布吉河生物修复过程中氮循环功能菌群分布研究
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摘要
随着环境的日益恶化,河流治理工作已刻不容缓。生物修复以其低耗、高效和无污染等优点已成为河流治理的热点技术之一。而微生物在生物修复技术中扮演着极其重要的角色。
本课题以深圳市布吉河为研究对象,采用最大可能数(MPN)法和变性梯度凝胶电泳(DGGE)法研究分析了2007年10月至2008年9月期间布吉河主要微生物功能菌群(包括氨化菌、亚硝化菌、硝化菌和反硝化菌)的结构、数量分布以及菌群变化规律,同时考察了菌剂投加对河水中微生物主要菌群的数量变化和氮含量的影响。论文开展的主要工作和结论包括:
1)采用MPN法分析了布吉河水体和底泥中微生物主要功能菌群的数量分布及其随季节变化的情况,同时也采用PCR-DGGE技术对布吉河水体和底泥中微生物多样性进行了初步探讨。结果表明:氨化菌数量最多,高达8-12个数量级。亚硝化细菌和硝化细菌数量极少,仅0-3个数量级。氮循环功能菌群存在严重的数量不均衡。DGGE结果显示,布吉河中微生物多样性随着时间的变化而变化。水质指标分析结果表明布吉河中氨氮是布吉河的主要氮存在形式。
2)对布吉河的土著微生物进行分离筛选和鉴定,发现在布吉河中起氨化作用的菌属主要是芽孢杆菌属(Bacillus)和微小杆菌属(Exiguobacterium) ,起反硝化作用的菌属主要是土壤杆菌属(Agrobacterium)和假单胞菌属(Pseudomonas)。
3)为强化河流对氮的自净能力,实验通过对布吉河底泥中的优势功能菌群进行富集驯化,分离出高效菌种。通过菌剂复配得到以硝化、亚硝化为主要功能菌群的高效复配菌剂。
4)以布吉河水为原水,在三段串联接触氧化工艺小试装置中开展了菌剂投加试验。采用MPN法和PCR-DGGE技术探讨了菌剂投加前后该反应器中微生物菌群的数量和多样性变化,同时比较了菌剂投加前后氨氮及总氮的去除效果。结果表明,通过菌剂投加,可以明显提高反应器中硝化和亚硝化菌群数量,使氮循环中不同菌群的比例分布更加合理,大大提高了系统对氨氮和总氮的去除率。菌剂投加后,氨氮的去除率由
原来的34.48%增加到70.09%,总氮的去除率由原来的25.86%增加到50.33%。
With increasingly deterioration of the surface water environment, the treatment of the river is no time to lose. Bioremediation has become one of the hot technology, as its advantage in low energy consume, efficient, and no secondary pollution. Microorganisms play an extremely important role in bioremediation.
By taking Buji River of Shenzhen as the research object, this issue analysis the distribution, number and changes of main functional bacteria(ammonifying bacteria, ammonia-oxidizing bacteria, nitrobacteria, denitrifying bacteria, phosphorus accumulation bacteria)from October 2007 to September 2008 ,using the methods of MPN and DGGE(denaturing gradient gel electrophoresis). At the same time, investigate microbial community and nitrogen changes after adding mixed bacteria .The main work and conclusions, including:
1)The investigation of functional microbial communities in Buji River by MPN and PCR-DGGE methods show that, the number of ammonifying bacteria is the largest, as much as 8-12 orders. The result of water quality analysis shows that, ammonia content is high in Buji River. The result of DGGE analysis showed that, microbial diversity of Buji river changes with time and location.
2)After isolated and identified indigenous microorganisms in Buji river, we found that Bacillus and Exiguobacterium play major role in the ammoniation. The main denitrifying bacteria are Agrobacterium and Pseudomonas.
3)To improve the capacity of nitrogen removal of Buji River, we screen high effective bacterium, and then compound it. The mixed bacteria is identified as a high effective group, in which ammonia-oxidizing bacteria, nitrobacteria are main group.
4)Add the mix-bacteria into small scale devise, using water of Buji River as raw water. After adding, analysis the distribution and diversity changes of main functional bacteria using the method of MPN and DGGE; and quality of in-let water and out-let water. We find that, the amount of ammonia-oxidizing bacteria and nitrobacteria have an obvious increase. The ratio of Nitrogen cycle bacteria distribution is more reasonable; The NH3-N and TN removal capacity of the systems has been improved, the ammonia nitrogen removal rate increase from 34.48% to 70.09% ,the total nitrogen removal rate increase from 25.86% to 50.33%.
本课题以深圳市布吉河为研究对象,采用最大可能数(MPN)法和变性梯度凝胶电泳(DGGE)法研究分析了2007年10月至2008年9月期间布吉河主要微生物功能菌群(包括氨化菌、亚硝化菌、硝化菌和反硝化菌)的结构、数量分布以及菌群变化规律,同时考察了菌剂投加对河水中微生物主要菌群的数量变化和氮含量的影响。论文开展的主要工作和结论包括:
1)采用MPN法分析了布吉河水体和底泥中微生物主要功能菌群的数量分布及其随季节变化的情况,同时也采用PCR-DGGE技术对布吉河水体和底泥中微生物多样性进行了初步探讨。结果表明:氨化菌数量最多,高达8-12个数量级。亚硝化细菌和硝化细菌数量极少,仅0-3个数量级。氮循环功能菌群存在严重的数量不均衡。DGGE结果显示,布吉河中微生物多样性随着时间的变化而变化。水质指标分析结果表明布吉河中氨氮是布吉河的主要氮存在形式。
2)对布吉河的土著微生物进行分离筛选和鉴定,发现在布吉河中起氨化作用的菌属主要是芽孢杆菌属(Bacillus)和微小杆菌属(Exiguobacterium) ,起反硝化作用的菌属主要是土壤杆菌属(Agrobacterium)和假单胞菌属(Pseudomonas)。
3)为强化河流对氮的自净能力,实验通过对布吉河底泥中的优势功能菌群进行富集驯化,分离出高效菌种。通过菌剂复配得到以硝化、亚硝化为主要功能菌群的高效复配菌剂。
4)以布吉河水为原水,在三段串联接触氧化工艺小试装置中开展了菌剂投加试验。采用MPN法和PCR-DGGE技术探讨了菌剂投加前后该反应器中微生物菌群的数量和多样性变化,同时比较了菌剂投加前后氨氮及总氮的去除效果。结果表明,通过菌剂投加,可以明显提高反应器中硝化和亚硝化菌群数量,使氮循环中不同菌群的比例分布更加合理,大大提高了系统对氨氮和总氮的去除率。菌剂投加后,氨氮的去除率由
原来的34.48%增加到70.09%,总氮的去除率由原来的25.86%增加到50.33%。
With increasingly deterioration of the surface water environment, the treatment of the river is no time to lose. Bioremediation has become one of the hot technology, as its advantage in low energy consume, efficient, and no secondary pollution. Microorganisms play an extremely important role in bioremediation.
By taking Buji River of Shenzhen as the research object, this issue analysis the distribution, number and changes of main functional bacteria(ammonifying bacteria, ammonia-oxidizing bacteria, nitrobacteria, denitrifying bacteria, phosphorus accumulation bacteria)from October 2007 to September 2008 ,using the methods of MPN and DGGE(denaturing gradient gel electrophoresis). At the same time, investigate microbial community and nitrogen changes after adding mixed bacteria .The main work and conclusions, including:
1)The investigation of functional microbial communities in Buji River by MPN and PCR-DGGE methods show that, the number of ammonifying bacteria is the largest, as much as 8-12 orders. The result of water quality analysis shows that, ammonia content is high in Buji River. The result of DGGE analysis showed that, microbial diversity of Buji river changes with time and location.
2)After isolated and identified indigenous microorganisms in Buji river, we found that Bacillus and Exiguobacterium play major role in the ammoniation. The main denitrifying bacteria are Agrobacterium and Pseudomonas.
3)To improve the capacity of nitrogen removal of Buji River, we screen high effective bacterium, and then compound it. The mixed bacteria is identified as a high effective group, in which ammonia-oxidizing bacteria, nitrobacteria are main group.
4)Add the mix-bacteria into small scale devise, using water of Buji River as raw water. After adding, analysis the distribution and diversity changes of main functional bacteria using the method of MPN and DGGE; and quality of in-let water and out-let water. We find that, the amount of ammonia-oxidizing bacteria and nitrobacteria have an obvious increase. The ratio of Nitrogen cycle bacteria distribution is more reasonable; The NH3-N and TN removal capacity of the systems has been improved, the ammonia nitrogen removal rate increase from 34.48% to 70.09% ,the total nitrogen removal rate increase from 25.86% to 50.33%.
引文
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2卜伟.城市河流污染治理的新观念探讨.环境科学管理. 2006, 31(3):34~35
3王涛.自然水体水质改善与修复技术中国环保产业. 2004, (1):34~35
4 E. Lmon. Determining in Situ Biodegradation: Facts and Challenges. Environmental Science and Technology. 1991, 25(10):1663~1672
5丁吉震. CBS水体修复技术.洁净煤技术. 2000, 6(4):36~38
6张铨昌,杨华蕊,韩成.天然沸石离子交换性能及其应用.科学出版社, 1986:73~98
7 N. Tije. Effects of Wastewater Discharge on Microbial Populations and Enzyme Activities in Mangrove Soils. Environmental Pollution. 2005, (102):45~66
8 L. Piirtola, B. M. Hultman. Effects of Detergent Zeolite in a Nitrogen Removal Activated Sludge Proces. Water Science and Technology. 1998, 38(2):41~48
9 O. G. Lahav. Ammonium Removal Using Ion Exchange and Biological Regeneratio. Water Researcch. 1998, 32(7):2019~2028
10 A. Zorpas, T. Constantinides, A. G. Vlyssides. Heavy Metal Uptake by Natural Zeolite and Metals Partitioning in Sewage Sludge Compost. Bioresource Technology. 2000, (72):113~119
11 A. Haralambous, E. M. Maliou. Use of Zeolite for Ammonium Uptake. Water Science and Technology. 1992, 25(1):139~145
12 G. T. Mill, N. A. Mitkowski. Methods Foassessing the Composition and Diversity of Soil Microbial Communities. Applied Soil Ecology. 2000, (15):25~36
13 E. J. Park, W. J. Sul. Glucose Additions to Aggregates Subjected to Drying/Wetting Cycles Promote Carbon Sequestration and Aggregate Stability. Soil Biology and Biochemistry. 2007, 39(11):2758~2768
14胡洪营,童中华.微生物醌指纹法在环境微生物群体组成研究中的应用.微生物学通报. 2002, 29(4):95~98
15 B. S. Griffiths, K. Ritz. Broad-Scale Approaches to the Determination of Soil Microbial Community Structure: Application of the Community DNA Hybridization Technique. Microbial Ecology. 1996, (31):269~280
16 W. E. Holben, D. Harris. DNA-Based Monitoring of Total Bacterial Community Structure in Environmental Samples. Molecular Ecology. 1995, (4):627~631
17 M. Annette, G. Ulfb. Fluorescence in Situ Hybridization(FISH) for Direct Visualization of Microorganisms. Journal of Microbiological Methods. 2005, (41):85~112
18 G. Muyzer. DGGE/TGGE a Method for Identifying Genes from Natural Ecosystems. Current Opinion in Microbiology. 1999, (2):317~322
19 J. Vivesrego, P. Lebaron, G. N. Caron. Current and Future Applications of Low Cytometry in Aquatic Microbiology. FEMS Microbiology Reviews. 2000, (24):429~448
20 A. Schmalenberger, F. Schwieger. Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling. Applied Environmental Microbiology. 2001, (67):3 557~3563
21 T. Hoshino, N. Noda, A. S. Tsuned. Direct Detection by Insitu PCR of the amoA Gene in Biofilm Resulting From a Nitrogen Removal Process. Applied Environmental Microbiology. 2004, (67):5261~5266
22 A. E. Mccaig, L. A. Glover. Molecular Analysis of Bacterial Community Structure and Diversity in Unimproved and Improved Upland Grass Pastures. Applied Environmental Microbiology. 1999, (65):1721~1730
23 C. D.esnuesa, V. D. Michoteya. Seasonal and Diel Distributions of Denitrifying and Bacterial Communities in a Hypersaline Microbial Mat. Water Research. 2007, (41):3407~3419
24 E. Smit, P. Leeflang. Detection of Shifts in Microbial Community Structure and Diversity in Soil Caused by Copper Contamination Using Amplified Ribosomal DNA Restriction Analysis. FEMS Microbiology Ecology. 1997, 23(3):249~261
25吴少慧,张成刚,张忠泽. RAPD技术在微生物生物多样鉴定中的应用.微生物学杂志.2000, 20(2):44~47
26姚健,杨永华,沈晓蓉等.农用化学品污染对土壤微生物群落DNA序列多样性影响研究.生态学报. 2001, 20(6):1021~1027
27 C. S. Hulton, C. F. Higgins, P. M. Sharp. ERIC Sequences: a Novel Family of Repetitive Element in the Genomes of Escherichia Coli., Salmonella Ttyphimurium and Other Enterobacteria. Molecular Microbiology. 1991, (5):825~834
28 M. Gillings, M. Holley. Repetitive Element PCR Fingerprinting (rep-PCR) Using Enterobacterial Repetitive Intergenic Consensus (ERIC) Primers is Not Necessarily Directed at ERIC Elements. Letters in Applied Microbiology. 1997, (25):17~21
29潘莉,杜惠敏,黄海东等.腹泻儿童肠道菌群结构特征的ERIC-PCR指纹图分析.中国微生态学杂志. 2003, 15(3):141~143
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2卜伟.城市河流污染治理的新观念探讨.环境科学管理. 2006, 31(3):34~35
3王涛.自然水体水质改善与修复技术中国环保产业. 2004, (1):34~35
4 E. Lmon. Determining in Situ Biodegradation: Facts and Challenges. Environmental Science and Technology. 1991, 25(10):1663~1672
5丁吉震. CBS水体修复技术.洁净煤技术. 2000, 6(4):36~38
6张铨昌,杨华蕊,韩成.天然沸石离子交换性能及其应用.科学出版社, 1986:73~98
7 N. Tije. Effects of Wastewater Discharge on Microbial Populations and Enzyme Activities in Mangrove Soils. Environmental Pollution. 2005, (102):45~66
8 L. Piirtola, B. M. Hultman. Effects of Detergent Zeolite in a Nitrogen Removal Activated Sludge Proces. Water Science and Technology. 1998, 38(2):41~48
9 O. G. Lahav. Ammonium Removal Using Ion Exchange and Biological Regeneratio. Water Researcch. 1998, 32(7):2019~2028
10 A. Zorpas, T. Constantinides, A. G. Vlyssides. Heavy Metal Uptake by Natural Zeolite and Metals Partitioning in Sewage Sludge Compost. Bioresource Technology. 2000, (72):113~119
11 A. Haralambous, E. M. Maliou. Use of Zeolite for Ammonium Uptake. Water Science and Technology. 1992, 25(1):139~145
12 G. T. Mill, N. A. Mitkowski. Methods Foassessing the Composition and Diversity of Soil Microbial Communities. Applied Soil Ecology. 2000, (15):25~36
13 E. J. Park, W. J. Sul. Glucose Additions to Aggregates Subjected to Drying/Wetting Cycles Promote Carbon Sequestration and Aggregate Stability. Soil Biology and Biochemistry. 2007, 39(11):2758~2768
14胡洪营,童中华.微生物醌指纹法在环境微生物群体组成研究中的应用.微生物学通报. 2002, 29(4):95~98
15 B. S. Griffiths, K. Ritz. Broad-Scale Approaches to the Determination of Soil Microbial Community Structure: Application of the Community DNA Hybridization Technique. Microbial Ecology. 1996, (31):269~280
16 W. E. Holben, D. Harris. DNA-Based Monitoring of Total Bacterial Community Structure in Environmental Samples. Molecular Ecology. 1995, (4):627~631
17 M. Annette, G. Ulfb. Fluorescence in Situ Hybridization(FISH) for Direct Visualization of Microorganisms. Journal of Microbiological Methods. 2005, (41):85~112
18 G. Muyzer. DGGE/TGGE a Method for Identifying Genes from Natural Ecosystems. Current Opinion in Microbiology. 1999, (2):317~322
19 J. Vivesrego, P. Lebaron, G. N. Caron. Current and Future Applications of Low Cytometry in Aquatic Microbiology. FEMS Microbiology Reviews. 2000, (24):429~448
20 A. Schmalenberger, F. Schwieger. Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling. Applied Environmental Microbiology. 2001, (67):3 557~3563
21 T. Hoshino, N. Noda, A. S. Tsuned. Direct Detection by Insitu PCR of the amoA Gene in Biofilm Resulting From a Nitrogen Removal Process. Applied Environmental Microbiology. 2004, (67):5261~5266
22 A. E. Mccaig, L. A. Glover. Molecular Analysis of Bacterial Community Structure and Diversity in Unimproved and Improved Upland Grass Pastures. Applied Environmental Microbiology. 1999, (65):1721~1730
23 C. D.esnuesa, V. D. Michoteya. Seasonal and Diel Distributions of Denitrifying and Bacterial Communities in a Hypersaline Microbial Mat. Water Research. 2007, (41):3407~3419
24 E. Smit, P. Leeflang. Detection of Shifts in Microbial Community Structure and Diversity in Soil Caused by Copper Contamination Using Amplified Ribosomal DNA Restriction Analysis. FEMS Microbiology Ecology. 1997, 23(3):249~261
25吴少慧,张成刚,张忠泽. RAPD技术在微生物生物多样鉴定中的应用.微生物学杂志.2000, 20(2):44~47
26姚健,杨永华,沈晓蓉等.农用化学品污染对土壤微生物群落DNA序列多样性影响研究.生态学报. 2001, 20(6):1021~1027
27 C. S. Hulton, C. F. Higgins, P. M. Sharp. ERIC Sequences: a Novel Family of Repetitive Element in the Genomes of Escherichia Coli., Salmonella Ttyphimurium and Other Enterobacteria. Molecular Microbiology. 1991, (5):825~834
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