沼泽红假单胞菌（NS-04）的分离、鉴定及其在暗纹东方鲀养殖上的应用
|
摘要
本文以从水塘污泥中分离出一株红假单胞菌(Rhodopseudomonas)NS-04菌株为材料，对它进行了初步的菌种鉴定、营养成分分析、发酵条件的优化，并研究了不同浓度的NS-04菌株培养液在暗纹东方鲍养殖中的作用。
通过富集培养水塘污泥筛选光合细菌，经电镜观察形态学特征、活细胞扫描以及碳源利用试验了解菌株的生理生化特征，并通过16S rRNA的同源性比较，了解序列保守性。结果表明NS-04菌株革兰氏反应阴性，单个细胞为杆状至卵形，菌体大小为0.6-0.9×1.2-2.0μm，二分分裂，极生或亚极生鞭毛，片层状光合内膜，位于细胞质膜之下并与之平行，菌落为棕红色，光照厌氧和黑暗好氧条件下均可生长，菌体富含类胡萝卜素和细菌叶绿素a，能利用多种碳源，酵母膏对其生长有明显刺激作用，16S rRNA基因的分子系统学分析表明，该菌株与GenBank中的沼泽红假单胞菌菌株的同源性为97％，初步确定筛选出的NS-04菌株为沼泽红假单胞菌(Rhodopseudomonas palustris)。
通过对菌株的营养成分的分析可知，蛋白质、脂肪、灰分、可溶性糖类分别占细胞干重的61.24％、8.11％、7.5％和5.73％，类胡萝卜素占细胞干重的4.24mg／g，富含维生素B_2、B_6和烟酸，氨基酸比值达92.38％，必需氨基酸总含量达40.92％，必需氨基酸与非必需氨基酸比值为79.52％，符合FAO／WHO对饲料的理想模式，是一种比较优质的饲料蛋白源。
菌株的发酵条件优化试验表明，比较理想的发酵培养基配方(g／L)为：黄豆芽100，苹果酸1.5，乙酸钠2，微量元素1，pH 7.0。最适发酵条件；接种量1％，装液量为80mL／250mL，40001x光照下，30℃好氧培养36h后转入厌氧培养24h。在此条件下生物量的活菌数可达到1.4×10~9cfu／mL，符合农业部的标准。
将光合细菌应用于特种水产养殖上，初步研究了施加不同浓度光合细菌在暗纹东方鲀养殖中的效果。试验表明添加光合细菌浓度在50μg／L以上时能有效的改善养殖水环境，使DO浓度增加，COD、BOD_5、氨态氮、亚
南京师范大学20(冈届硕士学位论文
沼泽红假单胞菌(NS一04)的分离、鉴定及其在暗纹东方纯养殖上的应用研究
硝基盐浓度下降并保持在正常范围，与不投加光合细菌的对照池相比，鱼
苗成活率增加，体长体重增加，减轻了病原微生物的感染。
This article elaborately studied the identification, analysis and evaluation of nutrient components the optimum fermentation of Photosynthetic bacteria NS-04 which isolated from the sludge of the pond in Nanjing. In addition, the effect of different concentrations of strain NS-04 on the aquaculture on Takif u gu obscurus is also studied .
Photosynthetic bacteria were screened by enrichment culture from the sludge. Transmission electron microstcope, tests of using different carbon sources of PSB and of absorption of live cell suspension were used to see the biological character of the PSB. In addition, we also analysed the sequence of 16S rRNA of the PSB to know the homology. The strain NS-04 screened from the sludge is a Gram-negative bacterium. Cell is ovoid or rod in shape, 0.6-0.9 um wide and 1.2-2.0 um long, multriplication by binary fission. The photosynthetic membrance was lamellar type, which is under and parallel to intro-cytoplasmic membrance. The clone appeared brown with major pigments being Bchll a and carotenoids. PSB NS-04 can grow both under anaerobic condition in the light and aerobic condition in the dark. The growth of cells can also prompted by yeast extract. Phylotenetic tree of 16S rRNA sequences indicated that the similarity of strain NS-04 and standard bacterium of Rhodopseudomonas palustris in GenBank is 97%. According
to Bergey's Manual of Systematic Bacteriology, strain NS-04 was identified as R. palustris.
The nutrient components of strain NS-04 were analysised. The result indicated that the content of protein, crude fat, ash and soluble carbohydrate in dried bacteria were 61.24%, 8.11%, 7.5% and 5.73% respectively. The content of carotenoids and amino acid was 4.24mg/g and 92.38%. In addition VitaminB2 VitaminB6 and nicotinic acid were abound in PSB. In all, the R.palustris is a good resource of protein in feedingstuff industry.
To obtain the high level of cfu/mL, the media components and cultivation
conditions were investigated. The tests showed the optimum fermentation medium was consisted of soybean sprout 100, malic acid 1.5, NaAc 2, trace elements 1, pH 7.0. The incubata condition is: inoculation 1%, 80mL/250mL flask, 40001x, 30C in aerobiotic condition for 36h and then in anaerobic condition for 24h In this condition the titre was 1.4 X 10 cfu/mL and it measured up to the national standard of PSB.
As far as the effect of the PSB on the aquiculture the effect of different concentrations of PSB on the Takifu.gu obscurus were studied. The experiment showed that the PSB can improve the water quality and make the numerical value or the concentration of DO, COD, BOD,N-NH3 and NO2 decrease when that of the PSB is over 50 u g/L.And the livability, weight and incidence of a disease decreased compared with the control.
通过富集培养水塘污泥筛选光合细菌，经电镜观察形态学特征、活细胞扫描以及碳源利用试验了解菌株的生理生化特征，并通过16S rRNA的同源性比较，了解序列保守性。结果表明NS-04菌株革兰氏反应阴性，单个细胞为杆状至卵形，菌体大小为0.6-0.9×1.2-2.0μm，二分分裂，极生或亚极生鞭毛，片层状光合内膜，位于细胞质膜之下并与之平行，菌落为棕红色，光照厌氧和黑暗好氧条件下均可生长，菌体富含类胡萝卜素和细菌叶绿素a，能利用多种碳源，酵母膏对其生长有明显刺激作用，16S rRNA基因的分子系统学分析表明，该菌株与GenBank中的沼泽红假单胞菌菌株的同源性为97％，初步确定筛选出的NS-04菌株为沼泽红假单胞菌(Rhodopseudomonas palustris)。
通过对菌株的营养成分的分析可知，蛋白质、脂肪、灰分、可溶性糖类分别占细胞干重的61.24％、8.11％、7.5％和5.73％，类胡萝卜素占细胞干重的4.24mg／g，富含维生素B_2、B_6和烟酸，氨基酸比值达92.38％，必需氨基酸总含量达40.92％，必需氨基酸与非必需氨基酸比值为79.52％，符合FAO／WHO对饲料的理想模式，是一种比较优质的饲料蛋白源。
菌株的发酵条件优化试验表明，比较理想的发酵培养基配方(g／L)为：黄豆芽100，苹果酸1.5，乙酸钠2，微量元素1，pH 7.0。最适发酵条件；接种量1％，装液量为80mL／250mL，40001x光照下，30℃好氧培养36h后转入厌氧培养24h。在此条件下生物量的活菌数可达到1.4×10~9cfu／mL，符合农业部的标准。
将光合细菌应用于特种水产养殖上，初步研究了施加不同浓度光合细菌在暗纹东方鲀养殖中的效果。试验表明添加光合细菌浓度在50μg／L以上时能有效的改善养殖水环境，使DO浓度增加，COD、BOD_5、氨态氮、亚
南京师范大学20(冈届硕士学位论文
沼泽红假单胞菌(NS一04)的分离、鉴定及其在暗纹东方纯养殖上的应用研究
硝基盐浓度下降并保持在正常范围，与不投加光合细菌的对照池相比，鱼
苗成活率增加，体长体重增加，减轻了病原微生物的感染。
This article elaborately studied the identification, analysis and evaluation of nutrient components the optimum fermentation of Photosynthetic bacteria NS-04 which isolated from the sludge of the pond in Nanjing. In addition, the effect of different concentrations of strain NS-04 on the aquaculture on Takif u gu obscurus is also studied .
Photosynthetic bacteria were screened by enrichment culture from the sludge. Transmission electron microstcope, tests of using different carbon sources of PSB and of absorption of live cell suspension were used to see the biological character of the PSB. In addition, we also analysed the sequence of 16S rRNA of the PSB to know the homology. The strain NS-04 screened from the sludge is a Gram-negative bacterium. Cell is ovoid or rod in shape, 0.6-0.9 um wide and 1.2-2.0 um long, multriplication by binary fission. The photosynthetic membrance was lamellar type, which is under and parallel to intro-cytoplasmic membrance. The clone appeared brown with major pigments being Bchll a and carotenoids. PSB NS-04 can grow both under anaerobic condition in the light and aerobic condition in the dark. The growth of cells can also prompted by yeast extract. Phylotenetic tree of 16S rRNA sequences indicated that the similarity of strain NS-04 and standard bacterium of Rhodopseudomonas palustris in GenBank is 97%. According
to Bergey's Manual of Systematic Bacteriology, strain NS-04 was identified as R. palustris.
The nutrient components of strain NS-04 were analysised. The result indicated that the content of protein, crude fat, ash and soluble carbohydrate in dried bacteria were 61.24%, 8.11%, 7.5% and 5.73% respectively. The content of carotenoids and amino acid was 4.24mg/g and 92.38%. In addition VitaminB2 VitaminB6 and nicotinic acid were abound in PSB. In all, the R.palustris is a good resource of protein in feedingstuff industry.
To obtain the high level of cfu/mL, the media components and cultivation
conditions were investigated. The tests showed the optimum fermentation medium was consisted of soybean sprout 100, malic acid 1.5, NaAc 2, trace elements 1, pH 7.0. The incubata condition is: inoculation 1%, 80mL/250mL flask, 40001x, 30C in aerobiotic condition for 36h and then in anaerobic condition for 24h In this condition the titre was 1.4 X 10 cfu/mL and it measured up to the national standard of PSB.
As far as the effect of the PSB on the aquiculture the effect of different concentrations of PSB on the Takifu.gu obscurus were studied. The experiment showed that the PSB can improve the water quality and make the numerical value or the concentration of DO, COD, BOD,N-NH3 and NO2 decrease when that of the PSB is over 50 u g/L.And the livability, weight and incidence of a disease decreased compared with the control.
引文
[1] Nagadomi Hisashi, Kitamura Tomohiro, Watanabe Masanori, et al. Simultaneous removal of chemical oxygen demand(COD), phosphate, nitrate and H2S in the synthetic swage wastewater using porous ceramic immobilized photosynthetic bacteria[J]. Biotechnology Letters, 2000,22(17): 1369-1374
[2] Nagadomi H, Takahasi T, Saski K, et al. Simultaneous removal of chemical oxygen demand and nitrate in aerobic treatment of sewage wastewater using an immobilized photosynthetic bacterium of porous ceramic plates[J] World Journal of Microbiology and Biotechnology,2000,16(1): 57-62
[3] 小林正泰 发酵工業 36(9)，753
[4] Kodayasi M. Utilization and disposal of water by photosynthetic bacteria[J]Peramanpress ,1997,39-45
[5] Sawada H. Photosynthetic bacteria in waste treatment[J] Ferment Technol, 1997,55:311-316
[6] Tao Ho Lee, Ji-young Park, Sunghoon Park et al. Growth of Rhodopseudomonas palustris under phototrophic and non-phototrophic conditions and its CO-dependent H2 production[J] Biotechnology Letters, 2002,24:91-96
[7] Fedorov A.S, Tsygankov A.A, Rao K.K et al. Hydrogen photoproduction by Rhodobacter sphaeroides immobilized on polyurethane foam[J] Biotechnology letters, 1998,20(11):1007-1009
[8] Lee Chimei, Chen Peichung, Wang Chunchin et al. Photohydrogen production using purple nonsulfur bacteria with hydrogen fermentation reactor effluent[J] Intenational Journal of Hydrogen Energy, 2002,27:1309-1313
[9] Gest H, Ormerod JG, Ormerod KS. Photomitabolism of R. rubrum light-dependent dismimilation of organic compounds to carbon dioxide and molecular hydrogen by an anaerobic critric acid cycle[J]Arch
Biochem Biophys, 1962,97:21
[10] Ike A, Saimura C, Hirata K, et al. Hydrogen photo-production form CO2-fixing microalgal biomass: application of halo-torelant photosynthetic bacteria[J] J Ferment Bioeng, 1997,84:606-609
[11] Wakayama T, Asada Y, Miyake J. Effect of light/dark cycle on baterial hydrogen production by Rhodobacter sphaeroides RV from hour to second range[J] Appiled Biochem Biotech, 2000,84-86:431-440
[12] Toda, Yoshimoto, Kudo. Purple photosynthetic bacteria and health food. Patent: EP1172434AI 020116(Basic) WO 20016077 010823
[13] Yoshida H et al. Journal of general and applied microbiology. 1998,44(1):19-26
[14] Weaver PF. Patent:CA2075161 A 1993,2,3;US 7739368 A 1994,3,15
[15] Hisashi Nagadomi, Takako Hiromitsu, Kenji Takcno. Treatment of aquaculture water by denitrifying photosynthetic bacteia using immobilized Polyvinyl Alcohol beads[J]Journal of Bioscience and Bioengineering, 1999,87(2):189-193
[16] 小川静夫，光合细菌在养殖上的应用[J]，养殖，1985，(5)：66-68
[17] 刘如林、刁虎欣、梁凤来、赵大健等，光合细菌及其应用[M]，中国农业科技出版社，1991，北京
[18] Jhon G. Holt, Noel R. Kieg, et al. Bergey's manual of determination bacteriology[M], 9th Edition, Willimas & wilkings, Baltimore London, 1992
[19] Pereg-Gerk Lily, Sar Nehemia, hipkin Yaakov. In situ nitrogen fixation associated with seagrasses in the Gulf of Elat (Red Sea)[J], 2002,36(3):387-394
[20] Ludden Paul W. Roberts Gary P. Nitrogen fixation by photosynthetic bacteria[J], 2002,73(1/3):115-118
[21] Zbigniew S. Kolber, F. gerald PlumLey, Andrew S. Lang, et al. Contribution of Aerobic Photoheterotrophic Bacteria to the Carbon Cycle in the Ocean[J] Science, 2001, 292:2492-2495
[22] Chalam A V et al. Bull. Environ. Contam. Toxicol. 1997, 58(3):463-468
[23] Stonell M H B, Mcphillips T M, Rees D C, et al. Lighet induced structural changes and the mechanism of electron/proton tranfer in the photosynthetic reaction center[J] Science, 1997,276:812-816
[24] Abrensch E C, Paddock M L, Stonell M H B. Identifiction of proton transfer pathways in the Ⅹ-ray crystal structure of the bacterial reaction center from R. sphareoides[J]. Photosyn Res, 1998,55:119
[25] 张纯喜、樊红军、李良璧、匡廷云等，光合细菌原初反应的理论研究[J]，中国科学(C辑)，1999，29(3)276-282
[26] 光合作用高效光能转化的机理及其在农业中的应用[J]，中国科学院院刊—国家重点基础研究发展规划项目，2002，1：43-44
[27] Ludden PW et al. Science 1976, 194-424
[28] 吴永强、末鸿遇等，光合细菌固氮分子生物学研究进展[J]，植物生理学通讯，1991，27(3)：161-166
[29] Yasuhiro Oda, Simen-Jan Slagman, Wim G Meijer, et al. Influence of growth rate and starvation on luoreseent in situ hubridization of Rbodopseudomonas palustris[J]. FEMS Microbiology Ecology, 2000,32(3):205-213
[30] Chemical & Engineering News, 2001,8,6,32
[31] 杨惠芳，黄志勇，张德民，刘志恒等，紫色非硫光合细菌的空间生物学效应[J]，航天医学与医学工程，1999，12(1)：46-50
[32] 刘中等，光合细菌在淡水养殖中的应用研究[J]，水产科学，1995，14(1)：13-17
[33] 黄钧、韦勇刚、周毅、柯永恩、李志文等，在饲料中添加光合细菌饲养月鳢鱼种的初步试验[J]，广西畜牧兽医，2000，16(1)：17-18
[34] 陈德华等，光合细菌添加饲料养鸡蛋试验[J]，饲料工业，1994，15(5)：11-12
[35] 田维熙、王叶、赵荣芝、杨宪忠等，光合细菌在奶牛、肉牛饲养中应用的试验[J]，中国乳业，2001，6：14-15
[36] 夏宏、夏青、夏成敏、张清元等，光合细菌在早熟甘蓝、油菜上的应用研究[J]，山西农业大学学报，2000，39(2)，116-118
[37] 王晖等，光合细菌肥料对植物的影响[J]，山西教育学院学报，2000，3(3)：36-38
[38] 李俊峰，王梦亮等，光合细菌对农田生态系统的影响[J]，山西农业科学，2002，30(1)：52～56
[39] 徐亚同、史家梁、张明等，污染控制微生物王程[M]，化学工业出版社，2001，北京
[40] Khanna P et al. Current Microbiol. 1992,25:63-67
[41] Caroline S Harwood, Gerhard Burchhardt, Herrmanm, et al. Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway[J] FEMS Microbiology Reviews, 1999,22(5):439-458
[42] Kroomeman J et al. Compostsience & utiligation. 1998, 6(3):42-52
[43] 周洪茂、赵肖为、吴雪昌等，光和细菌沼泽红假单胞菌同化磷能力的研究[J]，科技通报，2002，18(2)：142-146
[44] 刘双江、杨惠芳、周培瑾、孙燕、岑运华等，固定化光合细菌处理豆制品废水产氢研究[J]，环境科学，1995，15(1)：42-44
[45] Joong Kyun Kim, Bum-Kyu Lee, Sang-Hee Kim, et al. A characterization of denitrifying photosynthetic bacteria isolated from photosynthetic sludge[J]. Aquacultural Engineering, 1999,19(3):179-193
[46] 牟海津、王宇新、钱新民等，光合细菌类胡萝卜素的研究[J]，海洋湖沼通报，1998，4：32-38
[47] 刘春朝等，光合细菌发酵产类胡萝卜素的中试研究[J]，食品与发酵工业，1994，6：47-49
[48] 曾宇、秦松、梁明山等，光合细菌综合应用新进展[J]，水产科学，2000，19(5)：34-36
[49] 焦瑞身、周德庆等，微生物生理代谢实验技术[M]，北京，科学出版社，1990
[50] Marmur J. J Mol Biol, 1961,3:208～218.
[51] 林万明，细菌分子遗传学分类鉴定法[M]，上海，上海科学技术出版社，1990，86～92
[52] 张德民、黄志勇、杨惠芳等，几株红假单胞菌属细菌的表观特征及其遗传多样性研究[J]，微生物学报，2000，40(3)：229-236
[53] 张德民，黄志勇，杨惠芳等，紫色非硫细菌Rhodocista属一新分离株的鉴定及其系统学微生物学报；2000，40(1)：14～20．
[54] Thomas Hurek, Blanca Wagner&Barbara Reinhold-Hurek, Applied And Environmental Microbiology, 1997, 63(11): 4331-4339
[55] Stackebrandt E、Gobel B、M Taxonomic note:A place for DNA-DNA reassociation And 16S rRNA sequence analysis in the present species definition in bacteriology, Int J syst bacterial, 1994,44:846-849
[56] 黄伟坤，食品检验与分析[M]，北京，中国轻工业出版社，2000，18-103
[57] 杨祖英，马永健，常凤启等，食品检验[M]，北京，化学工业出版社，2001，40-42
[58] 张龙翔，生化实验方法和技术[M]北京，高等教育出版社，1985，7-10，55-59
[59] Gyoo Yeol Jung, Hyun Ok Jung, Jung Rae Kim et al, Isolation and characterization of Rhodopseudomonas palustris P_4 which utilizes CO with the production of H_2
[60] 方益舫、俞吉安、林志新等，PSBAD菌体蛋白生物效应评价[J]，上海交通大学学报，1991：25(5)：65-67
[61] 张新英、莫天砚，红螺菌的细胞成分分析及在水产养殖上的初步应用试验[J]，广西畜牧兽医，2000，16(6)：8-10
[62] 范文询、李泽英、赵熙和等，蛋白质食物的营养评价[M]北京，人民出版社，1984，7
[63] 金龙飞等，食品与营养学[M]，北京，中国轻工业出版社，1999，68
[64] 刘勇、张德咏，光合细菌PSB-1菌株的分离鉴定及其生物学特征的研究[J]，生命科学研究，2000，4(4)：334
[65] 樊凌雯、张肇铭、张德咏等，利用光合细菌处理糖蜜酒精发酵废液中
试研究[J] 中国环境科学，1998，18(2)：173～175
[66] 闰琦涛、王维坚、韩克文等，玉米淀粉黄浆水PSB法资源化开发的应用前景[J] 吉林粮食高等专科学校学报，2000，15(4)：18-21
[67] Isamu Maeda, Tadashi Mizoguchi, Youshiharu Miura, et al. Influence of sulfate reducingbacteria on outdoorhydrogen production by photosyn the ticbacterium with seawater [J] Current Microbiology, 2001, 40(3):210-213.
[68] 李勤生，王业勤．水产养殖与微生物[M]．武汉：武汉出版社，2000，53-133
[69] 张道南，孙其焕，陈乃松等，红螺菌科光合细菌的分离、培养及其作为鱼虾类饵料添加剂的初步研究[J]．水产学报，1988，12(4)：367-369
[70] 国家环保局、《水和废水检测分析方法》编委会等，水和废水监测分析方法(第三版)[M]．北京：中国环境科学出版社，1989，246-366
[71] 罗世忠、沈纬，废水中痕量亚硝酸盐的光度法测定[J]．青岛化工学院学报，1997，18(1)：63-66
[2] Nagadomi H, Takahasi T, Saski K, et al. Simultaneous removal of chemical oxygen demand and nitrate in aerobic treatment of sewage wastewater using an immobilized photosynthetic bacterium of porous ceramic plates[J] World Journal of Microbiology and Biotechnology,2000,16(1): 57-62
[3] 小林正泰 发酵工業 36(9)，753
[4] Kodayasi M. Utilization and disposal of water by photosynthetic bacteria[J]Peramanpress ,1997,39-45
[5] Sawada H. Photosynthetic bacteria in waste treatment[J] Ferment Technol, 1997,55:311-316
[6] Tao Ho Lee, Ji-young Park, Sunghoon Park et al. Growth of Rhodopseudomonas palustris under phototrophic and non-phototrophic conditions and its CO-dependent H2 production[J] Biotechnology Letters, 2002,24:91-96
[7] Fedorov A.S, Tsygankov A.A, Rao K.K et al. Hydrogen photoproduction by Rhodobacter sphaeroides immobilized on polyurethane foam[J] Biotechnology letters, 1998,20(11):1007-1009
[8] Lee Chimei, Chen Peichung, Wang Chunchin et al. Photohydrogen production using purple nonsulfur bacteria with hydrogen fermentation reactor effluent[J] Intenational Journal of Hydrogen Energy, 2002,27:1309-1313
[9] Gest H, Ormerod JG, Ormerod KS. Photomitabolism of R. rubrum light-dependent dismimilation of organic compounds to carbon dioxide and molecular hydrogen by an anaerobic critric acid cycle[J]Arch
Biochem Biophys, 1962,97:21
[10] Ike A, Saimura C, Hirata K, et al. Hydrogen photo-production form CO2-fixing microalgal biomass: application of halo-torelant photosynthetic bacteria[J] J Ferment Bioeng, 1997,84:606-609
[11] Wakayama T, Asada Y, Miyake J. Effect of light/dark cycle on baterial hydrogen production by Rhodobacter sphaeroides RV from hour to second range[J] Appiled Biochem Biotech, 2000,84-86:431-440
[12] Toda, Yoshimoto, Kudo. Purple photosynthetic bacteria and health food. Patent: EP1172434AI 020116(Basic) WO 20016077 010823
[13] Yoshida H et al. Journal of general and applied microbiology. 1998,44(1):19-26
[14] Weaver PF. Patent:CA2075161 A 1993,2,3;US 7739368 A 1994,3,15
[15] Hisashi Nagadomi, Takako Hiromitsu, Kenji Takcno. Treatment of aquaculture water by denitrifying photosynthetic bacteia using immobilized Polyvinyl Alcohol beads[J]Journal of Bioscience and Bioengineering, 1999,87(2):189-193
[16] 小川静夫，光合细菌在养殖上的应用[J]，养殖，1985，(5)：66-68
[17] 刘如林、刁虎欣、梁凤来、赵大健等，光合细菌及其应用[M]，中国农业科技出版社，1991，北京
[18] Jhon G. Holt, Noel R. Kieg, et al. Bergey's manual of determination bacteriology[M], 9th Edition, Willimas & wilkings, Baltimore London, 1992
[19] Pereg-Gerk Lily, Sar Nehemia, hipkin Yaakov. In situ nitrogen fixation associated with seagrasses in the Gulf of Elat (Red Sea)[J], 2002,36(3):387-394
[20] Ludden Paul W. Roberts Gary P. Nitrogen fixation by photosynthetic bacteria[J], 2002,73(1/3):115-118
[21] Zbigniew S. Kolber, F. gerald PlumLey, Andrew S. Lang, et al. Contribution of Aerobic Photoheterotrophic Bacteria to the Carbon Cycle in the Ocean[J] Science, 2001, 292:2492-2495
[22] Chalam A V et al. Bull. Environ. Contam. Toxicol. 1997, 58(3):463-468
[23] Stonell M H B, Mcphillips T M, Rees D C, et al. Lighet induced structural changes and the mechanism of electron/proton tranfer in the photosynthetic reaction center[J] Science, 1997,276:812-816
[24] Abrensch E C, Paddock M L, Stonell M H B. Identifiction of proton transfer pathways in the Ⅹ-ray crystal structure of the bacterial reaction center from R. sphareoides[J]. Photosyn Res, 1998,55:119
[25] 张纯喜、樊红军、李良璧、匡廷云等，光合细菌原初反应的理论研究[J]，中国科学(C辑)，1999，29(3)276-282
[26] 光合作用高效光能转化的机理及其在农业中的应用[J]，中国科学院院刊—国家重点基础研究发展规划项目，2002，1：43-44
[27] Ludden PW et al. Science 1976, 194-424
[28] 吴永强、末鸿遇等，光合细菌固氮分子生物学研究进展[J]，植物生理学通讯，1991，27(3)：161-166
[29] Yasuhiro Oda, Simen-Jan Slagman, Wim G Meijer, et al. Influence of growth rate and starvation on luoreseent in situ hubridization of Rbodopseudomonas palustris[J]. FEMS Microbiology Ecology, 2000,32(3):205-213
[30] Chemical & Engineering News, 2001,8,6,32
[31] 杨惠芳，黄志勇，张德民，刘志恒等，紫色非硫光合细菌的空间生物学效应[J]，航天医学与医学工程，1999，12(1)：46-50
[32] 刘中等，光合细菌在淡水养殖中的应用研究[J]，水产科学，1995，14(1)：13-17
[33] 黄钧、韦勇刚、周毅、柯永恩、李志文等，在饲料中添加光合细菌饲养月鳢鱼种的初步试验[J]，广西畜牧兽医，2000，16(1)：17-18
[34] 陈德华等，光合细菌添加饲料养鸡蛋试验[J]，饲料工业，1994，15(5)：11-12
[35] 田维熙、王叶、赵荣芝、杨宪忠等，光合细菌在奶牛、肉牛饲养中应用的试验[J]，中国乳业，2001，6：14-15
[36] 夏宏、夏青、夏成敏、张清元等，光合细菌在早熟甘蓝、油菜上的应用研究[J]，山西农业大学学报，2000，39(2)，116-118
[37] 王晖等，光合细菌肥料对植物的影响[J]，山西教育学院学报，2000，3(3)：36-38
[38] 李俊峰，王梦亮等，光合细菌对农田生态系统的影响[J]，山西农业科学，2002，30(1)：52～56
[39] 徐亚同、史家梁、张明等，污染控制微生物王程[M]，化学工业出版社，2001，北京
[40] Khanna P et al. Current Microbiol. 1992,25:63-67
[41] Caroline S Harwood, Gerhard Burchhardt, Herrmanm, et al. Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway[J] FEMS Microbiology Reviews, 1999,22(5):439-458
[42] Kroomeman J et al. Compostsience & utiligation. 1998, 6(3):42-52
[43] 周洪茂、赵肖为、吴雪昌等，光和细菌沼泽红假单胞菌同化磷能力的研究[J]，科技通报，2002，18(2)：142-146
[44] 刘双江、杨惠芳、周培瑾、孙燕、岑运华等，固定化光合细菌处理豆制品废水产氢研究[J]，环境科学，1995，15(1)：42-44
[45] Joong Kyun Kim, Bum-Kyu Lee, Sang-Hee Kim, et al. A characterization of denitrifying photosynthetic bacteria isolated from photosynthetic sludge[J]. Aquacultural Engineering, 1999,19(3):179-193
[46] 牟海津、王宇新、钱新民等，光合细菌类胡萝卜素的研究[J]，海洋湖沼通报，1998，4：32-38
[47] 刘春朝等，光合细菌发酵产类胡萝卜素的中试研究[J]，食品与发酵工业，1994，6：47-49
[48] 曾宇、秦松、梁明山等，光合细菌综合应用新进展[J]，水产科学，2000，19(5)：34-36
[49] 焦瑞身、周德庆等，微生物生理代谢实验技术[M]，北京，科学出版社，1990
[50] Marmur J. J Mol Biol, 1961,3:208～218.
[51] 林万明，细菌分子遗传学分类鉴定法[M]，上海，上海科学技术出版社，1990，86～92
[52] 张德民、黄志勇、杨惠芳等，几株红假单胞菌属细菌的表观特征及其遗传多样性研究[J]，微生物学报，2000，40(3)：229-236
[53] 张德民，黄志勇，杨惠芳等，紫色非硫细菌Rhodocista属一新分离株的鉴定及其系统学微生物学报；2000，40(1)：14～20．
[54] Thomas Hurek, Blanca Wagner&Barbara Reinhold-Hurek, Applied And Environmental Microbiology, 1997, 63(11): 4331-4339
[55] Stackebrandt E、Gobel B、M Taxonomic note:A place for DNA-DNA reassociation And 16S rRNA sequence analysis in the present species definition in bacteriology, Int J syst bacterial, 1994,44:846-849
[56] 黄伟坤，食品检验与分析[M]，北京，中国轻工业出版社，2000，18-103
[57] 杨祖英，马永健，常凤启等，食品检验[M]，北京，化学工业出版社，2001，40-42
[58] 张龙翔，生化实验方法和技术[M]北京，高等教育出版社，1985，7-10，55-59
[59] Gyoo Yeol Jung, Hyun Ok Jung, Jung Rae Kim et al, Isolation and characterization of Rhodopseudomonas palustris P_4 which utilizes CO with the production of H_2
[60] 方益舫、俞吉安、林志新等，PSBAD菌体蛋白生物效应评价[J]，上海交通大学学报，1991：25(5)：65-67
[61] 张新英、莫天砚，红螺菌的细胞成分分析及在水产养殖上的初步应用试验[J]，广西畜牧兽医，2000，16(6)：8-10
[62] 范文询、李泽英、赵熙和等，蛋白质食物的营养评价[M]北京，人民出版社，1984，7
[63] 金龙飞等，食品与营养学[M]，北京，中国轻工业出版社，1999，68
[64] 刘勇、张德咏，光合细菌PSB-1菌株的分离鉴定及其生物学特征的研究[J]，生命科学研究，2000，4(4)：334
[65] 樊凌雯、张肇铭、张德咏等，利用光合细菌处理糖蜜酒精发酵废液中
试研究[J] 中国环境科学，1998，18(2)：173～175
[66] 闰琦涛、王维坚、韩克文等，玉米淀粉黄浆水PSB法资源化开发的应用前景[J] 吉林粮食高等专科学校学报，2000，15(4)：18-21
[67] Isamu Maeda, Tadashi Mizoguchi, Youshiharu Miura, et al. Influence of sulfate reducingbacteria on outdoorhydrogen production by photosyn the ticbacterium with seawater [J] Current Microbiology, 2001, 40(3):210-213.
[68] 李勤生，王业勤．水产养殖与微生物[M]．武汉：武汉出版社，2000，53-133
[69] 张道南，孙其焕，陈乃松等，红螺菌科光合细菌的分离、培养及其作为鱼虾类饵料添加剂的初步研究[J]．水产学报，1988，12(4)：367-369
[70] 国家环保局、《水和废水检测分析方法》编委会等，水和废水监测分析方法(第三版)[M]．北京：中国环境科学出版社，1989，246-366
[71] 罗世忠、沈纬，废水中痕量亚硝酸盐的光度法测定[J]．青岛化工学院学报，1997，18(1)：63-66