一株红酵母Y11吸附镉的特性研究及其金属硫蛋白的分离纯化
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摘要
微生物在治理镉污染中有着广阔的应用前景，其中微生物吸附剂的研究已成为目前研究的热点之一。本文对一株可在高镉浓度(2000mg／L)下存活的酵母菌Y11吸附镉的特性作了详细的研究，并对其吸附镉的机制进行初步探讨，为实现微生物的镉污染治理奠定了基础。
根据形态学观察和葡萄糖发酵实验，脲酶实验以及重氮基蓝B实验结果，结合《酵母菌的特征与鉴定手册》，确定Y11为一株红酵母(Rhodotorula sp．)。
生长曲线结果表明菌体液体培养7小时进入对数期，18小时左右进入稳定期，因此初步确定菌体培养时间为20小时。在此基础上，研究了吸附时间、菌体加入量及pH对Y11吸附效果的影响。结果表明菌体对镉的吸附在7～8分钟达到峰值，30分钟基本平衡。在镉浓度为48.1mg／L的溶液中，菌体加入量为6g／L时，镉的去除率最大，达到95.6％，菌体中镉含量为干重的9‰。pH对吸附效率有决定性的影响，随着pH的升高，菌体吸附镉的能力增强，但低pH条件有利于镉的解吸。
Y11菌体具有重复吸附镉的能力。经五次吸附循环，其对50.7mg／L含镉溶液中镉的去除能力保持在90.87％～93.23％之间。将菌体用明胶—海藻酸钠固定后吸附镉，最大去除率达到77.91％。
研究了不同处理的菌体吸附的镉在细胞内的分布情况，结果发现在菌体培养过程中添加浓度为100mg／L镉时，菌体吸附的镉有3.27％进入细胞内；但是用培养好的菌体，冻干后吸附镉，菌体吸附的镉只有1％可进入原生质体，其余99％都分布在细胞壁周围。
探讨了Y11菌产金属硫蛋白(MT)的情况，结果表明镉诱导能明显促进Y11菌MT的产量，菌体经诱导后的MT产量达到638.8μg／g，是非诱导菌体的85倍，远大于报道的酵母菌中MT的产量。采用玻璃珠破碎细胞，经煮沸除去不耐热蛋白，再通过乙醇沉淀得到MT粗品。依次经过Sephadex G-50凝胶过滤，DEAE-52离子交换，再通过Sephadex G-25凝胶过滤脱盐纯化MT，经SDS-PAGE检测MT周围没有其它杂蛋白，分子量为6500 Da。
Environmental cadmium pollution is of increasing concern. Microorganisms are deemed to have great potential in treating cadmium pollution. Biosorption has emerged as one of the most promising process because of its high efficiency and favorable economics. A Yeast strain, isolated and designated as Y11 in our lab, could survive 2000mg/L of cadmium. Its characteristics and mechanism of cadmium removal were studied in this paper.
According to its morphological characteristics and the ability of glucose fermentation, urease production, Fast Blue BB Salt(DBB) experimental 1 was identified as a Rhodotorula sp.
Growth curve of Y11 showed the logarithmic phase began from 7 hr and stationary phase began from about 18 hr. 20hr was consided as the optimal culture time for Y11. The effect of adsorption time, the amount of biomass, and the pH on the Cd2+-adsorption by Y11 were studied. The results showed that the adsorption peak value is about 7 min and equilibrium time was about 30 min. When the initial cadmium concentrations is 48.1mg/L, with the optimum biomass quantum of 6g/L, the best removal ratio was 95.6% and Cd content of biomass reached 9‰. pH play a key effect on adsorption. Absorption of Cd2+ effectively enhanced with increasing pH. While low pH had good effect on Cd2+ desorption from biomass.
Y11 had good repetitious ability of cadmium adsorption. With initial Cd2+ concentrations of 48.1mg/L in solution, after 5 absorption and desorption cycles, the cadmium absorptivity kept 90.87%~93.23%. Immobilized Yl 1 cell with gelatin-alginate can removal 77.91% Cd2+.
The distribution of accumulated Cd2+ in different disposed Y11 is different too. 3.29% of the total Cd2+ can entered cytoplasm during growth culture with 100mg/L of Cd2+ in medium. But only 1% entered cytoplasm and 99% existed around cell wall when used the lyophilized biomass to adsorb cadmium.
Metallothionein (MT) was found in Y11, and Cd could induce a high output of MT. When Y11 was cultured with 100mg/L cadmium, the MT content was 638.8ug/g biomass, which is 85 times of non-induced Y11 (7.51ug/g) and by far more than other Yeast strains reported to this day. After breaking up Y11 cell by glass beadings, seething solution to remove heat-sensitive proteins, MT was deposited by alcohol. The purification procedure involved gel filtration on Sephadex G-50, ion exchange chromatography on DEAE-52, and gel filtration on Sephadex G-25 and discontinuous polyacrylamide gel electrophoresis. It exhibited a clear MT stripe with molecular weight of 6500 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
根据形态学观察和葡萄糖发酵实验，脲酶实验以及重氮基蓝B实验结果，结合《酵母菌的特征与鉴定手册》，确定Y11为一株红酵母(Rhodotorula sp．)。
生长曲线结果表明菌体液体培养7小时进入对数期，18小时左右进入稳定期，因此初步确定菌体培养时间为20小时。在此基础上，研究了吸附时间、菌体加入量及pH对Y11吸附效果的影响。结果表明菌体对镉的吸附在7～8分钟达到峰值，30分钟基本平衡。在镉浓度为48.1mg／L的溶液中，菌体加入量为6g／L时，镉的去除率最大，达到95.6％，菌体中镉含量为干重的9‰。pH对吸附效率有决定性的影响，随着pH的升高，菌体吸附镉的能力增强，但低pH条件有利于镉的解吸。
Y11菌体具有重复吸附镉的能力。经五次吸附循环，其对50.7mg／L含镉溶液中镉的去除能力保持在90.87％～93.23％之间。将菌体用明胶—海藻酸钠固定后吸附镉，最大去除率达到77.91％。
研究了不同处理的菌体吸附的镉在细胞内的分布情况，结果发现在菌体培养过程中添加浓度为100mg／L镉时，菌体吸附的镉有3.27％进入细胞内；但是用培养好的菌体，冻干后吸附镉，菌体吸附的镉只有1％可进入原生质体，其余99％都分布在细胞壁周围。
探讨了Y11菌产金属硫蛋白(MT)的情况，结果表明镉诱导能明显促进Y11菌MT的产量，菌体经诱导后的MT产量达到638.8μg／g，是非诱导菌体的85倍，远大于报道的酵母菌中MT的产量。采用玻璃珠破碎细胞，经煮沸除去不耐热蛋白，再通过乙醇沉淀得到MT粗品。依次经过Sephadex G-50凝胶过滤，DEAE-52离子交换，再通过Sephadex G-25凝胶过滤脱盐纯化MT，经SDS-PAGE检测MT周围没有其它杂蛋白，分子量为6500 Da。
Environmental cadmium pollution is of increasing concern. Microorganisms are deemed to have great potential in treating cadmium pollution. Biosorption has emerged as one of the most promising process because of its high efficiency and favorable economics. A Yeast strain, isolated and designated as Y11 in our lab, could survive 2000mg/L of cadmium. Its characteristics and mechanism of cadmium removal were studied in this paper.
According to its morphological characteristics and the ability of glucose fermentation, urease production, Fast Blue BB Salt(DBB) experimental 1 was identified as a Rhodotorula sp.
Growth curve of Y11 showed the logarithmic phase began from 7 hr and stationary phase began from about 18 hr. 20hr was consided as the optimal culture time for Y11. The effect of adsorption time, the amount of biomass, and the pH on the Cd2+-adsorption by Y11 were studied. The results showed that the adsorption peak value is about 7 min and equilibrium time was about 30 min. When the initial cadmium concentrations is 48.1mg/L, with the optimum biomass quantum of 6g/L, the best removal ratio was 95.6% and Cd content of biomass reached 9‰. pH play a key effect on adsorption. Absorption of Cd2+ effectively enhanced with increasing pH. While low pH had good effect on Cd2+ desorption from biomass.
Y11 had good repetitious ability of cadmium adsorption. With initial Cd2+ concentrations of 48.1mg/L in solution, after 5 absorption and desorption cycles, the cadmium absorptivity kept 90.87%~93.23%. Immobilized Yl 1 cell with gelatin-alginate can removal 77.91% Cd2+.
The distribution of accumulated Cd2+ in different disposed Y11 is different too. 3.29% of the total Cd2+ can entered cytoplasm during growth culture with 100mg/L of Cd2+ in medium. But only 1% entered cytoplasm and 99% existed around cell wall when used the lyophilized biomass to adsorb cadmium.
Metallothionein (MT) was found in Y11, and Cd could induce a high output of MT. When Y11 was cultured with 100mg/L cadmium, the MT content was 638.8ug/g biomass, which is 85 times of non-induced Y11 (7.51ug/g) and by far more than other Yeast strains reported to this day. After breaking up Y11 cell by glass beadings, seething solution to remove heat-sensitive proteins, MT was deposited by alcohol. The purification procedure involved gel filtration on Sephadex G-50, ion exchange chromatography on DEAE-52, and gel filtration on Sephadex G-25 and discontinuous polyacrylamide gel electrophoresis. It exhibited a clear MT stripe with molecular weight of 6500 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
引文
[1] 叶寒青，杨祥良，周井炎，等．环境污染物镉毒性作用机理研究进展．广东微量元素科学，200 1，8(3)：9～12
[2] Chien H.F.,Kao C.H.. Accumulation of ammonium in rice leaves in response to excess Cadmium. Plant Sci. ,2000,1 (56): 111～115
[3] 唐宜，李维信．镉对男性生殖系统的影响．重庆医科大学学报，1989，14：343～347
[4] 王夔．生命科学中的微量元素(第二版)．北京：中国计量出版社，1996，34～35
[5] 黄吉厚．镉对环境的污染及其对策研究．辽宁教育学院学报，2000，17(5)：29～31
[6] 王世俊．金属中毒(第二版)．北京：人民卫生出版社，1988，201～204
[7] 陈怀满．土壤—植物系统中的重金属污染．北京：科学出版社，1996，115～125
[8] 张秀丽，刘月英．贵重金属的生物吸附．应用与环境生物学报，2002，8(6)：668～671
[9] 帕特森，工业废水处理技术手册．北京：化学T业出版社，1993，38
[10] Cunningham S.C.. Phytoremediation of contaminated soils. Trend in Biotechnology, 1995,13(9): 393-397
[11] Ruchhofi C.C.. Sewage Works J, 1949,21 (5): 877
[12] Salt D.E., Blaylock M.. Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Biotechnology, 1995,3(6): 468～474
[13] Holan Z.R., Volsky B.. Biosorption of lead and nickel by biomass of marine algae. Biotechnology and bioengineering, 1994, 43(11): 1001～1009
[14] Kratochvil D.,Volesky B.. Advances in the biosorption of heavy metals. Trends in Biotechnology, 1998,16(7): 291～301
[15] Salinas E., Elorza M.. Removal of cadmium and lead from dilute aqueous solutions by Rhodotorula rubra. Bioresource Technology,2000,72: 107～112
[16] Kratochvil D.,Volesky B.. Advances in the biosorption of heavy metals. Trends in Biotechnology, 1998,16(7): 291～301
[17] Strandberg G.W.,Shumate S.E.,Parrot J.R.. Microbial Cells as Biosorbents for Heavy Metals: Accumulation of Uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa Appl and Environ Microbiol. 1981, 41 (1)237～245
[18] Schiewer S.,Volesky B.. Modeling Multi-Metal Ion Exchange in Biosorption. Environmental Science & Technology, 1996,30(10): 2921～2927.
[19] Schiewer S..Modelling complexation and electrostatic attraction in heavy metal biosorption by Sargassum biomass. Journal of Applied Phycology, 1999, 11 (1): 79～87
[20] Holmes J.D.,Richardson D.J.,Saed S.. Cadmium-specific formation of metal sulfide Q-particles by Klebsiella pneumoniae. Microbiology, 1997,143(81): 2521～2530
[21] Volesky B.,May H.,Holan Z.R.. Cadmium biosorption by Saccharomyces cerevisiae. Biotechnology and bioengineering. 1993,41 (8): 826～829
[22] Tsezos M.,Volesky B.. Metabolic-heat relation for aerobic yeast respiration and fermentation. J.Chem tech Biotechnol, 1982,32(6): 650～559
[23] Tabja K.,Ralph J. Silver based crystalline nanoparticles microbially fabricated. Applied Physical Science and Microbiology, 1999,96(24): 13611～13614
[24] Kuyucak N., Volesky B.. Biosorbents for recovery of metals from industrial solutions. Biotechnol.Lett. 1988,10(2): 137-142
[25] 田建民．用微生物外红硫螺菌属形成的生物聚合物去除废水中的重金属．太原理工大学学报，1999，30(2)：175～178
[26] Ashkenazy R.,Gottlieb L.,Yannai S.. Characterization of acetone-washed yeast biomass functional groups involved in lead biosorption Biotechnology and Bioengineering, 1997,55, 1, 1～10
[27] 吴涓．黄孢展齿革菌菌丝球吸附铅离于的研究：[硕士论文]，厦门，厦门大学：1998
[28] Sharma P.K.,Salkwill D.L., Frenkei A.,et al. A new Klebsiella plantcola strain(Cd-1) grows anaerobically at high cadmium concentrations and precipitates cadmium sulfide. Applied and Environmental Microbiology, 2000,66(7): 3083～3087
[29] Wang C.L.,Michels P.C.,Dawson S.C.,et al. Cadmium removal by a new strain of Pseudomonas aeruginosa in aerobic culture. Applied and Environmental Microbiology, 1997,63(10): 4075～4078
[30] Allan V J M, Callow M E, Macaskie L E, et al. Effect of nutrient limitation on biofilm formation and phosphatase activity of a Citrobacter sp. Microbiology Reading, 2002,148(1): 277～288
[31] Belly R.T.,Kydd G.C.. Silver resistance in microorganisms Dev. Ind. Microbiol., 1982, 23: 567～577
[32] Remacle J.,Muguruza I.,Fransolet M. Cadmium removal by a strain of Alcaligenes denitrificans isolated from a metal-polluted pond. Water Resourses, 1992,26(7): 923～926
[33] Nies D.H.. Microbial heavy-metal resistance. Appl Microbiol Bioteehnol. 1999,51: 730～750
[34] Rensing C, Mitra B, Rosen B P.. Insertional inactivation of dsbA produces sensitivity to cadmium and zinc in Escherichia coll. J. Bacteriol., 1997,179: 2769～2771
[35] Daniels M.J.,TurnerCavet J.S.,Seikirk R.,et al. The cadmium-stress stimulon of Escherichia coli K-12. Microbiolog, 1998,144: 1045-1050
[36] Nies D.H, Silver S.. Metal ion uptake by a plasmid-free metal-sensitive Alcaligenes eutrophus strain. J.Bacterioi., 1989, 171: 4073～4075
[37] Liu X.F., Supek F., Nelson N.,et al. Negative control of heavy metal uptake by the Saccharomyces cerevisiae BSD2 gene,J. Biol. Chem., 1997,272: 11763～11769
[38] Burke B.E,Pfister R.M.. Cadmium transport by a Cd~(2+)-sensitive and a Cd~(2+)-resistant strain of Bacillus sub#lis. Can. J.Microbiol., 1986,32: 539～542
[39] Tynecka Z., Maim A.. Energetic basis of cadmium toxicity in Staphylococcus aureus. Biometals, 1995,8: 197～204
[40] Clemens S.,Antosiewicz D.M.,Ward J.M.,et al. The plant cDNA LCTI mediates the uptake of calcium and cadmium in yeast,Proc.Natl. Acad. Sci. USA., 1998, 95: 12043～12048
[41] Olafson R.W.,Abel K.,Sim R.S..Prokaryotic metallothionein: preliminary characterization of a
blue-green algae heavy-metal binding protein. Biochem. Biophys. Res. Commun.,1979,89: 36～43
[42] Gupta A.,Whitton B.A.,Morby A.P.,et al. Amplification and rearrangement of a prokaryotic metallothionein locus smt in Synechococcus PCC-6301 selected for tolerance to cadmium,Proc.R.Soc. Lond. Ser. B.Biol. Sci., 1992, 248: 273～281
[43] Gupta A.,Morby A.P,Turner J.S,et al. Deletion within the metallothionein locus of cadmium-tolerant Synechococcus PCC 6301 involving a highly iterated palindrome (HIP1). Mol. Microbiol., 1993, 7: 189～195
[44] Turner J.S.,Glands P.D.,Samson A.C.,et al. Zn~(2+) sensing by the cyanobacterial metallothionein repressor SmtB: different motifs mediate metal-induced protein-DNA dissociation. Nucleic.Acids. Res., 1996,24: 3714～3721
[45] Thelwell C.,Robinson N.J.,Turner C.J.S.. An SmtB-like repressor from Synechocystis PCC 6803 regulates a zinc exporter, Proc.NatI.Acad. Sci.USA., 1998, 95: 10728～10733
[46] Nies D.H. The cobalt, zinc, and cadmium effiux system CzcABC from dlcaligenes eutrophus functions as a cation-proton-antiporter in Escherichia coB. J.Bacteriol, 1995, 177: 2707～2712
[47] Schmidt T, Schlegel H G. Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A. J.Bacteriol., 1994, 176: 7045～7054
[48] Nucifora G.,Chu L.,Misra T.K.,et al. Cadmium resistance from Staphylococcus aureus plasmid pI258 cadA gene results from a cadmium-efflux ATPase. Proc.Natl. Acad. Sci. USA., 1989, 86: 3544～3548
[49] Liu C.Q., Khunajakr N., Chia L.G., et al. Genetic analysis of regions involved in replication and cadmium resistance of the plasmid pND302 from Lactococcus lactis. Plasmid, 1997,38: 79～90
[50] Li Z.S., Szczypka M, Lu Y.P., et al. The yeast cadmium factor protein (YCF1) is a vacuolar glutathione-S-conjugate pump. J.Bio. Chem., 1996,271: 6509～6517
[51] Tommasini R., Evers R., Vogt E., et al. The human multidrug resistance associated protein functionally complements the yeast cadmium resistance factor 1. Proc. Natl. Acad. Sci. USA., 1996,93: 6743～4748
[52] Inouhe M., Sumiyoshi M., Tohoyama H., et al. Resistance to cadmium ions and formation of a cadmium-binding complex in various wild-type yeasts. Plant Cell Physiol., 1996,37: 341～346
[53] Ortiz D.F., Ruscitti T., Mccue K.F., et al. Transport of metal-binding peptides by HMTI, a fission yeast ABC-type vacuolar membrane protein. J.Biol. Chem., 1995,270: 4721～4728
[54] Tohoyama, H., Inouhe, M., Joho. M.. Production of metallothionein in copper and cadmium resistant strains of Saccharomyces cerevisiae J. IND. Microbiol., 1995,14(2): 126～131
[55] Jayna Chan, Zuyun Huang, Maureen E. Merrifield, Maria T. Salgado, Martin J.. Stillman Studies of metal binding reactions in metallothioneins by spectroscopic, molecular biology, and molecular modeling techniques: Coordination Chemistry Reviews, 2002,319～339
[56] Narula S. S., Brouwer g., Hum Y., Armitage I M.. Three-dimensional solution structure of Callinectes sapidus metallothionein-1 determined by homonuclear and heteronuclear magnetic resonance spectroscopy. Biochemistry (Washington), 1995, 34 (2): 620～631
[57] D.E.Borne F.D,E.L.Mayan N.T.,D.E.Roton C.. Cadmium partitioning in transgenic tobacco plants expressing a mammalian metallothionein gene. Mol Breed, 1998,4(2): 8390
[58] Vaák M., Kgi. JHR: Metallothioneins. In Encyclopedia of Inorganic Chemistry. Edited by King RB. New York: J Wiley & Sons Ltd; 1994: 2229～2241.
[59] Carolina S., Pavel K., Tomas R. et al. Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer membrane protein LamB. Journal of Bacteriology, 1998, 2280～2284
[60] Romeyer F.M., Jacobs F.A., Brousseau R. Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli. Applied and Environmental Microbiology, 1990, 2748～2754
[61] Pazirandeh M., Wells B.M., Ryan R.I.. Develomental of bacterium-Based heavy metal biosorbents: enhanced uptake of cadmium and mercury by Escherichia coli expression a metal binding motif. Applied and Environmental Microbiology, 1998,64(10): 4068～4072
[62] Samuelson P., Wernerus H., Svedberg M., et al. Staphylococcal surface display of meatal-binding polyhistidyl peptides. Applied and Environmental Microbiology, 2000, 66(3): 1243～1248
[63] Rensing C., Ku es U., Stahl U., et al. Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae. J. Bacteriol., 1992,174: 1288～1292
[64] Ahuja P.,Gupta R.,Sixema R.K..Zn~(2+) biosorption by Oscillatoria anguistissima Process Biochemistry, 1999,34 (1): 77～85
[65] Ozer D.,Aksu Z.,Kutsal T.,Caglar A.. A comparative study of various biosorbents for removal of chromium(Ⅵ) ions from industrial waste waters. Process biochemistry, 1994,29(1): 1～5
[66] Aksu Z.,Kutsal T., A bioseparation process for removing lead(Ⅱ) ions from waste water by using vulgaris. Journal of chemical technology and biotechnology. 1991,52(1): 109～118
[67] Schiewer S.,Volesky B.. Mathematical evaluation of the experimental and modeling errors in biosorption. Biotechnology Techniques, 1995,9(11)843～848
[68] Crist R.H.,Martin J.R.,Carr D.,Watson J.R.,Clarke H.J.. Interaction of metals and protons with algae.4 Ion exchange vs adsorption models and a reassessment of scatchard plots;ion-exchange rates and equilibria compared with calcium alginate. Environmental science & technology. 1994, 28(11): 1859～1866
[69] Ting Y.P.,Lawson F.,Prince I.G.. Uptake of cadmium and zinc by the alga Chlorella vulgaris: Part 1. Individual ion species. Biotechnology and Bioengineering. 1989,34(7): 990～999
[70] 牛慧，许学书，王建华．第五届全国生物化工学术会议论文集，化学工业出版社，1993，399
[71] Yin P.H.,Yu Q.M.,Jin B.,Lins Z.. Heavy metal uptake capacities of common marine macro algal biomass. Water Research,1999,33 (6): 1534～1537
[72] 居乃琥．固定化细胞技术研究和应用的现状与展望．工业微生物．1989，19(1)：251
[73] 桥本．用水废水．水处理技术，1985，27(11)：31
[74] 杨麒，李小明．固定化微生物技术及其在生物脱氮中的应用．江苏环境科技，第15卷第1期
2002,15(2)：19～21
[75] 赵力，等．明胶包埋黑根霉菌丝体对水中Pb~(2+)吸附性能的研究．离子交换与吸附，1996，12(5)：4181
[76] Geoffrey W.. Accumulation of colbalt, zinc and manganese by the estuarine green microalgae Chioralla salina immobilized in alginate microbeads. Environ. Sci Technol, 1992,26: 1764～1770
[77] Wong P.K.. Removal and recovery of Cu(Ⅱ)from industrial effluent by immobilized cells of Psedomonas Putida-11. Applied Microbiology Biotechnology, 1993, 39: 1279～1311
[78] Barkley N.P.. Extraction of mercury from groundwater using immobilized algae. Journal of the Air & Waste Management Association, 1991,41 (10): 1381～1392
[79] USEPA. Phytoremediation of Contaminated Soil and Water at Hazardous Waste Sites [M]. EPA/540/S-01/ 500,Washington D C,2001.
[80] Glass D.. Associations, Inc. US and International Markets for Phytoremediation, 1999-2000[Z].Washington D C, 1999
[81] 应娇妍，一株酵母菌富集镉的特性和机制研究：[硕士论文]，北京，中国农业大学：2003
[82] J．A．巴尼特，R．W．佩恩，D．亚罗．酵母菌的特征与鉴定手册，青岛：青岛海洋大学出版社，1991，45
[83] Shengxi Hu, Ken W.K.L., Madeline Wu. Cadmium sequestration in Chlamydomonas reinhardtii. Plant Science, 2001,161: 987～996.
[84] Sharron McEldowney. The impact of surface attachment on cadmium accumulation by Pseudomonas fluorescens H2. FEMS Microbiology Ecology,2000, 33: 121～128.
[85] 北京师范大学，华中师范大学，南京师范大学．无机化学(第四版，上册)，北京：高等教育出版社，2002．419～420
[86] Abdullah M.F.. Comparative toxicity of heavy metals to some yeasts isolated from Saudi Arabian soil. Bioresource Technology, 1998,64: 193～198.
[87] Yongming L.,Ebtisam W.. Heavy metal removal by caustic-treated yeast immobilized in alginate. Journal of Hazardous Materials, 1996,49: 165～179
[88] Zhiqi Hao, Heinz R.R.,David B.W.. Characterization of Cadmium Uptake in Lactobacillus lantarum and Isolation of Cadmium and Manganese Uptake Mutants. Applied and Environmental Microbiology, 1999,65(11): 4741～4745
[89] Martin S., Rene K., Jan V., Libus T., Miroslav N.. Electrochemical study of heavy metals and metallothionein in yeast Yarrowia lipolytica. Bioelectrochemistry,2003, 60: 29～36
[90] Rufus L.C., Minnie M., Yin M.L., Sally L.B., Eric P.B., J Scott Angle, Alan J.M..Phytoremediation of soil metals. Current Opinion in Biotechnology 1997,8: 279～284
[91] 林稚兰，马国栋，李福荣，舒建芬，常立梅．异常汉逊酵母BD102金属硫蛋白的分离纯化和鉴定．微生物学报，2001，41(2)：216～221
[92] Masahiro I.,Masato H., Hiroshi T., et al. Cadmium-binding protein in a cadmiun-resistant strain of Saccaromyces cerevisiae. Biochimica et Biophysica Acta,1989, 993: 51～55
[2] Chien H.F.,Kao C.H.. Accumulation of ammonium in rice leaves in response to excess Cadmium. Plant Sci. ,2000,1 (56): 111～115
[3] 唐宜，李维信．镉对男性生殖系统的影响．重庆医科大学学报，1989，14：343～347
[4] 王夔．生命科学中的微量元素(第二版)．北京：中国计量出版社，1996，34～35
[5] 黄吉厚．镉对环境的污染及其对策研究．辽宁教育学院学报，2000，17(5)：29～31
[6] 王世俊．金属中毒(第二版)．北京：人民卫生出版社，1988，201～204
[7] 陈怀满．土壤—植物系统中的重金属污染．北京：科学出版社，1996，115～125
[8] 张秀丽，刘月英．贵重金属的生物吸附．应用与环境生物学报，2002，8(6)：668～671
[9] 帕特森，工业废水处理技术手册．北京：化学T业出版社，1993，38
[10] Cunningham S.C.. Phytoremediation of contaminated soils. Trend in Biotechnology, 1995,13(9): 393-397
[11] Ruchhofi C.C.. Sewage Works J, 1949,21 (5): 877
[12] Salt D.E., Blaylock M.. Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Biotechnology, 1995,3(6): 468～474
[13] Holan Z.R., Volsky B.. Biosorption of lead and nickel by biomass of marine algae. Biotechnology and bioengineering, 1994, 43(11): 1001～1009
[14] Kratochvil D.,Volesky B.. Advances in the biosorption of heavy metals. Trends in Biotechnology, 1998,16(7): 291～301
[15] Salinas E., Elorza M.. Removal of cadmium and lead from dilute aqueous solutions by Rhodotorula rubra. Bioresource Technology,2000,72: 107～112
[16] Kratochvil D.,Volesky B.. Advances in the biosorption of heavy metals. Trends in Biotechnology, 1998,16(7): 291～301
[17] Strandberg G.W.,Shumate S.E.,Parrot J.R.. Microbial Cells as Biosorbents for Heavy Metals: Accumulation of Uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa Appl and Environ Microbiol. 1981, 41 (1)237～245
[18] Schiewer S.,Volesky B.. Modeling Multi-Metal Ion Exchange in Biosorption. Environmental Science & Technology, 1996,30(10): 2921～2927.
[19] Schiewer S..Modelling complexation and electrostatic attraction in heavy metal biosorption by Sargassum biomass. Journal of Applied Phycology, 1999, 11 (1): 79～87
[20] Holmes J.D.,Richardson D.J.,Saed S.. Cadmium-specific formation of metal sulfide Q-particles by Klebsiella pneumoniae. Microbiology, 1997,143(81): 2521～2530
[21] Volesky B.,May H.,Holan Z.R.. Cadmium biosorption by Saccharomyces cerevisiae. Biotechnology and bioengineering. 1993,41 (8): 826～829
[22] Tsezos M.,Volesky B.. Metabolic-heat relation for aerobic yeast respiration and fermentation. J.Chem tech Biotechnol, 1982,32(6): 650～559
[23] Tabja K.,Ralph J. Silver based crystalline nanoparticles microbially fabricated. Applied Physical Science and Microbiology, 1999,96(24): 13611～13614
[24] Kuyucak N., Volesky B.. Biosorbents for recovery of metals from industrial solutions. Biotechnol.Lett. 1988,10(2): 137-142
[25] 田建民．用微生物外红硫螺菌属形成的生物聚合物去除废水中的重金属．太原理工大学学报，1999，30(2)：175～178
[26] Ashkenazy R.,Gottlieb L.,Yannai S.. Characterization of acetone-washed yeast biomass functional groups involved in lead biosorption Biotechnology and Bioengineering, 1997,55, 1, 1～10
[27] 吴涓．黄孢展齿革菌菌丝球吸附铅离于的研究：[硕士论文]，厦门，厦门大学：1998
[28] Sharma P.K.,Salkwill D.L., Frenkei A.,et al. A new Klebsiella plantcola strain(Cd-1) grows anaerobically at high cadmium concentrations and precipitates cadmium sulfide. Applied and Environmental Microbiology, 2000,66(7): 3083～3087
[29] Wang C.L.,Michels P.C.,Dawson S.C.,et al. Cadmium removal by a new strain of Pseudomonas aeruginosa in aerobic culture. Applied and Environmental Microbiology, 1997,63(10): 4075～4078
[30] Allan V J M, Callow M E, Macaskie L E, et al. Effect of nutrient limitation on biofilm formation and phosphatase activity of a Citrobacter sp. Microbiology Reading, 2002,148(1): 277～288
[31] Belly R.T.,Kydd G.C.. Silver resistance in microorganisms Dev. Ind. Microbiol., 1982, 23: 567～577
[32] Remacle J.,Muguruza I.,Fransolet M. Cadmium removal by a strain of Alcaligenes denitrificans isolated from a metal-polluted pond. Water Resourses, 1992,26(7): 923～926
[33] Nies D.H.. Microbial heavy-metal resistance. Appl Microbiol Bioteehnol. 1999,51: 730～750
[34] Rensing C, Mitra B, Rosen B P.. Insertional inactivation of dsbA produces sensitivity to cadmium and zinc in Escherichia coll. J. Bacteriol., 1997,179: 2769～2771
[35] Daniels M.J.,TurnerCavet J.S.,Seikirk R.,et al. The cadmium-stress stimulon of Escherichia coli K-12. Microbiolog, 1998,144: 1045-1050
[36] Nies D.H, Silver S.. Metal ion uptake by a plasmid-free metal-sensitive Alcaligenes eutrophus strain. J.Bacterioi., 1989, 171: 4073～4075
[37] Liu X.F., Supek F., Nelson N.,et al. Negative control of heavy metal uptake by the Saccharomyces cerevisiae BSD2 gene,J. Biol. Chem., 1997,272: 11763～11769
[38] Burke B.E,Pfister R.M.. Cadmium transport by a Cd~(2+)-sensitive and a Cd~(2+)-resistant strain of Bacillus sub#lis. Can. J.Microbiol., 1986,32: 539～542
[39] Tynecka Z., Maim A.. Energetic basis of cadmium toxicity in Staphylococcus aureus. Biometals, 1995,8: 197～204
[40] Clemens S.,Antosiewicz D.M.,Ward J.M.,et al. The plant cDNA LCTI mediates the uptake of calcium and cadmium in yeast,Proc.Natl. Acad. Sci. USA., 1998, 95: 12043～12048
[41] Olafson R.W.,Abel K.,Sim R.S..Prokaryotic metallothionein: preliminary characterization of a
blue-green algae heavy-metal binding protein. Biochem. Biophys. Res. Commun.,1979,89: 36～43
[42] Gupta A.,Whitton B.A.,Morby A.P.,et al. Amplification and rearrangement of a prokaryotic metallothionein locus smt in Synechococcus PCC-6301 selected for tolerance to cadmium,Proc.R.Soc. Lond. Ser. B.Biol. Sci., 1992, 248: 273～281
[43] Gupta A.,Morby A.P,Turner J.S,et al. Deletion within the metallothionein locus of cadmium-tolerant Synechococcus PCC 6301 involving a highly iterated palindrome (HIP1). Mol. Microbiol., 1993, 7: 189～195
[44] Turner J.S.,Glands P.D.,Samson A.C.,et al. Zn~(2+) sensing by the cyanobacterial metallothionein repressor SmtB: different motifs mediate metal-induced protein-DNA dissociation. Nucleic.Acids. Res., 1996,24: 3714～3721
[45] Thelwell C.,Robinson N.J.,Turner C.J.S.. An SmtB-like repressor from Synechocystis PCC 6803 regulates a zinc exporter, Proc.NatI.Acad. Sci.USA., 1998, 95: 10728～10733
[46] Nies D.H. The cobalt, zinc, and cadmium effiux system CzcABC from dlcaligenes eutrophus functions as a cation-proton-antiporter in Escherichia coB. J.Bacteriol, 1995, 177: 2707～2712
[47] Schmidt T, Schlegel H G. Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A. J.Bacteriol., 1994, 176: 7045～7054
[48] Nucifora G.,Chu L.,Misra T.K.,et al. Cadmium resistance from Staphylococcus aureus plasmid pI258 cadA gene results from a cadmium-efflux ATPase. Proc.Natl. Acad. Sci. USA., 1989, 86: 3544～3548
[49] Liu C.Q., Khunajakr N., Chia L.G., et al. Genetic analysis of regions involved in replication and cadmium resistance of the plasmid pND302 from Lactococcus lactis. Plasmid, 1997,38: 79～90
[50] Li Z.S., Szczypka M, Lu Y.P., et al. The yeast cadmium factor protein (YCF1) is a vacuolar glutathione-S-conjugate pump. J.Bio. Chem., 1996,271: 6509～6517
[51] Tommasini R., Evers R., Vogt E., et al. The human multidrug resistance associated protein functionally complements the yeast cadmium resistance factor 1. Proc. Natl. Acad. Sci. USA., 1996,93: 6743～4748
[52] Inouhe M., Sumiyoshi M., Tohoyama H., et al. Resistance to cadmium ions and formation of a cadmium-binding complex in various wild-type yeasts. Plant Cell Physiol., 1996,37: 341～346
[53] Ortiz D.F., Ruscitti T., Mccue K.F., et al. Transport of metal-binding peptides by HMTI, a fission yeast ABC-type vacuolar membrane protein. J.Biol. Chem., 1995,270: 4721～4728
[54] Tohoyama, H., Inouhe, M., Joho. M.. Production of metallothionein in copper and cadmium resistant strains of Saccharomyces cerevisiae J. IND. Microbiol., 1995,14(2): 126～131
[55] Jayna Chan, Zuyun Huang, Maureen E. Merrifield, Maria T. Salgado, Martin J.. Stillman Studies of metal binding reactions in metallothioneins by spectroscopic, molecular biology, and molecular modeling techniques: Coordination Chemistry Reviews, 2002,319～339
[56] Narula S. S., Brouwer g., Hum Y., Armitage I M.. Three-dimensional solution structure of Callinectes sapidus metallothionein-1 determined by homonuclear and heteronuclear magnetic resonance spectroscopy. Biochemistry (Washington), 1995, 34 (2): 620～631
[57] D.E.Borne F.D,E.L.Mayan N.T.,D.E.Roton C.. Cadmium partitioning in transgenic tobacco plants expressing a mammalian metallothionein gene. Mol Breed, 1998,4(2): 8390
[58] Vaák M., Kgi. JHR: Metallothioneins. In Encyclopedia of Inorganic Chemistry. Edited by King RB. New York: J Wiley & Sons Ltd; 1994: 2229～2241.
[59] Carolina S., Pavel K., Tomas R. et al. Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer membrane protein LamB. Journal of Bacteriology, 1998, 2280～2284
[60] Romeyer F.M., Jacobs F.A., Brousseau R. Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli. Applied and Environmental Microbiology, 1990, 2748～2754
[61] Pazirandeh M., Wells B.M., Ryan R.I.. Develomental of bacterium-Based heavy metal biosorbents: enhanced uptake of cadmium and mercury by Escherichia coli expression a metal binding motif. Applied and Environmental Microbiology, 1998,64(10): 4068～4072
[62] Samuelson P., Wernerus H., Svedberg M., et al. Staphylococcal surface display of meatal-binding polyhistidyl peptides. Applied and Environmental Microbiology, 2000, 66(3): 1243～1248
[63] Rensing C., Ku es U., Stahl U., et al. Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae. J. Bacteriol., 1992,174: 1288～1292
[64] Ahuja P.,Gupta R.,Sixema R.K..Zn~(2+) biosorption by Oscillatoria anguistissima Process Biochemistry, 1999,34 (1): 77～85
[65] Ozer D.,Aksu Z.,Kutsal T.,Caglar A.. A comparative study of various biosorbents for removal of chromium(Ⅵ) ions from industrial waste waters. Process biochemistry, 1994,29(1): 1～5
[66] Aksu Z.,Kutsal T., A bioseparation process for removing lead(Ⅱ) ions from waste water by using vulgaris. Journal of chemical technology and biotechnology. 1991,52(1): 109～118
[67] Schiewer S.,Volesky B.. Mathematical evaluation of the experimental and modeling errors in biosorption. Biotechnology Techniques, 1995,9(11)843～848
[68] Crist R.H.,Martin J.R.,Carr D.,Watson J.R.,Clarke H.J.. Interaction of metals and protons with algae.4 Ion exchange vs adsorption models and a reassessment of scatchard plots;ion-exchange rates and equilibria compared with calcium alginate. Environmental science & technology. 1994, 28(11): 1859～1866
[69] Ting Y.P.,Lawson F.,Prince I.G.. Uptake of cadmium and zinc by the alga Chlorella vulgaris: Part 1. Individual ion species. Biotechnology and Bioengineering. 1989,34(7): 990～999
[70] 牛慧，许学书，王建华．第五届全国生物化工学术会议论文集，化学工业出版社，1993，399
[71] Yin P.H.,Yu Q.M.,Jin B.,Lins Z.. Heavy metal uptake capacities of common marine macro algal biomass. Water Research,1999,33 (6): 1534～1537
[72] 居乃琥．固定化细胞技术研究和应用的现状与展望．工业微生物．1989，19(1)：251
[73] 桥本．用水废水．水处理技术，1985，27(11)：31
[74] 杨麒，李小明．固定化微生物技术及其在生物脱氮中的应用．江苏环境科技，第15卷第1期
2002,15(2)：19～21
[75] 赵力，等．明胶包埋黑根霉菌丝体对水中Pb~(2+)吸附性能的研究．离子交换与吸附，1996，12(5)：4181
[76] Geoffrey W.. Accumulation of colbalt, zinc and manganese by the estuarine green microalgae Chioralla salina immobilized in alginate microbeads. Environ. Sci Technol, 1992,26: 1764～1770
[77] Wong P.K.. Removal and recovery of Cu(Ⅱ)from industrial effluent by immobilized cells of Psedomonas Putida-11. Applied Microbiology Biotechnology, 1993, 39: 1279～1311
[78] Barkley N.P.. Extraction of mercury from groundwater using immobilized algae. Journal of the Air & Waste Management Association, 1991,41 (10): 1381～1392
[79] USEPA. Phytoremediation of Contaminated Soil and Water at Hazardous Waste Sites [M]. EPA/540/S-01/ 500,Washington D C,2001.
[80] Glass D.. Associations, Inc. US and International Markets for Phytoremediation, 1999-2000[Z].Washington D C, 1999
[81] 应娇妍，一株酵母菌富集镉的特性和机制研究：[硕士论文]，北京，中国农业大学：2003
[82] J．A．巴尼特，R．W．佩恩，D．亚罗．酵母菌的特征与鉴定手册，青岛：青岛海洋大学出版社，1991，45
[83] Shengxi Hu, Ken W.K.L., Madeline Wu. Cadmium sequestration in Chlamydomonas reinhardtii. Plant Science, 2001,161: 987～996.
[84] Sharron McEldowney. The impact of surface attachment on cadmium accumulation by Pseudomonas fluorescens H2. FEMS Microbiology Ecology,2000, 33: 121～128.
[85] 北京师范大学，华中师范大学，南京师范大学．无机化学(第四版，上册)，北京：高等教育出版社，2002．419～420
[86] Abdullah M.F.. Comparative toxicity of heavy metals to some yeasts isolated from Saudi Arabian soil. Bioresource Technology, 1998,64: 193～198.
[87] Yongming L.,Ebtisam W.. Heavy metal removal by caustic-treated yeast immobilized in alginate. Journal of Hazardous Materials, 1996,49: 165～179
[88] Zhiqi Hao, Heinz R.R.,David B.W.. Characterization of Cadmium Uptake in Lactobacillus lantarum and Isolation of Cadmium and Manganese Uptake Mutants. Applied and Environmental Microbiology, 1999,65(11): 4741～4745
[89] Martin S., Rene K., Jan V., Libus T., Miroslav N.. Electrochemical study of heavy metals and metallothionein in yeast Yarrowia lipolytica. Bioelectrochemistry,2003, 60: 29～36
[90] Rufus L.C., Minnie M., Yin M.L., Sally L.B., Eric P.B., J Scott Angle, Alan J.M..Phytoremediation of soil metals. Current Opinion in Biotechnology 1997,8: 279～284
[91] 林稚兰，马国栋，李福荣，舒建芬，常立梅．异常汉逊酵母BD102金属硫蛋白的分离纯化和鉴定．微生物学报，2001，41(2)：216～221
[92] Masahiro I.,Masato H., Hiroshi T., et al. Cadmium-binding protein in a cadmiun-resistant strain of Saccaromyces cerevisiae. Biochimica et Biophysica Acta,1989, 993: 51～55