摘要
根据历年《中国环境状况公报》显示,我国水体中氮素污染日益严重,对自然环境和人体健康产生极大危害,氮素在水体中主要以有机氮和无机氮两种形式存在。废水脱氮方法很多,其中生物脱氮是目前最经济有效的方法之一。因传统生物脱氮中各脱氮步骤的不同细菌菌群对营养物质及溶解氧的要求不同,工艺复杂,极大限制了生物脱氮在实际工程中的应用。近年国内外不少研究充分证明了好氧反硝化菌的存在,突破了传统生物脱氮的理论,为生物脱氮技术及其应用提供了崭新的思路与发展。与传统的缺氧反硝化脱氮相比,利用好氧反硝化菌发展生物脱氮技术具有独特优势,已成为废水脱氮研究的热点。
本实验采用极限稀释、滴加格里斯试剂及二苯胺试剂鉴别、平板划线相结合的方法,从校园草坪土壤中分离筛得一株能以硝酸钾为氮源进行好氧反硝化作用的好氧反硝化菌LD3。对菌株LD3进行种属鉴定,并研究其反硝化能力及各种环境因素的影响情况,包括碳源、氮源、溶解氧、C/N、pH值和温度等。研究结果表明,菌株LD3初步鉴定为脂肪杆菌属,其硝酸盐氮去除率高达97.67%,最佳脱氮时间为5d,最适合菌液剂量比例为5%;除过高浓度的亚硝酸盐对菌株LD3生长繁殖有一定的抑制作用外,菌株LD3具有较广泛的碳源谱和氮源谱;菌株LD3对生长环境的溶解氧要求不高,浓度达到1.8mg/L即可,其好氧反硝化作用最佳的初始C/N为8,最佳的初始pH值为7.5,最适宜的温度为30℃左右。正交实验确定菌株LD3好氧反硝化作用最优的环境因素条件为:转速为160r/min,初始C/N为10,初始pH值为7.5,温度为30℃。菌株LD3的分离筛选及其脱氮特性的实验研究对实现好氧反硝化菌脱氮技术利用提供了理论参考。
According to the Report on the State of Environment in China over the years, increasingly severe nitrogen pollution of water bodies does great harm to the natural environment and human health, in which nitrogen was present in the organic and inorganic forms.One of economic and effective approachs among numerous nitrogen removal processes was biodenitrification, yet which was confined in practice due to the complex process, the various nutriment and dissolved oxygen (DO) taken by corresponding bacterias in the diverse traditional nitrogen removal steps. The existence of aerobic denitrifier, proved by the domestic and international research in recent decades, made a breakthrough at the tradition denitrification theory, provided a new perspective to view the biodenitrification technology and application. Compared with the tradition anoxic denitrification, aerobic denitrification had became a hotspot of nitrogen removal from wastewater owing to its unique superiority.
The aerobic denitrifier strain LD3with potassium nitrate as nitrogen source was isolated and screened from the campus lawn soil via limiting concentration, adding Chris reagents and diphenylamine and combined with plate streaking, and then expertized species identification, investigated denitrification ability and the effect of environment factors including carbon source, nitrogen source, DO, carbon nitrogen ratio (C/N), pH,temperature, etc. It was concluded that strain LD3was preliminarily identified to be pimelobacter, The removal rate of nitratenitrogen reached97.67%, the best denitrification time was5days, the optimum bacterium fluid does ratio was5%.Strain LD3lacked advantage of rich carbon and nitrogen sources except that high concentration nitrite was a disincentive. Strain LD3wasn't strict with DO, which could meet the demands at1.8mg/L. The optimum initial C/N, pH and temperature was8,7.5and30℃respectively for aerobic denitrification. the best environmental factors determined by orthogonal test was as follows:rotate speed, initial C/N, initial pH, temperature was160r/min,10,7.5and30℃respectively. Experiemntal investigation on identification and denitrification characteration of strain LD3provided the theoretical reference for achieving nitrogen removal by aerobic denitrifier.
引文
[1]郑兴灿,李亚森.污水脱氮除磷技术[M].北京:中国建筑工业出版社,1998,11-19.
.[2]王士芬.湖泊水藻类的去除方法[J].污染防治技术.2000,13(1):23-25.
[3]王红兵,朱惠光.淡水浮游藻类污染及其毒性[J].上海环境科学.1995,14(8):38-41.
[4]庞金钊.异养硝化菌的筛选及其在污水脱氮中的应用[D].天津:天津科技大学.2006.
[5]项慕飞.好氧反硝化菌的分离筛选和鉴别研究[D].北京:北京工商大学.2007.
[6]冯叶成,王建龙等.同时硝化反硝化的实验研究[J].上海环境科学,2002,21(9):527-529.
[7]Otani Y, Hasegawa K, Hanaki K. Comparison of aerobic denitrifying activity among three cultural species with various carbon sources[J].Water Sci.Technol,2004,50 (8):15-22.
[8]Lukow T,Diekmann H. Aerobic denit rification by a newly isolated heterot rophic bacterium strain T[J].Biotechnology Letters,1997,11 (9):1157-1159.
[9]Stevension F.J,Origin and distribution of nitrogen in soil. In:soil nitrogen,Bartholome W.V., Clark F E eds,Madesin.,Wisconsin Am.Soc.Agron.,1965:1-42.
[10]张弛,李生秀等.氮素在地球生物中的分布及循环[J].西北农业大学学报,1995,23(1):93-97.
[11]何芳.好氧反硝化菌的筛选[D].扬州:扬州大学.2006.
[12]陈坚.环境生物技术(第一版)[M].北京:中国轻工业出版社.1999.
[13]郭海娟.亚硝酸型硝化和反硝化除磷的生物脱氮除磷技术研究[D].哈尔滨:哈尔滨工业大学.2003.
[14]张自杰.排水工程下册[M].北京:中国建筑工业出版社.2000.
[15]李勇智.短程生物脱氮和反硝化除磷的基础研究[D].哈尔滨:哈尔滨工业大学.2003.
[16]耿朝安,张洪林.废水生物处理发展与实践[M].沈阳:东北大学出版社.1997.
[17]沈耀良,王宝贞.废水生物处理新技术——理论与应用(第一版)[M].北京:中国环境科学出版社.1997.
[18]周少奇,周吉林.生物脱氮新技术研究进展[J].环境污染治理技术与设备.2000,1(6):11-18.
[19]Robertson L A, Kuenen J G. Aerubic Denitrification:a Controversy Revived[J].Arch Microbiol,1984,139(5):351-354.
[20]P.Klangduen, K.Jurg. Model Development For Simultaneous Nitrification and Denitrification. Wat.Sci.Tech.,1999.39(1):235-243.
[21郑平,徐向阳等.新型生物脱氮理论与技术[M].北京:科学出版社.2004.
[22]张自杰.排水工程下册(第四版)[M].北京:中国建筑工业出版社.2006.
[23]L.A.Robertson, et al.Nitrogen Removal from Water.In:Microbial Control of Pollution.Cambridge, United Kingdom:Cambridge University Press,1992:227-267.
[24]Wilson L P, Bouwer E J.Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions:a review[J].Journal of Industrial Microbiology & Biotechnology, 1997,18:116-130.
[25]L A.Robertson, Van Neil E WJ. Simultaneous Nitrifiction and Denitrification inAerobic Chemostat Cultures ofThiosphaera Pantotropha[J].Applied Environmental Microbiology,1988, 54(1):2812-2818.
[26]王薇,蔡祖聪等.好氧反硝化菌的研究进展[J].应用生态学报.2007,18(11):2618-2625.
[27]Bell C,Richardson D J, Ferguson S J.Periplasmic and membrane-bound nitrate reductases in Thiosphaerapuntotropha[J].FEBS Letters,1990,265:85-87.
[28]孔庆鑫,李君文.微生物好氧反硝化脱氮作用研究进展[J].环境与健康杂志.2004,21(3):189-191.
[29]Richardson D J, Ferguson S.The influence of carbon substrate on the activity of the periplasmic nitrate reductase in aerobically grown Thiosphaera pantotropha[J].Archives of Microbiology,1992,157:535-537.
[30]Fulop V, Moir J W, Ferguson S J, et al.The anatomy of a bifunctional enzyme:Structural basis for reduction of oxygen to water and synthesis of nitric oxide by cytochrome cd[J]. Cell, 1995,81:369-377.
[31]Komer H, Zumfit W G. Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonus stufzeri[J].Applied and Environmental Microbiology,1989,55:1670-1676.
[32]Shapleigh J P, Payne W J.Differentiation of cdl cytochrome and copper nitrite reductase production in denitrifiers[J].FEMS Microbiology Letters,1985,26:275-279.
[33]刘懿.好氧反硝化菌的诱变分离及其对硝酸盐废水处理的研究[D].长春:吉林大学.2009.
[34]李平,张山等.细菌好氧反硝化研究进展[J].微生物学杂志.2005,25(1):60-64.
[35]Berks B C, Baratta D, Richardson D J, et al. Purification and characterisation of a nitrous oxide reductase from Thiosphtrera pantotropha[J].European Journal of Biochemistry,1993, 272:467-476.
[36]Gupta A B.Thiosphaera pantotropha:A sulphur bacterium capable of simultaneous heterotrophic nitrification and aerobic denitrification[J].Enzyme and Microbial Technology, 1997,21:589-595.
[37]马放,王弘宇等.好氧反硝化生物脱氮机理分析及研究进展[J].工业用水与废水.2005,36(2):11-14、59.
[38]B.C.Berks, D.J.Rchardson, C.Robinson, et al.Pyrification and Characterization of the Periplasmic Nitrate Reductase from Thiosphaera pantotropha[J].Biochem J,1995,309:983-992.
[39]B.C.Berks, D.J.Rochardson, A.Reilly, et al.The napEDABCgene.Cluster Encoding the Periplasmic Nitrate Reductase System of Thiosphaera pantotropha[J].Eur.J.Biochem,1994, 220:117-124.
[40 L.C.Bell,D.J.Rochardson, S.J.Ferguson. Periplasmic and Membrane-bound Respiralary Nitrate Reductase in Thiosphaera pantotropha[J].FWBSLell,1990,265:85-87.
[41]S.Zhou, H.H.P.Fang. Simultaneous Nitrification and Denitrification of High Ammonia Organic Waste Water Treatment.In:CDR of 1999 Asian Industrial Technology Congress[J].April 1999, Hongkong.
[42]程翔.好氧颗粒污泥同步硝化反硝化处理高浓度氨氮废水研究[D].哈尔滨:哈尔滨工业大学.2006.
[43]赵丹丹.好氧反硝化菌株的快速筛选及其在硝态氮废水处理中的应用研究D].长春:吉林大学.2007.
[44]E.Scholten, T.Lukow, G.Auling, et al.Thaneramec henichensis sp nor:DAnaerobic Denitrification from Denitrifier from a Leachate Treatment Plant[J].Int J Syst Bacteriol,1999, 49:1045-1051.
[45]吕锡武,李丛娜等.溶解氧及活性污泥浓度对同步硝化反硝化的影响[J].城市环境与城市生态.2001,14(1):33-35.
[46]A B Gupta, S K Gupta. Simultaneous Carbon and Nitrogen Removal in a Mixed Culture Aerobic RBC Biofilm[J].Water Resources Research,1999,33(2):555-561.
[47]Shwu-Ling Pai, Nyuk-Min Chong, Chei-Hsiang Chen. Potential Applications of Aerobic Denitrifiying Bacteria as Bioagents in Wastewater Treatment[J].Bioresource Technology,1999, 68(2):179-185.
[48]Seung-Hun Baek, Cheng-Ri Yin, Sung-Taik Lee. Aerobic Nitrate Respiration by a Newly Isolated Phenol-degrading Bacterium,Alcaligenes strain P5[J].Biotechology Letters,2001, 23(8):627-630.
[49]Suzuki T, Ichihara Y, Yamada M, et al.Some characteristics of pseudomonas O-3 which utilities polyvinyl alcohol[J].Agricultural and Biological Chemistry,1973,37(4):747-756.
[50]于爱茸,李尤等.一株耐氧反硝化细菌的筛选及脱氮特性研究[J].微生物学杂志.2005,25(3):77-81.
[51]L.Kuai, W.Verstrartr, Autotrophic denitfification with elemental sulphur insmall scale wastewater treatment facilities[J].Environ. Tech,1999,20:201-209.
[52]M.Mike Jenen, Susanne logemann, Gerard Muyzer, et al.Novel principle in the microbial conversion ofnitrogen compounds[J].Antonie van Leeuwenhoek,1997,71:75-93.
[53]I.Mchmidt, O.Sliekers, M.Schmid, et al.New COIlCepts of microbial treatmentprocesses for the nitrogen removal in wastewater[J].Ferns Microbiology Reviews,2003,27:481-492.
[54]姚樱,张文等.新型生物脱氮工艺的研究进展[J].环境科学与技术.2005,28:138-140.
[55]Votes J E. Removal of nitrogen from highly nitrogenous wastewater[J].JW-PCF,1975, 47(4):394-098.
[56]Hellinga C, Schellena J C, Mulder J W, et al.The SHARON process:an innovativemethod for nitrogen removal from ammonium rich wastewater[J].Wat. Sci.Tech,1998,37(9):135-142.
[57]Kuallp, Verstraete w. Ammonium removal by the oxygen limited autotrophicnitrification denitdficmion system[J].Appl.Environ.Microliol,1998,64:4500-450.
[58]E W J Van Neil.Nitrification by Heterotrophic Denitrifiers and Its Relationship toAutotrophic Nitrification[Ph.D Thesis].Delft University ofTechnology, Delft,1991,78-80.
[59]Alexander M. Nitrification In soil[J]. Microbiology. New York,1977,251-271.
[60]Papen H,R. A.Most Probable Number(MPN)method for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil[J].Plant and soil,1998,199:123-130.
[61]Robertson LA, Kuenen JG.Thiospaera pantotropha gen.nov. sp. nov, afacultatively anaerobic, facultatively autotrophic sulphur bacterium[J].Journal of GeneralM icrobiology,1983, 129(8):2847-2855.
[62]Lone E, Bo G, Peter W. Aerobic denitrifiers isolated from an alternating activated sludge system[J].FEMS Microbiology Ecology,1997,24(4):363-370.
[63]周丹丹,马放等.关于好氧反硝化菌筛选方法的研究[J].微生物学报.2004,44(6):837-839.
[64]张凤君,戴宁等.活性污泥法驯化筛选好氧反硝化菌的试验研究[J].环境科学学报.2006,26(11):1084-1088.
[65]吴美仙,张萍华等.好氧反硝化细菌的筛选及培养条件的初步研究[J].浙江海洋学院学报(自然科学版).2008,27(4):406-409.
[66]于大禹,郭威等.好氧反硝化菌的筛选及其脱氮除磷性质的研究[J].微生物学通报.2009,36(4):598-603.
[67]何伟,王薇等.一株好氧反硝化菌的分离鉴定及其混合应用特性研究[J].生态与农村环境学报.2009,25(2):88-93.
[68]Mike SM Jetten, Susanne Logemann, GerardMuyzer, et al.Novel principles in the microbial conversion of nitrogen compounds[J].Antonie van Leeuwenhoek.1997,71:75-93.
[69]孔庆鑫,李君文等.一种新的好氧反硝化菌筛选方法的建立及新菌株的发现[J].应用与环境生物学报.2005,11(2):222-225.
[70]Meiberg JBM, Bruinenberg PM, HarderW. Effect of dissolved oxygen tension on the metabolism of methylated amines in Hyphom icrobium X in the absence and p resence of nitrate: Evidence for aerobic denitrification[J].Journal of GeneralM icrobiology.1980,120:453-463.
[71]Gupta SK, Mona K. Quantitative estimation of Thiosphaeta pantotropha from aerobic mixed culture[J].Water Research.2000,34(15):3765-3768.
[72]张光亚,陈培钦.好氧反硝化菌的分离鉴定及特性研究[J].微生物学杂志.2005,25(6):23-26.
[73]朱月琪,卫晋波等.一株好氧反硝化菌的分离及特性研究[J].微生物学通报.2009,36(4):616-619.
[74]左薇.一株好氧反硝化菌的筛选鉴定及其脱氮特性分析[D].哈尔滨:哈尔滨工业大学.2006.
[75]李阜棣.农业微生物学实验技术[M].北京:中国农业出版社.1996.
[76]周立祥,黄峰源等.好氧反硝化菌的分离及其在土壤氮素转化过程中的作用[J].土壤通报.2006,43(3):6430-435.
[77]朱晓宇,王世梅等.两株高效好氧反硝化细菌的分离鉴定及其脱氮效率[J].环境科学学报.2009,29(1):111-117.
[78]张小玲,张卫东等.好氧反硝化菌的选育及其初步应用[J].微生物学通报.2008,35(10):1556-1561.
[79]翟茜,汪萍等.活性污泥中好氧反硝化菌的富集筛选及鉴别[J].环境科学与技术.2007,30(1):11-13.
[80]关文君.好氧反硝化菌在养殖池塘中的初步应用[D].广州:华南师范大学.2010.
[81]马放,王弘宇等.好氧反硝化菌株X31的反硝化特性[J].华南理工大学学报(自然科学版).2005,33(7):42-46.
[82]Naoki z, Maria-Antonina B.,Catalan S.,Yasnshi S.,et al.Aerobic denitrifying bacteria that produce low levels of nitrous oxide[J].Appl.Envir. MicrobiOl.2003,69(6):3152-3157.
[83]Kester R.A, Boer D.,and Laanbroek w..Production of NO and N20 by Pure Cultures of Nitrifying and Denitrifying Bacteria during Changes in Aeration[J].Appl.Envir. MicrobiOl., 1997,63(10):3872.
[84]徐亚同.pH值、温度对反硝化的影响[J].中国环境科学.1994,14(4):308-313.
[85]阎胜利,温淑瑶等.土壤中反硝化作用的试验研究[J].河海大学学报.1998,26(2):91-94.
[86]徐亚同.不同碳源对生物反硝化的影响[J].环境科学.1994,15(2):29-32、44.
[87]Stouthamer A H,de Boer A P, van Der O J, et al.Emerging principles of inorganic nitrogen metabolism in Paracoccus denitrificans and related bacteria[J].Antonie van Leeuw enhoek,1997, 71:33-41.
[88]郑平.环境微生物实验指导[M].杭州:浙江大学出版社.2006.
[89]东秀珠,蔡妙英等.常见细菌系统鉴定手册[M].北京:科学出版社.2001.
[90]翟茜.优选高性能的好氧反硝化菌株[D].北京:北京工商大学.2007.
[91]王弘宇,马放等.好氧反硝化菌株的鉴定及其反硝化特性研究[J].环境科学.2007,28(7):1548-1552.
[92]高铎,靳先云.好氧反硝化菌的脱氮特性研究与探讨.内蒙古环境科学[J].2008,20(6):60-63.
[93]刘晶晶.N20的生物控逸及好氧反硝化菌的反硝化性能研究[D].北京:北京工商大学.2009.
[94]魏巍,黄廷林,苏俊峰等.1株贫营养好氧反硝化菌的分离鉴定及其脱氮特性[J].生态环境学报.2010,19(9):2166-2171.
[95]魏巍,黄廷林,苏俊峰等.1株贫营养好氧反硝化菌的分离鉴定及其脱氮特性[J].生态环境学报.2010,19(9):2166-2171.
[96]王娟,王萍,项慕飞等.2株好氧反硝化菌还原能力测定及菌种鉴定[J].环境与健康杂志.2008,25(6):525-528.
[97]孔强,任宗明,付荣恕.北运河沉积物中好氧反硝化菌的分离鉴定及脱氮效率研究[J].供水技术.2010,4(3):1-4.
[96]林娜,郭楚玲,柯林等.富营养化池塘中好氧反硝化菌的分布及脱氮研究[J].中国科技论文在线.2010,4(5):369-372、376.
[97]孙菲,方金武,蔡昌凤等.好氧反硝化茵群的筛选及其培养条件的研究[J].安徽工程科技学院学报.2009,25(2):36-39、72.
[98]修海峰,朱仲元,丁爱中等.好氧反硝化菌种DF2的分离鉴定及生理生化特性分析[J].生态环境学报.2011,20(8-9):1307-1314.
[99]黄廷林,苏俊峰,李倩.好氧反硝化菌株的筛选培养及其反硝化性能研究[J].西安建筑科技大学学报.2009,41(5):704-707.
[100]李静,王文文,梁磊等.耐盐好氧反硝化菌筛选及其反硝化特性的研究[J].环境科学与技术.2011,34(6):48-52.
[101]王莹,周巧红,梁威等.人工湿地高效好氧反硝化菌的分离鉴定及反硝化特性研究[J].农业环境科学学报.2010,29(6):1193-1198.
[102]李慧颖,黄少斌,范利荣.一株好氧反硝化菌的反硝化性能研究[J].环境科学与技术.2009,32(8):9-12.
[103]许尚莹,张华,李娜.一株好氧反硝化菌的鉴定及反硝化特性研究[J].环境科学与技术.2010,32(2):10-13.
[104]杨基先,高珊珊,马放等.一株好氧反硝化细菌的分离鉴定及反硝化能力[J].环境科学学报.2008,28(7):1302-1307.
[105]黄运红,龙中儿,许杨.一株好氧反硝化细菌的筛选及初步鉴定[J].食品科学.2007,28(8):266-268.