一株新型脱氮微生物的分离鉴定及其脱氮机制
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摘要
氮素是导致水体污染的一类重要污染物，可引起水体的富营养化。生物脱氮是目前污水脱氮处理中最常用的方法，但工艺复杂，一般经过好氧的硝化过程和缺氧的反硝化过程才能够将氨氮转化为氮气。近年来，一些异养硝化菌的发现为生物脱氮提供了新的思路，可以实现同步硝化反硝化。但已报道的异养硝化细菌脱氮过程中往往会产生亚硝酸盐及N_2O，不利于实际应用。本课题筛选出一株直接将氨氮转化为氮气，没有或少量产生亚硝酸盐、N_2O的异养硝化菌，为新型脱氮技术的发展打下基础。
通过极限稀释法及平板划线分离法，共筛选到了6株异养硝化菌，其中YY-5脱氮效果最好，24 h内可将58mg／L氨氮全部脱除。经鉴定，为不动杆菌属，命名为Acinetobacter sp.YY-5。
通过单因素实验以及正交实验确定了Acinetobacter sp.YY-5最佳脱氮条件：30℃，C／N=12，pH=10.0；并发现YY-5在脱氮过程中有N_2产生，但没有硝酸盐、亚硝酸盐及N_2O的产生；培养基pH值明显升高，且氨氮脱除不是由于高碱环境下氨挥发造成的。这些特点跟传统的异养硝化细菌具有很大差别。
通过对YY-5脱氮过程中各种含氮代谢物的定量以及相关基因、蛋白的初步分析，发现YY-5中的羟胺氧化酶(HAO)在好氧条件下可将羟胺氧化，氧化产物中未检测出亚硝酸盐；而已报道的其它异养硝化细菌好氧条件下HAO催化羟胺氧化产物为亚硝酸盐，两者差别较大；而且实验证明，YY-5没有好氧反硝化功能，却能够产生大量氮气。因此，认为YY-5脱氮途径跟已报道的异养硝化途径不同，据此提出了YY-5脱氮假设途径，即氨氮经氨单加氧酶(AMO)转化为羟胺，羟胺经YY-5特有的HAO的作用直接转化为氮气。
将YY-5接种于生活污水、工业废水、食品加工废水以及垃圾渗滤液中，检验其在实际污水中脱氮效果。发现YY-5对小分子碳源具有较强的依赖性；在加入小分子碳源的情况下，YY-5对各类污水都具有较强的脱氮能力，氨氮去除率为82.93％～94.35％。
Nitrogen is one of major pollutants of water pollution,and can cause water eutrophication.Biological denitrogenation is a method used most widely in wastewater treatment. But it needs two procession,} .e. aerobic nitrification and anoxic denitrification,to change ammonia to dinitrogen,which makes this method more complex. The discovery of heterotrophic nitrification bacteria recently years,which can nitrify and denitrify simultaneously, gives a new principle to biological denitrogen.But nitrite or nitrous oxide can be a harmful by product in nitrogen removal by most heterotrophic nitrifier as yet reported. In this study a strain of heterophic nitrifier was screened which could directly convert ammonia to dinitrogen and produced little nitrite and nitrous oxide.
Six strains of bacteria which could heterotrophic nitrify was isolated. Strain YY-5 could' remove nitrogen most rapidly in these 6 strains,which could reduce ammonia from 58mg/l to zero in 24 h Strain YY-5 was identified as Acinetobacter genus,and named as Acinetobacter sp.YY-5.
The most optimal nitrogen removal condition was 30℃, C/N=12, pH=10.0. Little nitrite and nitrous oxide,as expected,was produced during the nitrogen removal by Acinetobacter sp.YY-5, and the quantity of dinitrogen in the headspace and pH of the culture media were increased in evidence,without ammonia gas volatilization.These characters were much different from autotrophic nitrifier.
Nitrogen removal mechanisms of Acinetobacter sp.YY-5 were studied by determining the nitrogen compound production during nitrogen removal and analysising some related gene and protein. The hydroxylamine oxidase(HAO) in Acinetobacter sp.YY-5 extract could remove hydroxylamine and yield Na but not nitrite ,which made it much different from those reported ones. Acinetobacter sp.YY-5 could not make aerobic denitrification but could produce dinitrogen .It's believed that the nitrogen removal
mechanisms of Acinetobacter sp.YY-5 were different from the reported ones,and led us to develop a working model for the nitrogen removal of Acinetobacter sp.YY-5,i.e. ammonia was converted to hydroxylamine by ammonia monooxygenase(AMO) and then produced hydroxylamine was converted directly to dinitrogen by HAO.
Nitrogen removal ability of Acinetobacter sp.YY-5 was evaluated in the domestic sewage N chemical industrial wastewater. food industrial wastewater and garbage percolate.It's found that the growth and nitrogen removal ability of Acinetobacter sp.YY-5 depended much on the quantity of small molecule carbon source ,and nitrogen removal rate of Acinetobacter sp.YY-5 in every wastewater with small molecule carbon source was evidently and ranged from 82.93%to 94.35%.
通过极限稀释法及平板划线分离法，共筛选到了6株异养硝化菌，其中YY-5脱氮效果最好，24 h内可将58mg／L氨氮全部脱除。经鉴定，为不动杆菌属，命名为Acinetobacter sp.YY-5。
通过单因素实验以及正交实验确定了Acinetobacter sp.YY-5最佳脱氮条件：30℃，C／N=12，pH=10.0；并发现YY-5在脱氮过程中有N_2产生，但没有硝酸盐、亚硝酸盐及N_2O的产生；培养基pH值明显升高，且氨氮脱除不是由于高碱环境下氨挥发造成的。这些特点跟传统的异养硝化细菌具有很大差别。
通过对YY-5脱氮过程中各种含氮代谢物的定量以及相关基因、蛋白的初步分析，发现YY-5中的羟胺氧化酶(HAO)在好氧条件下可将羟胺氧化，氧化产物中未检测出亚硝酸盐；而已报道的其它异养硝化细菌好氧条件下HAO催化羟胺氧化产物为亚硝酸盐，两者差别较大；而且实验证明，YY-5没有好氧反硝化功能，却能够产生大量氮气。因此，认为YY-5脱氮途径跟已报道的异养硝化途径不同，据此提出了YY-5脱氮假设途径，即氨氮经氨单加氧酶(AMO)转化为羟胺，羟胺经YY-5特有的HAO的作用直接转化为氮气。
将YY-5接种于生活污水、工业废水、食品加工废水以及垃圾渗滤液中，检验其在实际污水中脱氮效果。发现YY-5对小分子碳源具有较强的依赖性；在加入小分子碳源的情况下，YY-5对各类污水都具有较强的脱氮能力，氨氮去除率为82.93％～94.35％。
Nitrogen is one of major pollutants of water pollution,and can cause water eutrophication.Biological denitrogenation is a method used most widely in wastewater treatment. But it needs two procession,} .e. aerobic nitrification and anoxic denitrification,to change ammonia to dinitrogen,which makes this method more complex. The discovery of heterotrophic nitrification bacteria recently years,which can nitrify and denitrify simultaneously, gives a new principle to biological denitrogen.But nitrite or nitrous oxide can be a harmful by product in nitrogen removal by most heterotrophic nitrifier as yet reported. In this study a strain of heterophic nitrifier was screened which could directly convert ammonia to dinitrogen and produced little nitrite and nitrous oxide.
Six strains of bacteria which could heterotrophic nitrify was isolated. Strain YY-5 could' remove nitrogen most rapidly in these 6 strains,which could reduce ammonia from 58mg/l to zero in 24 h Strain YY-5 was identified as Acinetobacter genus,and named as Acinetobacter sp.YY-5.
The most optimal nitrogen removal condition was 30℃, C/N=12, pH=10.0. Little nitrite and nitrous oxide,as expected,was produced during the nitrogen removal by Acinetobacter sp.YY-5, and the quantity of dinitrogen in the headspace and pH of the culture media were increased in evidence,without ammonia gas volatilization.These characters were much different from autotrophic nitrifier.
Nitrogen removal mechanisms of Acinetobacter sp.YY-5 were studied by determining the nitrogen compound production during nitrogen removal and analysising some related gene and protein. The hydroxylamine oxidase(HAO) in Acinetobacter sp.YY-5 extract could remove hydroxylamine and yield Na but not nitrite ,which made it much different from those reported ones. Acinetobacter sp.YY-5 could not make aerobic denitrification but could produce dinitrogen .It's believed that the nitrogen removal
mechanisms of Acinetobacter sp.YY-5 were different from the reported ones,and led us to develop a working model for the nitrogen removal of Acinetobacter sp.YY-5,i.e. ammonia was converted to hydroxylamine by ammonia monooxygenase(AMO) and then produced hydroxylamine was converted directly to dinitrogen by HAO.
Nitrogen removal ability of Acinetobacter sp.YY-5 was evaluated in the domestic sewage N chemical industrial wastewater. food industrial wastewater and garbage percolate.It's found that the growth and nitrogen removal ability of Acinetobacter sp.YY-5 depended much on the quantity of small molecule carbon source ,and nitrogen removal rate of Acinetobacter sp.YY-5 in every wastewater with small molecule carbon source was evidently and ranged from 82.93%to 94.35%.
引文
[1] 郑兴灿，李亚新．污水除磷脱氮技术．北京：中国建筑工业出版社，1998．11-19．
[2] Rotthauwe JH, Deboer W, Liesack W. Comparative analysis of gene sequences encoding ammonia monooxygenase of Nitrosospira sp. AHB1 and Nitrosolobus multiformisC-71. FEMS Microbiol Lett, 1995, 133(1-2): 131-135.
[3] Sayavedra Soto LA, Hommes NG, Alzerreca JJ, Arp DJ, Norton JM, Klotz MG. Transcription of the amoC, amoA and amo B genes in Nitrosomonas europaea and Nitrosospira sp. NpAV. FEMS Microbiol Lett, 1998, 167: 81-88.
[4] Bonin P, Gilewicz M, Bertrand JC. Effects of oxygen on each step of denitrification on Pseudomonas nautical. Can J Microbiol, 1989, 35: 1061~1064.
[5] Shrestha NK, Hadano S, Kamachi T, Okura I.. Conversion of ammonia to dinitrogen in wastewater by Nitrosomonas europaeaa. Appl Biochem Biotechnol, 2001, 90: 221-232.
[6] Focht DD, Verstraete W. Biochemical ecology of nitrification and dinitrification. Adv Microbial Ecol, 1977, 1: 135-214.
[7] Mobarry BK, Wagner M, Urbain V, Rittmann BE, Stahl DA. Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl Environ Microbiol 1996, 62(6): 2156-2162.
[8] 郑兴灿，李亚新．污水除磷脱氮技术．北京：中国建筑工业出版社，1998．48-49．
[9] Allgeier RJ, Peterson WH, Juday C, Birge EA. The anaerobic fermentation of lake deposits. Int Rev Hydrobiol, 1932, 26: 444-461.
[10] Van Luijn F, Boers Paul CMA, Lambertus L. Anoxic N_2 fluxes from freshqater sedimentr in the absence of oxidized nitrogen compounds. Water Res, 1998, 32(2): 407-409.
[11] Lloyd JMA, Liehr SKA, Classen JJA, Robarge W. Mechanisms of dinitrogen gas formation in anaerobic lagoons. Adv Environ Res, 2000, 4(4): 133-139.
[12] Van de Graaf A A, Mulder A, de Bruijn P, Jetten MS, Roberson LA, Kuenen JG. Anaerobic oxidation of ammonium is a biologically mediated process. Appl Environ Microbiol, 1995, 61(4): 1246-1251.
[13] Van de Graaf AA, Mulder A, de Bruijn P, Jetten MS, Robertson LA, Kuenen JG. The SHARON-Anammox process for treatment of ammonium rich wastewater. War Sei Tech, 2001, 44(1): 153-160.
[14] Verstraete W1, Alexander M. Heterotrophic nitrification by Arthrobacter sp. J Bacteriol, 1972, 110(3): 955-961.
[15] Genevieve Mével, Prieur D. Heterotrophic nitrification by a thermophilic Bacillus species as influenced by different culture conditions. Can J Microbiol, 2000, 46(5): 465-473.
[16] Robertson LA, Kuenen JG. Thiosphaera pantotropha gen. nov. sp. nov., a faeultatively anaerobic, faeultatively autotrophie sulphur bacterium. J Gen Microbial, 1983, 129: 2847-2855.
[17] Su JJ, Liu BY, Liu CY. Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system. J Appl Microbiol, 2001, 90(3): 457-462.
[18] Robertson LA, Van Nell EWJ, Torremans, Rob AM, Kuenen JG. Simultaneous nitrification and denitrification in aerobic chemostat cultures of Thiosphaera pantotropha. Appl Envion Mierobiol, 1988, 54(1): 2812-2818.
[19] Van't Riet J, Stouthamer AH, Planta RJ. Regulation of nitrate assimilation and nitrate respiration in Aerobater aerogenes. Bacteriol, 1968, 96: 1455-1464.
[20] Rand MC, Greenberg AE, Taras MJ(ed.). Standard methods for the examination of water and wastewater. Washington, D. C. American Public Health association, 1975, 14th ed.: 434-436.
[21] 国家环保局《水和废水监测分析方法》编委会．水和废水监测分析方法．第三版．北京：中国环境科学出版社，1989．252-255．
[22] RE．布坎南，NE．吉本斯．伯杰细菌鉴定手册 第八版．北京：科学出版社，1984．中国科学院微生物研究所伯杰细菌鉴定手册翻译组译．
[23] 东秀珠，蔡妙英．《常见细菌系统鉴定手册》北京：科学出版社，2001．409-412．
[24] Poth M. Dinitrogen production from nitrite by a Nitromonas isolate. Appl Environ Microbiol, 1986, 52: 957-959.
[25] Mike SMJ, Logemann S, Muyzer G, Robertson LA, Simon de Vries, Mark CML, Kuenen JG. Novel principles in the microbial conversion of nitrogen compounds. Antinie van heeuwenhoek, 1997, 71: 75-93.
[26] 郑兴灿，李亚新．污水除磷脱氮技术．北京：中国建筑工业出版社，1998．45-46．
[27] Ebina J, Tsutsui T, Shriai T. Simultaneous determination of total nitrogen and total phosphorus in water using peroxodisulfate oxidation. Wat Res, 1983, 17(12): 1721-1726.
[28] Otte S, Grobben NG, Robertson LA, Jetten MS, Kuenen JG. Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions. Appl Environ Microbiol. 1996, 62(7): 2421-2426.
[29] Wehrfritz JM, Reilly A, Spiro S, Richardson DJ. Purification of hydroxylamine oxidase from Thiosphaera pantotropha. Identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification. FEBS Lett, 1993 Dec 6; 335(2): 246-250.
[30] Yamanaka T, Shinra M. Cytochrome c-552 and cytochrome c-554 derived from nitrosomonas europaea. J Biochem, 1974, 75: 1265-1273.
[31] Frear DS, Burrell RC. Spectrophotometric method for determining hydroxylamine reductase activity in higher plants. Anal Chem, 1955, 27(10): 1664-1665.
[32] van Niel EJW, Braber KJ, Robertson LA, Kuenen JG. Heterotrophic nitrification and aerobic denitrification in Alcaligenes faecalis strain TUD. Antonie van Leeuwenhoek, 1992, 62: 231-237.
[33] Mike SMJ, Peter de Bruijin, Kuenen JG. Hydroxylamine metablolism in pseudomonas PB16: involvement of a novel hydroxylamine oxidorductase. Antonie van Leeuwenhoek, 1997, 71: 69-74.
[34] Arts PAM, Robertson LA, Kuenen J G. Nitrification and denitrification by Thiosphaera pantotropha in aerobic batch and chemostat cultures. FEMS Microbiol Ecol 1996, 18: 305-315.
[35] Ohashi A, Viraj de Silva DG, Mobarry B, Manem JA, Stahl DA, and Rittmann BE. Influence of substrate C/N ratio on the structure of multi-species biofilms consisting of
nitrifiers and heterotrophs. Wat Sci Tech., 1996, 32: 75-84.
[36] Kuenen JG, Roberson LA. Combined nitrification and denitrification processes. FEMS Microbiol Rev, 1994, 15: 109-117.
[37] Castignetti D and Thomas C. H. Heterotrophic nitrification among denitrifier. Appl Environ Micobio1, 1984, 47(4): 620-623.
[38] Robertson LA, Dalsgaard T, Revsbeck N P, Kuenen J G. Confirmation of 'aerobic denitrification' in batch cultures, using gas chromatography and 15N mass spectrometry. FEMS Microbiol Ecol, 1995, 18: 113-120.
[39] Takaya N, Catalan-Sakairi MA, Sakaguchi Y, Kato I, Zhou Z, Shoun H. Aerobic denitrifying bacteria that produce low levels of nitrous oxide, Appl Environ Microbiol, 2003, 69(6): 3152-3157.
[40] Richardson DJ, Wehrfritz JM, Keech A, Crossman LC, Roldan MD, Sears HJ, Butler CS, Reilly A, Moir JW, Berks BC, Ferguson SJ, Thomson AJ, Spiro S. The diversity of redox proteins involved in bacterial heterotrophic nitrification and aerobic denitrification. Bibchem Soc Trans, 1998, 26(3): 401-408.
[41] Terry KR, Hooper AB. Hydroxylamine oxidoreductase: a 20-heme, 200000 molecular weight cytochrome c with unusual denaturation properties which forms a 63000 molecular weight monomer after heme removal, Biochemistry, 1981, 20(24): 7026-7032.
[42] Arciero DM, Hooper AB. Hydroxylamine oxidoreductase from Nitrosomonas europaea is a multimer of an octa-heme subunit. J Biol Chem, 1993, 268(20): 14645-14654.
[43] Kurokawa M, Fukumori Y, Yamanaka T. A hydroxylamine- cytochrome C reductase occurs in the heterotrophic nitrifier Arthrobacter globiformis. Plant Cell Physiol, 1985, 26: 1438-1442.
[44] Moir JWB, Josa MW, Spiro S, Richardson D J. The biochemical characterization of a novel non-haem-iron hydroxylamine oxidase from Paracoccus denitrificans GB17. Biochem J, 1996, 319: 823-827.
[45] Bell LC, Richardson DJ, Ferguson SJ. Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha. The periplasmic enzyme catalyzes the first step in aerobic denitrification. FEBS Left, 1990, 265(1-2): 85-87.
[46] Bell LC, Page MD, Berks BC, Richardson DJ, Ferguson SJ. Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity, J Gen Microbiol. 1993, 139(Pt 12): 3205-3214.
[47] Denis K, Dias F M, Rowe J J. Oxygen regulation of nitrate transport by diversion of electron flow in Escherichia co11. J Biol Chem, 1990, 265: 18095-18097.
[48] Brons HJ, Zehnder AJ. Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4. Arch Microbiol, 1990, 153(6): 531-536.
[2] Rotthauwe JH, Deboer W, Liesack W. Comparative analysis of gene sequences encoding ammonia monooxygenase of Nitrosospira sp. AHB1 and Nitrosolobus multiformisC-71. FEMS Microbiol Lett, 1995, 133(1-2): 131-135.
[3] Sayavedra Soto LA, Hommes NG, Alzerreca JJ, Arp DJ, Norton JM, Klotz MG. Transcription of the amoC, amoA and amo B genes in Nitrosomonas europaea and Nitrosospira sp. NpAV. FEMS Microbiol Lett, 1998, 167: 81-88.
[4] Bonin P, Gilewicz M, Bertrand JC. Effects of oxygen on each step of denitrification on Pseudomonas nautical. Can J Microbiol, 1989, 35: 1061~1064.
[5] Shrestha NK, Hadano S, Kamachi T, Okura I.. Conversion of ammonia to dinitrogen in wastewater by Nitrosomonas europaeaa. Appl Biochem Biotechnol, 2001, 90: 221-232.
[6] Focht DD, Verstraete W. Biochemical ecology of nitrification and dinitrification. Adv Microbial Ecol, 1977, 1: 135-214.
[7] Mobarry BK, Wagner M, Urbain V, Rittmann BE, Stahl DA. Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl Environ Microbiol 1996, 62(6): 2156-2162.
[8] 郑兴灿，李亚新．污水除磷脱氮技术．北京：中国建筑工业出版社，1998．48-49．
[9] Allgeier RJ, Peterson WH, Juday C, Birge EA. The anaerobic fermentation of lake deposits. Int Rev Hydrobiol, 1932, 26: 444-461.
[10] Van Luijn F, Boers Paul CMA, Lambertus L. Anoxic N_2 fluxes from freshqater sedimentr in the absence of oxidized nitrogen compounds. Water Res, 1998, 32(2): 407-409.
[11] Lloyd JMA, Liehr SKA, Classen JJA, Robarge W. Mechanisms of dinitrogen gas formation in anaerobic lagoons. Adv Environ Res, 2000, 4(4): 133-139.
[12] Van de Graaf A A, Mulder A, de Bruijn P, Jetten MS, Roberson LA, Kuenen JG. Anaerobic oxidation of ammonium is a biologically mediated process. Appl Environ Microbiol, 1995, 61(4): 1246-1251.
[13] Van de Graaf AA, Mulder A, de Bruijn P, Jetten MS, Robertson LA, Kuenen JG. The SHARON-Anammox process for treatment of ammonium rich wastewater. War Sei Tech, 2001, 44(1): 153-160.
[14] Verstraete W1, Alexander M. Heterotrophic nitrification by Arthrobacter sp. J Bacteriol, 1972, 110(3): 955-961.
[15] Genevieve Mével, Prieur D. Heterotrophic nitrification by a thermophilic Bacillus species as influenced by different culture conditions. Can J Microbiol, 2000, 46(5): 465-473.
[16] Robertson LA, Kuenen JG. Thiosphaera pantotropha gen. nov. sp. nov., a faeultatively anaerobic, faeultatively autotrophie sulphur bacterium. J Gen Microbial, 1983, 129: 2847-2855.
[17] Su JJ, Liu BY, Liu CY. Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system. J Appl Microbiol, 2001, 90(3): 457-462.
[18] Robertson LA, Van Nell EWJ, Torremans, Rob AM, Kuenen JG. Simultaneous nitrification and denitrification in aerobic chemostat cultures of Thiosphaera pantotropha. Appl Envion Mierobiol, 1988, 54(1): 2812-2818.
[19] Van't Riet J, Stouthamer AH, Planta RJ. Regulation of nitrate assimilation and nitrate respiration in Aerobater aerogenes. Bacteriol, 1968, 96: 1455-1464.
[20] Rand MC, Greenberg AE, Taras MJ(ed.). Standard methods for the examination of water and wastewater. Washington, D. C. American Public Health association, 1975, 14th ed.: 434-436.
[21] 国家环保局《水和废水监测分析方法》编委会．水和废水监测分析方法．第三版．北京：中国环境科学出版社，1989．252-255．
[22] RE．布坎南，NE．吉本斯．伯杰细菌鉴定手册 第八版．北京：科学出版社，1984．中国科学院微生物研究所伯杰细菌鉴定手册翻译组译．
[23] 东秀珠，蔡妙英．《常见细菌系统鉴定手册》北京：科学出版社，2001．409-412．
[24] Poth M. Dinitrogen production from nitrite by a Nitromonas isolate. Appl Environ Microbiol, 1986, 52: 957-959.
[25] Mike SMJ, Logemann S, Muyzer G, Robertson LA, Simon de Vries, Mark CML, Kuenen JG. Novel principles in the microbial conversion of nitrogen compounds. Antinie van heeuwenhoek, 1997, 71: 75-93.
[26] 郑兴灿，李亚新．污水除磷脱氮技术．北京：中国建筑工业出版社，1998．45-46．
[27] Ebina J, Tsutsui T, Shriai T. Simultaneous determination of total nitrogen and total phosphorus in water using peroxodisulfate oxidation. Wat Res, 1983, 17(12): 1721-1726.
[28] Otte S, Grobben NG, Robertson LA, Jetten MS, Kuenen JG. Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions. Appl Environ Microbiol. 1996, 62(7): 2421-2426.
[29] Wehrfritz JM, Reilly A, Spiro S, Richardson DJ. Purification of hydroxylamine oxidase from Thiosphaera pantotropha. Identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification. FEBS Lett, 1993 Dec 6; 335(2): 246-250.
[30] Yamanaka T, Shinra M. Cytochrome c-552 and cytochrome c-554 derived from nitrosomonas europaea. J Biochem, 1974, 75: 1265-1273.
[31] Frear DS, Burrell RC. Spectrophotometric method for determining hydroxylamine reductase activity in higher plants. Anal Chem, 1955, 27(10): 1664-1665.
[32] van Niel EJW, Braber KJ, Robertson LA, Kuenen JG. Heterotrophic nitrification and aerobic denitrification in Alcaligenes faecalis strain TUD. Antonie van Leeuwenhoek, 1992, 62: 231-237.
[33] Mike SMJ, Peter de Bruijin, Kuenen JG. Hydroxylamine metablolism in pseudomonas PB16: involvement of a novel hydroxylamine oxidorductase. Antonie van Leeuwenhoek, 1997, 71: 69-74.
[34] Arts PAM, Robertson LA, Kuenen J G. Nitrification and denitrification by Thiosphaera pantotropha in aerobic batch and chemostat cultures. FEMS Microbiol Ecol 1996, 18: 305-315.
[35] Ohashi A, Viraj de Silva DG, Mobarry B, Manem JA, Stahl DA, and Rittmann BE. Influence of substrate C/N ratio on the structure of multi-species biofilms consisting of
nitrifiers and heterotrophs. Wat Sci Tech., 1996, 32: 75-84.
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