25] 邢薇,左剑恶等.利用FISH和DGGE 对产甲烷颗粒污泥中微生物种群的研究环 境科学[J].2006,27(11):2268-2272.
[26] 沈永才, 袁佩娜.16S rRNA 荧光定量PCR 法检测双歧杆菌[J]. 中国微生态学杂志,2001,13(2):66-72.
[27] 叶立,黄卫星,杨平,伍勇.膜生物反应器及其在工业废水处理中的应用.化工环保,2003,32(3):142-146.
[28] Raju Sekar, Annelie Pernthaler, et al. An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization [J]. Applied And Environmental Microbiology,2003,69(5):2928-2935.
[29] Ryo Hondaa,Kensuke Fukushi,et al. Optimization of wastewater feeding for single-cell protein production in an anaerobic wastewater treatment process utilizing purple non-sulfur bacteria in mixed culture condition[J]. Journal of Biotechnology 2006,125:565–573.
[30] Suresh Kumar Dubey,Anil Kumar Tripathi,et al. Exploration of soil bacterial communities fortheir potential as bioresource [J]. Bioresource Technology 2006 ,97 :2217–2224.
[2] 严兴,徐正茂,冯晓西等.焦化废水工业处理装置和实验室装置硝化菌群的分子比较[N].微生物学报,47(2):301-30.
[3] 熊开容,张智明等.活性污泥总 DNA 提取方法的研究[J].生物技术,2005,15(4):43-45.
[4] 国家环境保护局.水和废水监测分析方法. 北京:中国环境科学出版社,1989.
[5]Yoshikazu Koizumi,Hisaya Kojima, et al. Characterization of depth-related microbial community structure in lake sediment by denaturing gradient gel electrophoresis of amplified 16rDNA and reversely transcribed 16S r RNA fragments. FEMS Microbiology Ecology,2003:147-157.
[6]Ruben Araya,Katsuji Tani, et al. Bacterial activity and community composition in stream water an iofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis. FEMS Microbiology Ecology,2003:111-119.
[7]Hans Henrik Larsen,Joseph A,Kovacs, et a .Development of a rapid real-time PCR assay for quantitation of Pneumocystis carinii f.sp.carinii. Journal of Clinical Microbiology,2002,40(8):2989-2993.
[8]K.E.Shannon,D.-Y.Lee, et al .Applicant of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment. Science of the Total Environment,2007,382:121-129.
[9]A.M.Ibekwe,C.m.Grieve.Changes in developing plant microbial community structure as affected by contaminated water.FEMS Microbiology Ecology,2004,48:239-248.
[10] Harms G, Layton AC, Dionisi HM, et al . Real2time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant .Environ Sci Technol,2003,37:343 -351.
[11] Markku J. Lehtola, Eila Torvinen, et al .Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis inPotable-Water Biofilms. Applied And Environmental Microbiology, Jan. 2006,72(1):848–853.
[12] 王爱杰,任南琪.环境中的分子生物学诊断技术[M].北京:化学工业出社,2004:1-14.
[13]朱彤,张洪军等.应用FISH 法对污水生物处理系统中细菌数量的计数[J].环境保护科学,2007,33(1):22-25.
[14] 孙寓姣,左剑恶等,厌氧颗粒污泥中微生物种群变化的分子生物学解析[J]. 中国环境科学,2006,26(2):183-187.
[15]孙淑斌,李宝珍等.水稻低丰度表达基因 OsAMT1;3 实时荧光定量 PCR 方法 的建立及其应用[J].中国水稻科学,2006,20(1):8-12.
[16] 邢德峰,任南琪等.不同16S rDNA靶序列对DGGE分析活性污泥群落的影响[J].环境科学,2006,27(7):1424-1428.
[17] 梁鹏,黄霞,钱易,丁国际.污泥减量化技术的研究进展[J].环境污染治理技术与设备,2003-1,4(1).
[18] 乐毅全,顾国维. 16SrRNA基因技术在环境科学领域中的应用[J].四川环境,2003,Vo(l22),No(6).
[19] ]Godon J J, Zumstein E, Dabert P, et al. Moecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis[J]. Appl. Environ. Microbiol.,1997,63(7):2802-2813.
[20] Pace N R, Stahl D A, Lane D J, Olsen GJ ,The analysis of natural microbial populations by ribosomal RNA sequence[J]. Advanced in Microbial Ecology,1986,(9):1 55.
[21] Skeiguchi Y, Kamagata Y, Syutsubo K, et al. Phylogenetic diversity of mesophilic and the rmophilic granular sludges determined by 16SrRNA gene analysis [J]. Microbiol.,1998,144(9):2655-2665.
[22] Lemarchand K, Berthiaume F, Maynard C et al. Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays[J].J Microbiological Methods, 2005, 63, 115-126.
[23] 叶芬霞,立波 活性污泥工艺中剩余污泥减量化技术 中国给水排水,2003.
[24] Gulnur Coskuner, Stuart J. Ballinger,et al. Agreement between Theory and Measurement in Quantification of Ammonia-Oxidizing Bacteria[J]. Applied And Environmental Microbiology,2005,71(10):6325-6334.
[25] 邢薇,左剑恶等.利用FISH和DGGE 对产甲烷颗粒污泥中微生物种群的研究环 境科学[J].2006,27(11):2268-2272.
[26] 沈永才, 袁佩娜.16S rRNA 荧光定量PCR 法检测双歧杆菌[J]. 中国微生态学杂志,2001,13(2):66-72.
[27] 叶立,黄卫星,杨平,伍勇.膜生物反应器及其在工业废水处理中的应用.化工环保,2003,32(3):142-146.
[28] Raju Sekar, Annelie Pernthaler, et al. An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization [J]. Applied And Environmental Microbiology,2003,69(5):2928-2935.
[29] Ryo Hondaa,Kensuke Fukushi,et al. Optimization of wastewater feeding for single-cell protein production in an anaerobic wastewater treatment process utilizing purple non-sulfur bacteria in mixed culture condition[J]. Journal of Biotechnology 2006,125:565–573.
[30] Suresh Kumar Dubey,Anil Kumar Tripathi,et al. Exploration of soil bacterial communities fortheir potential as bioresource [J]. Bioresource Technology 2006 ,97 :2217–2224.