两株异养硝化菌的筛选鉴定及其脱氮性能研究
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摘要
水体富营养化是当今我国水环境面临的重大问题,严重地阻碍着我国国民经济的发展。目前生物脱氮已经成为废水中去除氮素污染的最有效的方法之一。传统的生物脱氮由于作用于各脱氮步骤中的不同菌群对于营养及溶解氧的要求各异,造成了生物脱氮工艺复杂,限制了在实际工程中的应用。异养硝化的研究,是对传统硝化理论的丰富与突破,为污水脱氮引入全新的概念。异养硝化生物脱氮技术因具有缩短脱氮历程、节省碳源、提高处理能力、降低动力消耗等优点而被广泛关注。
本课题从生物陶粒脱氮反应器中筛选得到异养硝化效果比较好的两株菌Z5和Z8。采用丁二酸钠-硫酸铵培养基培养细菌进行研究,经2d培养,两株异养硝花菌对氨氮的去除率在65%以上,对COD的去除率在40%以上。对其进行生理生化指标鉴定及16SrDNA序列分析,将其鉴定到属,可基本确定前者为假单胞菌属的绿脓杆菌Pseudomonas aeruginosa ,后者为不动菌属Acinetobacter sp ,其相似性分别达到了100%和99%。
对菌Z5和Z8进行进一步的氮素轨迹分析,其最适碳氮源分别为丁二酸钠和硫酸铵,在最适碳氮源条件下,菌株Z5和Z8对氨态氮的去除率可分别达到57.95%和63.86%,同时这两株菌对硝态氮的去除率分别达到了86.93%和89.57%,氨氮的初期利用是促进菌体生长的关键因素,并且菌Z5和Z8具有很强的反硝化功能。菌的生长过程中,亚硝态氮和硝态氮没有明显积累,对所筛异养硝化菌的同步脱氮机理进行初步探讨。
将所筛选的异养硝化细菌扩大培养后建立SBR反应器进行了氨氮去除的试验研究。在SBR反应器进入稳定运行阶段时,可以观察到系统对于氨氮的去除率稳定在82.96%左右。表现出较好的氨氮去除效果,出水亚硝酸盐含量一直维持在较低的水平,其最大值不超过3.84mg/L。COD的平均去除率为54.72%,基本实现了同一反应器中的有机物和氨氮的共同去除。
PCR-DGGE图谱分析表明,微生物多样性与废水的处理效果出现协同变化的特征,1d与15d微生物群落结构相似程度高为75%,1d与30d相似性最低为52.94%,15d与30d相似性为70.59%,所筛选两株异氧硝化细菌Z5, Z8成为SBR反应器的优势菌群。
The nitrogen pollution is the main reason causing water eutrophication, and the removal of nitrogen has become a hot topic in wasterwater treatment field as water eutrophication become even worse. Because of the different requirement of the different nitrifiers and denitrifiers for nourishment , the process of removal of nitrogen is very complex and the application is limited. The find of heterotrophic nitrification provides a new method for developing the application of the biological removal of nitrogen. Because of their advantages such as enhancing the treatment effect, reducing the area and reducing the treatment process flow etc, heterotrophic nitrification process has been widely put attentions.
Two strains of heterotrophic nitrifiers, named Z5 and Z8, is isolated from bio-ceramic reactor. The analysis based on 16SrDNA showed that the strains are the relative of Pseudomonas aeruginosa and Acinetobacter sp. with 100% and 99% sequence similarity respectively. Under the conditions, the ammonium removal rate reached 57.95% and 63.86% respectively. The initial removal of ammonium is the critical factor to the organism growth. Meanwhile, 86.93% and 89.57% of the NO3—N removal rate can be gained, which indicated that Z5 and Z8 possessed high effective denitrification. During the whole growth of Z5 and Z8, there is no nitrite and nitrate accumulation.
The process of removing ammoniac nitrogen was done by setting the heterotrophic nitrification bacteria on the sequencing batch reactor. And the efficiency of ammoniac nitrogen removal can reach up to 82.96%, when the reactor works smoothly. The nitrite concentration of effluent was in a low level and the maxiuma value was no more than 3.84mg/L. At the same time, the efficiency of COD was 54.72%, which presents the coinstantaneous removing of both organic and ammoniac nitrogen.
The PCR-DGGE profiles showed that shift of microbial diversity correspond to the effect of nitrate removal.1d and the 15d similarity is 75%, which microbial diversity is the highest. 1d and the 30d similarity is the lowest which is just 52.94%, 15d and the 30d.similarity is 70.59%. Z5, Z8 were dominated bacteria in sequencing batch reactor.
本课题从生物陶粒脱氮反应器中筛选得到异养硝化效果比较好的两株菌Z5和Z8。采用丁二酸钠-硫酸铵培养基培养细菌进行研究,经2d培养,两株异养硝花菌对氨氮的去除率在65%以上,对COD的去除率在40%以上。对其进行生理生化指标鉴定及16SrDNA序列分析,将其鉴定到属,可基本确定前者为假单胞菌属的绿脓杆菌Pseudomonas aeruginosa ,后者为不动菌属Acinetobacter sp ,其相似性分别达到了100%和99%。
对菌Z5和Z8进行进一步的氮素轨迹分析,其最适碳氮源分别为丁二酸钠和硫酸铵,在最适碳氮源条件下,菌株Z5和Z8对氨态氮的去除率可分别达到57.95%和63.86%,同时这两株菌对硝态氮的去除率分别达到了86.93%和89.57%,氨氮的初期利用是促进菌体生长的关键因素,并且菌Z5和Z8具有很强的反硝化功能。菌的生长过程中,亚硝态氮和硝态氮没有明显积累,对所筛异养硝化菌的同步脱氮机理进行初步探讨。
将所筛选的异养硝化细菌扩大培养后建立SBR反应器进行了氨氮去除的试验研究。在SBR反应器进入稳定运行阶段时,可以观察到系统对于氨氮的去除率稳定在82.96%左右。表现出较好的氨氮去除效果,出水亚硝酸盐含量一直维持在较低的水平,其最大值不超过3.84mg/L。COD的平均去除率为54.72%,基本实现了同一反应器中的有机物和氨氮的共同去除。
PCR-DGGE图谱分析表明,微生物多样性与废水的处理效果出现协同变化的特征,1d与15d微生物群落结构相似程度高为75%,1d与30d相似性最低为52.94%,15d与30d相似性为70.59%,所筛选两株异氧硝化细菌Z5, Z8成为SBR反应器的优势菌群。
The nitrogen pollution is the main reason causing water eutrophication, and the removal of nitrogen has become a hot topic in wasterwater treatment field as water eutrophication become even worse. Because of the different requirement of the different nitrifiers and denitrifiers for nourishment , the process of removal of nitrogen is very complex and the application is limited. The find of heterotrophic nitrification provides a new method for developing the application of the biological removal of nitrogen. Because of their advantages such as enhancing the treatment effect, reducing the area and reducing the treatment process flow etc, heterotrophic nitrification process has been widely put attentions.
Two strains of heterotrophic nitrifiers, named Z5 and Z8, is isolated from bio-ceramic reactor. The analysis based on 16SrDNA showed that the strains are the relative of Pseudomonas aeruginosa and Acinetobacter sp. with 100% and 99% sequence similarity respectively. Under the conditions, the ammonium removal rate reached 57.95% and 63.86% respectively. The initial removal of ammonium is the critical factor to the organism growth. Meanwhile, 86.93% and 89.57% of the NO3—N removal rate can be gained, which indicated that Z5 and Z8 possessed high effective denitrification. During the whole growth of Z5 and Z8, there is no nitrite and nitrate accumulation.
The process of removing ammoniac nitrogen was done by setting the heterotrophic nitrification bacteria on the sequencing batch reactor. And the efficiency of ammoniac nitrogen removal can reach up to 82.96%, when the reactor works smoothly. The nitrite concentration of effluent was in a low level and the maxiuma value was no more than 3.84mg/L. At the same time, the efficiency of COD was 54.72%, which presents the coinstantaneous removing of both organic and ammoniac nitrogen.
The PCR-DGGE profiles showed that shift of microbial diversity correspond to the effect of nitrate removal.1d and the 15d similarity is 75%, which microbial diversity is the highest. 1d and the 30d similarity is the lowest which is just 52.94%, 15d and the 30d.similarity is 70.59%. Z5, Z8 were dominated bacteria in sequencing batch reactor.
引文
1邓英淘.中国的水危机.生态经济, 2002(7):5-10
2孙锦宜.含氮废水处理技术与应用.北京:化学工业出版社, 2003:33-35
3郑平,徐向阳,胡宝兰.新型生物脱氮理论与技术.北京:科学出版社,2004:5-7
4王磊,杨海真.生物脱氮的同步硝化反硝化.机电设备,2002(1).;12～15
5 K Killham. Heterotrophic nitrification. J I Prosser. Nitrification. IRL Press, 1986;117～126
6 J P Schimel, M K Firestone. Identification of heterotrophic nitrification in a sierran forest soil. App lied and Environmental Microbiology, 1984, 48 (4) : 802～806
7 J A Adam s. Identification of heterotrophic nitrification in strongly acid larch humus. Soil Biol. Bio-chemistry. , 1986, 18 (3) : 339～342
8 J A Duggin, G K Voigt. Autotrophic and heterotrophic nitrification in response to clear-cutting northern hardwood forest. Soil Biol. Bio-chemistry , 1991, 23 (8) : 779～787
9杜睿.内蒙古典型草原土壤N2O产生的机理探讨.中国环境科学, 2000, 20 (5) : 387～391
10 D Castignetii, T C Hollocher. Heterotrophic nitrification among denitrifiers. Applied and Environmental Microbiology, 1984,47 (4); 620～623
11 L A Robertson, J G Kuenen, R Kleijntjens. Aerobic denitrification and heterotrophic nitrification by thiosphaera pantotropha. Antonie Van Leeuwenhoek, 1985, 51 (4) : 445
12 L A Robertson, J G Kuenen. Heterotrophic nitrification in thiosphaera pantotropha: oxygen up take and enzyme studies. J Gen. Microbiology. 1988, 134 (4) : 857～863
13 D Castignetii. Bio-energetic examination of the heterotrophic nitrifier-denitrifier thiosphaera pantotropha. Antonie Van L eeuwenhoek, 1990, 58 (4) : 283～289
14 J M Wehrfritz, A Reilly, S Spiro, et al. Purification of hydroxylamine oxidase from thiosphaera pantotropha. Identification of Electron Acceptors that Couple Heterotrophic Nitrification to Aerobic Denitrification, Febs.Lett.,1993, 335 (2) : 246～250
15李良谟.土壤硝化作用研究概况.土壤学进展.1984, (5): 1～10
16林燕,孔海南,何义亮等.异养硝化细菌的分离及其硝化特性实验研究.环境学.2006,27(2);324～328
17林燕,何义亮,李春杰等.MBR同步硝化反硝化及异养硝化试验研究.环境科学与技术.2006,29(1);7～-9
18李良漠.中国土壤氮素.南京:江苏科学出版社1992, 94～120
19 Moir JWB, Wehrfritz J-M, et al. The biochemical characterization of novel non-heam-iron hydroxylamine oxidase from Paracoccus denitrifications GB17. Biochemistry. J.1996b, 319:823~827
20 Hynes RK, Knowles R. Effect of acetylene on autotrophic and heterotrophic nitrification. Can. J. Microbiology. 1982, 28: 334~340
21 Wehrfritz J-M, Carter JP, et al. Hydroxylamine oxidation in heterotrophic nitrate-reducing soil bacteria and purification of ahydroxylamine-cytochrome coxidoreductase from a Pseudomonas species. Arch. Microbiol.1997,166:421~424
22 Yamanaka T. Mechanisms of oxidation of inorganic electron donors in autotrophic bacteria. Plant Cell Physiology. 1996, 37:569~574
23 Ono Y, Makino N, et al. An iron dioxygenase from Alaligenes faecalis catalyzing the oxidation of pyruvic oxime to nitrite. FEMS Microbiol.Lett.1996,139:103~108
24 Geraats S GM ,Hooijmans C M ,Van Nicl ,et al.The use of a metabolically structured model in the study of growth , nitrification ,and denitrification by Thiosphaera pantotropha [J ]1Biotechnol Bio-eng.1990 ,36 :921~930.
25 Brierley E D R , Wood M. Heterotrophic nitrification in an acid forest soil; isolation and characterization of a nitrifying bacterium[J].Soil Biology &Biochemistry .2001,33 :1403~1409.
26 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.
27 Bock E , Schmidt I1Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonium or hydrogen as electron donors and nitrite as electron acceptor[J].Arch Microbiology .1995 ,163 :16~20.
28吕锡武,李峰,稻森悠平等.氨氮废水处理过程中的好氧反硝化研究[J].中国给水排水,2000 ,26(4) :17~20.
29 Patureau D, Helloin E1Combined phosphate and nitrogen removal in a sequencing batch reactor using the aerobic denitrifier[J].Micro virgule aero denitrifications. Wat Res,2000 ,35(1) :189~197.
30 Vidal S ,Rocha C , Galvao H. A comparison of organic and inorganic carbon controls over biological denitrification in aquaria [J]. Chemosphere,2002 ,48 :445~451.
31 Koeing A,Liu L H. Autotrophic denitrification of landfill leachate using elemental sulphur [J]. Wat Sci Tech .1996 ,34(526) :469~476.
32 Lesley A.Robertson&J.Gijs Kuenen. Combined heterotrophic nitrification and aerobic denitrification in Thiosphaera pantotropha and other bacteria. Antonie van Leeuwenhoek.1990, 57: 139~152.
33 E.W.J.van Niel, K.J.Braber, L.A. Robertson. Heterotrophic nitrification and aerobic denitrification in Alcaligenes faecalis. Antonie van Leeuwenhoek. 1992, 62: 231~237.
34 L.A.ROBERTSON, R.CORNELISSE, P.DE VOS, R.HADIOETOMO&J.G.KUENEN. Aerobic denitrification in various heterotrophic nitrifiers. Antonie van Leeuwenhoek.1989, 56: 289~299.
35林燕,孔海南,何义亮.异养硝化细菌的分离及其硝化特性试验研究[J].环境科学. 2006,27(2):324~328.
36张光亚,陈美,闵航.一株异养硝化细菌的分离及系统发育分析[J].微生物学报, 2003,43(2):156~161.
37 Kennish M J . Environmental threat s and environmental future of estuaries. [J] Environ. Conserv . 2002 , 29 :78-107
38 Rabalais N N , Turner R E , Scavia D. Beyond science into policy : Gulf of Mexico hypoxia and the Mississippi River [J] Bio- Science.2002 , 52 :129-142
39刘志培,贾省芬,俞志明等.新型异养氨氧化菌的分离鉴定及氨氧化特性环境污染与防治[J]. 2005,27(5):337-340
40 Papen H, von Berg 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
41 Stroo HF, Klein TM, et al. Heterotrophic Nitrification in an acid forest soil and by an acid-tolerant Fungus [J]. Appl Environ. Microbiology.. 1986, 52:1107~1111
42 Brierley EDR, Wood M. Heterotrophic nitrification in an acid forest soil: Isolation and characterization of a nitrifying bacterium [J]. Soil Biol. Biochemistry. 2001, 33:1403~1409
43 Verstraete W, Alexander M. Heterotrophic nitrification by Arthrobacter sp. [J]. Bacteriology .1972, 110:955~961
44 Robertson LA, Kuenen JG. Thiosphaera pantotropha : a facultatively anaerobic, facultatively autotrophic sulphur bacterium [J]. Gen. Microbiology .1983, 129: 2847~2855
45 Witzel K-P, Overbeck H J. Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions, growth state and acetate metabolism [J].Arch. Microbiology. 1979, 122:137~143
46 Mevel G, Prieur D. Thermophilic Heterotrophic nitrifiers isolated from Mid-Atlantic Ridge deep-sea hydrothermal vents. Can. [J]. Microbiology. 1998, 44:723~733
47 Lin Y, Kong H N , He Y L , et al . Simultaneous Nitrification and Denitrification in a Membrane Bioreactor and Isolation of heterotrophic Nitrifying Bacteria [J],Japanese Journal of Water Treatment Biology .2004 ,40 (3) : 105～114.
2孙锦宜.含氮废水处理技术与应用.北京:化学工业出版社, 2003:33-35
3郑平,徐向阳,胡宝兰.新型生物脱氮理论与技术.北京:科学出版社,2004:5-7
4王磊,杨海真.生物脱氮的同步硝化反硝化.机电设备,2002(1).;12～15
5 K Killham. Heterotrophic nitrification. J I Prosser. Nitrification. IRL Press, 1986;117～126
6 J P Schimel, M K Firestone. Identification of heterotrophic nitrification in a sierran forest soil. App lied and Environmental Microbiology, 1984, 48 (4) : 802～806
7 J A Adam s. Identification of heterotrophic nitrification in strongly acid larch humus. Soil Biol. Bio-chemistry. , 1986, 18 (3) : 339～342
8 J A Duggin, G K Voigt. Autotrophic and heterotrophic nitrification in response to clear-cutting northern hardwood forest. Soil Biol. Bio-chemistry , 1991, 23 (8) : 779～787
9杜睿.内蒙古典型草原土壤N2O产生的机理探讨.中国环境科学, 2000, 20 (5) : 387～391
10 D Castignetii, T C Hollocher. Heterotrophic nitrification among denitrifiers. Applied and Environmental Microbiology, 1984,47 (4); 620～623
11 L A Robertson, J G Kuenen, R Kleijntjens. Aerobic denitrification and heterotrophic nitrification by thiosphaera pantotropha. Antonie Van Leeuwenhoek, 1985, 51 (4) : 445
12 L A Robertson, J G Kuenen. Heterotrophic nitrification in thiosphaera pantotropha: oxygen up take and enzyme studies. J Gen. Microbiology. 1988, 134 (4) : 857～863
13 D Castignetii. Bio-energetic examination of the heterotrophic nitrifier-denitrifier thiosphaera pantotropha. Antonie Van L eeuwenhoek, 1990, 58 (4) : 283～289
14 J M Wehrfritz, A Reilly, S Spiro, et al. Purification of hydroxylamine oxidase from thiosphaera pantotropha. Identification of Electron Acceptors that Couple Heterotrophic Nitrification to Aerobic Denitrification, Febs.Lett.,1993, 335 (2) : 246～250
15李良谟.土壤硝化作用研究概况.土壤学进展.1984, (5): 1～10
16林燕,孔海南,何义亮等.异养硝化细菌的分离及其硝化特性实验研究.环境学.2006,27(2);324～328
17林燕,何义亮,李春杰等.MBR同步硝化反硝化及异养硝化试验研究.环境科学与技术.2006,29(1);7～-9
18李良漠.中国土壤氮素.南京:江苏科学出版社1992, 94～120
19 Moir JWB, Wehrfritz J-M, et al. The biochemical characterization of novel non-heam-iron hydroxylamine oxidase from Paracoccus denitrifications GB17. Biochemistry. J.1996b, 319:823~827
20 Hynes RK, Knowles R. Effect of acetylene on autotrophic and heterotrophic nitrification. Can. J. Microbiology. 1982, 28: 334~340
21 Wehrfritz J-M, Carter JP, et al. Hydroxylamine oxidation in heterotrophic nitrate-reducing soil bacteria and purification of ahydroxylamine-cytochrome coxidoreductase from a Pseudomonas species. Arch. Microbiol.1997,166:421~424
22 Yamanaka T. Mechanisms of oxidation of inorganic electron donors in autotrophic bacteria. Plant Cell Physiology. 1996, 37:569~574
23 Ono Y, Makino N, et al. An iron dioxygenase from Alaligenes faecalis catalyzing the oxidation of pyruvic oxime to nitrite. FEMS Microbiol.Lett.1996,139:103~108
24 Geraats S GM ,Hooijmans C M ,Van Nicl ,et al.The use of a metabolically structured model in the study of growth , nitrification ,and denitrification by Thiosphaera pantotropha [J ]1Biotechnol Bio-eng.1990 ,36 :921~930.
25 Brierley E D R , Wood M. Heterotrophic nitrification in an acid forest soil; isolation and characterization of a nitrifying bacterium[J].Soil Biology &Biochemistry .2001,33 :1403~1409.
26 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.
27 Bock E , Schmidt I1Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonium or hydrogen as electron donors and nitrite as electron acceptor[J].Arch Microbiology .1995 ,163 :16~20.
28吕锡武,李峰,稻森悠平等.氨氮废水处理过程中的好氧反硝化研究[J].中国给水排水,2000 ,26(4) :17~20.
29 Patureau D, Helloin E1Combined phosphate and nitrogen removal in a sequencing batch reactor using the aerobic denitrifier[J].Micro virgule aero denitrifications. Wat Res,2000 ,35(1) :189~197.
30 Vidal S ,Rocha C , Galvao H. A comparison of organic and inorganic carbon controls over biological denitrification in aquaria [J]. Chemosphere,2002 ,48 :445~451.
31 Koeing A,Liu L H. Autotrophic denitrification of landfill leachate using elemental sulphur [J]. Wat Sci Tech .1996 ,34(526) :469~476.
32 Lesley A.Robertson&J.Gijs Kuenen. Combined heterotrophic nitrification and aerobic denitrification in Thiosphaera pantotropha and other bacteria. Antonie van Leeuwenhoek.1990, 57: 139~152.
33 E.W.J.van Niel, K.J.Braber, L.A. Robertson. Heterotrophic nitrification and aerobic denitrification in Alcaligenes faecalis. Antonie van Leeuwenhoek. 1992, 62: 231~237.
34 L.A.ROBERTSON, R.CORNELISSE, P.DE VOS, R.HADIOETOMO&J.G.KUENEN. Aerobic denitrification in various heterotrophic nitrifiers. Antonie van Leeuwenhoek.1989, 56: 289~299.
35林燕,孔海南,何义亮.异养硝化细菌的分离及其硝化特性试验研究[J].环境科学. 2006,27(2):324~328.
36张光亚,陈美,闵航.一株异养硝化细菌的分离及系统发育分析[J].微生物学报, 2003,43(2):156~161.
37 Kennish M J . Environmental threat s and environmental future of estuaries. [J] Environ. Conserv . 2002 , 29 :78-107
38 Rabalais N N , Turner R E , Scavia D. Beyond science into policy : Gulf of Mexico hypoxia and the Mississippi River [J] Bio- Science.2002 , 52 :129-142
39刘志培,贾省芬,俞志明等.新型异养氨氧化菌的分离鉴定及氨氧化特性环境污染与防治[J]. 2005,27(5):337-340
40 Papen H, von Berg 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
41 Stroo HF, Klein TM, et al. Heterotrophic Nitrification in an acid forest soil and by an acid-tolerant Fungus [J]. Appl Environ. Microbiology.. 1986, 52:1107~1111
42 Brierley EDR, Wood M. Heterotrophic nitrification in an acid forest soil: Isolation and characterization of a nitrifying bacterium [J]. Soil Biol. Biochemistry. 2001, 33:1403~1409
43 Verstraete W, Alexander M. Heterotrophic nitrification by Arthrobacter sp. [J]. Bacteriology .1972, 110:955~961
44 Robertson LA, Kuenen JG. Thiosphaera pantotropha : a facultatively anaerobic, facultatively autotrophic sulphur bacterium [J]. Gen. Microbiology .1983, 129: 2847~2855
45 Witzel K-P, Overbeck H J. Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions, growth state and acetate metabolism [J].Arch. Microbiology. 1979, 122:137~143
46 Mevel G, Prieur D. Thermophilic Heterotrophic nitrifiers isolated from Mid-Atlantic Ridge deep-sea hydrothermal vents. Can. [J]. Microbiology. 1998, 44:723~733
47 Lin Y, Kong H N , He Y L , et al . Simultaneous Nitrification and Denitrification in a Membrane Bioreactor and Isolation of heterotrophic Nitrifying Bacteria [J],Japanese Journal of Water Treatment Biology .2004 ,40 (3) : 105～114.