本研究以由環境中自行篩選之抗重金屬菌體Enterobacter sp. J1與表現真核生物金屬鍵結蛋白Metallothionein (MT) 之基因重組大腸桿菌E. coli為生物吸附劑，探討其對工業污染中三種常見的重金屬 (鉛、銅、鎘) 之吸附行為與效果。首先以Enterobacter sp. J1吸附單一金屬，結果顯示菌株J1對鉛具有比較高的吸附能力，其吸附量超過50 mg/g dry cell，而銅與鎘之最大飽和吸附量則分別為32.5與46.2 mg/g dry cell。常用的Langmuir isotherm與Freundlich model皆可描述J1菌體對鉛、銅與鎘之吸附行為，然而Langmuir isotherm對鉛吸附之模擬偏差稍大。本研究所發展之combinative model則可準確地模擬三種金屬之吸附時間流程，同時也間接推測J1菌體對鉛的吸附機制，可能有胞內吸附的路徑存在。此外，可利用調降pH值的方式，將吸附在菌體上之金屬脫附出來。對鎘來說，pH＝3即可完全脫附；但對銅與鉛來說，在pH≦2時才能達到90%的脫附效果。該細胞經四次重複吸附/脫附操作後，對鉛、銅、鎘仍分別具有75 %、72 %、94 %以上之再生吸附能力。

　　接著以J1菌體為生物吸附劑，探討金屬離子共存時，彼此間之競爭吸附效應。本研究結合了反應曲面實驗設計法 (RSM)與成分組合設計法(Mixture design)完整地探討共存離子之吸附行為，且在不影響實驗的準確度下，成功地將實驗點從38組減少為26組。為了表達此多重金屬生物吸附之競爭反應實驗結果，本研究並以創新的三角等高線圖與三相立體曲面圖來檢測實驗值與理論值的差距以確定迴歸結果之準確性，進而解釋金屬離子之間的競爭吸附效應。結果發現金屬離子對J1菌體吸附位置之競爭力順序為：鉛＞銅＞鎘。為建構此實驗設計的反應曲面以利於吸附現象之預測，本研究嘗試以Langmuir-Freundich model來模擬多重金屬飽和吸附之結果，其預測的結果可用3D等高面圖成功地表達出來；藉由這些等高面圖，我們可正確地預測在多重金屬系統中任一金屬離子之吸附量。

　　最後，本研究以基因重組大腸桿菌E. coli做為重金屬生物吸附劑，主要是藉由表現真核細胞金屬鍵結蛋白Metallothionein (MT)來提高吸附金屬之效果。本研究將老鼠、人類以及吳郭魚之MT基因分別選殖至大腸桿菌宿主細胞中，以IPTG誘導MT蛋白質之大量表現後，評估該基因重組菌體對重金屬之吸附量與吸附速率是否提升。由重組菌全細胞分別對鉛、銅、鎘之恆溫吸附曲線可知，表現MT蛋白之基因重組菌比控制組具有較佳之吸附速率(約1.5-2.5倍)，且在低濃度時(小於100 mg/L)比控制組有較大之吸附容量。尤其是表現吳郭魚MT蛋白之重組菌(2T-OmMT1)，在起始金屬濃度為100 mg/L時，其對鉛、銅、鎘之吸附量可分別提升約6、14和89%。由模擬Langmuir isotherm所得之解離常數(Kd)與最大吸附量(qmax)值可知，各基因重組菌有較大之qmax/Kd值，顯示表現MT之大腸桿菌可提升該菌體對重金屬之整體吸附效率。

　　This study was undertaken to investigate biosorption kinetics of lead (Pb), copper (Cu) and cadmium (Cd) ions on the biomass of Enterobacter sp. J1 isolated from local industry wastewater treatment plant and recombinant Escherichia coli strains expressing eukaryotic metallothionein. Efficiency of metal ion recovery from metal-loaded biomass to regenerate the biosorbent was also determined. Enterobacter sp. J1 was able to uptake over 50 mg of Pb per gram of dry cell, while having lower adsorption capacities for Cu and Cd. The equilibrium adsorption of Enterobacter sp. J1 for Cu and Cd were 32.5 and 46.2 mg/g dry cell, respectively. Langmuir and Freundlich models were able to describe biosorption isotherm fairly well, except that prediction of Pb adsorption was relatively poor with Langmuir model, suggesting a different mechanism for Pb biosorption. Adjusting the pH value to 3.0 led to nearly complete desorption of Cd from metal-loaded biomass, and approximately 90% recovery of Pb and Cu ions was obtained at pH≦2. The acid-treated biomass can be reused for biosorption with 75 %、72 %、94 % of original adsorption capacity for Pb, Cu, and Cd, respectively. A new combinative model was developed to predict the kinetics of heavy metal adsorption with excellent agreement. The model simulation seemed to suggest that intracellular accumulation may occur during the uptake of Pb.

　　In order to evaluate the applicability of Enterobacter sp. J1 as a practical metal biosorbent, the interaction and competition of biosorption of lead, copper, and cadmium were investigated in multi-metal systems. A new experimental design method combining mixture design and response surface method (RSM) was developed. The experimental design can effectively reduce the number of experimental trials from 38 to 26. To present the three-metal biosorption equilibrium data in mixture design system, triangular contour diagrams and triangular three-dimensional (3D) biosorption surfaces were used. The results show that the biosorption preference of Enterobacter sp. J1 for the three metal adsorbates decreased in the order of Pb2+ > Cu2+ > Cd2+. The competitive metal biosorption in a multi-metal system can be predicted well by combined Langmuir-Freundlich model. The predicted results can be presented with a new contour-surface plot method developed in this study. Based on those contour-surface plots, the multi-metal biosorption results can be clearly and accurately predicted.

　　Finally, this study attempted to enhance the metal uptake by expressing eukaryotic metal-binding protein metallothionein (MT) on the biomass of recombinant Escherichia coli strains. The MT protein originating from eukaryotic organisms (such as animals and plants) is a low molecular weight cysteine-rich metal-binding motif, possessing peptide sequences that can enhance the capacity and specificity of metal binding. Thus, MT protein may have the potential to be used for the development of a novel and effective biosorbent for heavy metals. Recombinant Escherichia coli strains were constructed to overexpress MT proteins from murine, human and fish via induction of IPTG. From the biosorption isotherm results, the recombinant strains expressing MT proteins had better sorption affinity than the control strain (ca. 1.5-2.5 times) and also attained higher metal uptake capacities than control at low metal concentration (smaller than 100 mg/L). At an initial metal concentration of 100 mg/L, the strain expressing fish MT displayed a 6, 14, 89 % increase for biosorption of Pb, Cu, Cd, respectively. The values of sorption constant (Kd) and maximum sorption capacity (qmax) predicted from Langmuir isotherm model showed that recombinant E. coli strains expressing MT proteins had lower Kd and higher qmax/Kd ratio, indicating an improvement in the metal biosorption affinity and overall biosorption efficiency.

Beveridge, T.J. and Murray, R.G.E., Uptake and Retention of Metals by Cell Walls of Bacillus subtilis. J. Bacteriol. 127, 1502-1518 (1976)

Beveridge, T.J. and Murray, R.G.E., Sites of Metals Deposition in the Cell Wall of Bacillus subtilis. J. Bacteriol. 141, 876-887 (1980)

Brady, D. and Ducan, J. R. Bioaccumulation of metal cations by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 41, pp. 149-154 (1994)

Brady, J.M., Tobin, J.M., Adsorption of metal ions by Rhizopus arrhizus biomass: Characterization studies. Enzyme Microb. Technol. 16, 671-675 (1994)

Brierley, C. L., Brierley, J. A. and Davidson, M. S. Applied microbial processes for metals recovery and removal from wastewater. In Metal Ions and Bacteria (Edited by
Beveridge T. J. and Doyle R. J.), pp. 359-382, John Wiley, New York (1989)

Cabral, J.P.S., Selective binding of metal ions to Pseudomonas syringae cells. Microbios 71, 47-53 (1992)

Cetinkaya, D.G., Aksu, Z., Ozturk, A. and Kutsal, T. A comparative study on heavy metal biosorption characteristics of some algae. Process Biochem. 34, pp. 885-892 (1999)

Chang, J.S., Law, R. and Chang, C.C. Biosorption of lead, copper and cadmium by biomass of Pseudomonas aeruginosa

PU21. Wat. Res. Vol. 31, No. 7, pp. 1651-1658 (1997)
Chang, J. S. and Hong, J. Biosorption of mercury by the
inactivated cells of Pseudomonas aeruginosa PU21(Rip64).

Biotechnol. Bioeng. 44, pp. 999-1006 (1994)
Chang, J. S. and Huang, J. C., Selective adsorption/recovery of Pb, Cu, and Cd with multiple fixed beds containing immobilized bacterial biomass.

Biotechnology Progress, 14(5), 735-741 (1998)
Chen, B.Y., Utgikar, V.P., Harmon, S. M., Tabak, H.H.,

Bishop, D.F. and Govind, R. Studies on biosorption of zinc(II) and copper(II) on Desulfovibrio Desulfuricans. International Biodeterioration & Biodegradation, 46, pp. 11-18 (2000)

Chiou, M.S. and Li, H.Y. Adsorption behavior of reactive dye in aqueous solutionon chemical cross-linked chitosan beads. Chemosphere, 50, pp. 1095–1105 (2003)

Chong, K.H., Volesky, B., Description of two-metal Biosorption equilibria by Langmuir-type models. Biotechnol. Bioengng. 47, 451-460 (1995)

Diels, L., Roy, S. V., Mergeay, M., Doyen, W., Taghavi, S. and Leysen, R., Immobilization of bacteria in composite membranes and development of tubular membrane reactors for heavy metal recuperation. 275-293 (1993)

Fourest E. and Roux J.-C. Heavy metal biosorption by fungal mycelial by-products: mechanisms and influence of pH. Appl. Microbiol. Biotechnol .37, 399-403 (1992)

Gadd, G.M. Accumulation of metals by microorganisms and algae. Biotechnology, Vo l6b, pp. 401-430, VCH Weinheim, Germary (1988)

Harada, M., Minamata disease: methylmercury poisoning in Japan caused by environmental pollution., Crit. Rev. Toxicol., 25,1-24 (1995)

Hu, Z.C. and Reeves, M. Biosorption of Uranium by Pseudomonas aeruginosa Strain CSU immobilized in a Novel Matrix. Biotechnol. Prog., 13, pp. 60-67 (1997)

King, M. C., Metallothionein：Potential Biomarker for Monitoring Heavy Metal Pollution in Fish Around Hong Kong, Marine Pollution Bulletin, 31, 4-12, 411-415 (1995)

Kiff, R. J. and Little, D. R. Biosorption of heavy metals by immobilized fungal biomass. In Immobilization of Ions by Biosorption. pp.71-80. 1986

Kratochvil, D. and Volesky, B., Multicomponent biosorption in fixed beds. Water Research. 34, pp. 3186-3196 (2000)

Lu, W., Shi, J. and Chang, J., Biosorption of Lead, copper and cadium by a local isolate Enterobacter sp. J1 processing high heavy-metal resistance. Process Biochemistry (2004) (submitted).

Macaskie, L. E., and Dean, A. C. R., Metal sequestering biochemicals, 200-248. In B. Volesky (ed.), Biosorption of heavy metals. CRC Press, Boca Raton, Fla. (1990)

Macaskie, L. E., Dean, A. C. R., Cheetham, A. K., Jakeman, R. J. B. and Skarnulis, A. J., Cadmium accumulation by Citrobacter sp.: the chemical nature of the accumulated metal precipitate and its location on the bacterial cells. J. Gen. Microbiol., 133,539-544 (1987)

Martin J.S., Donald T., Colleen T., Xu G. and Micheal S., Circular dichroism, kinetic and mass spectrometric studies of copper and mercury binding to metallothionein. J. Inorganic Biochemistry, 79. pp.11-19 (2000)

Ma, W. and Tobin, J., Development of multimetal binding model and application to binary metal biosorption onto peat biomass. Water Research, 37. pp. 3967-3977 (2003)

McKay, G. and Ho, Y.S. The sorption of lead (II) on peat. Water Res. 33, pp. 578-584 (1999a)

McKay, G. and Ho, Y.S. Pseudo-second order model for sorption processes. Process Biochem. 34, pp. 451-465 (1999b)

Nakajima, A. and Sakaguchi, T. Accumulation of uranium by basidiomycetes. Appl. Microbiol. Biotechnol. 38, pp. 574-578 (1993)

Niu, H., Xu, X. S., Wang, J. H. and Volesky, B. Removal of lead from aqueous solutions by Penicilluium biomass. Biotechnol. Bioeng. 42, pp. 785-787 (1993)

Nogawa, K. and Kido, T., Biological monitoring of cadmium exposure in itai-itai disease epidemiology. Int. Arch. Occup. Environ. Health., 65,43-46 (1993)

Nordberg, M., Metallothioneins：historical review and state of knowledge, Talanta, 46, 243-254 (1998)

Pons M. P. and Fuste M. C. Uranium uptake by immobilized cells of Pseudomonas strain EPS 5028. Appl. Microbiol. Biotechnol. 39, pp.661-665 (1993)

Puranik, P. and Paknikar, K., Influence of co-cations on biosorption of lead and zinc – a comparative evalution in binary and multimetal systems. Bioresource Technology. 70, pp. 269-276 (1999)

Sag, Y., Akcael, B. and Kutsal, T., Evaluation, interpretation, and representation of three-metal biosorption equilibria using a fungal biosorbent. Process Biochemistry. 37, pp. 35-50 (2001)

Sips, R., On the structure of a catalyst surface. J. Chem. Phys. 16, pp.490-495 (1948)

Shumate S. E. II and Strandberg G. W. Accumulation of metals by microbial cells. In Comprehensive Biotechnology (Edited by Moo-Young M.), pp. 235-247, Pergamon Press, New York (1985)

Sousa, C., Kotrba, P., Ruml, T., Cebolla A. and De Lorenzo, V., Metalloadsorption by Escherichia coli cells displaying Yeast and mammalian metallothioneins anchored to the outer membrane protein LamB, J. Bacteriol., vol. 180, No. 9, pp. 2280-2284 (1998)

Sudha K., Lalitha R. G. and Karanth N.G., Studies on lactate dehydrogenase of Lactobacillus plantarum spp. involved in lactic acid biosynthesis using permeabilized cells. Process Biochemistry, 35, pp. 1191-1198 (2000)

Ting, Y.P., Teo, W.K., Uptake of cadmium and zinc by yeast: Effects of co-metal ion and physical/chemical treatments. Biores. Technol. 50, 113-117 (1994)

Tsezos M. The selective extraction of metals from solutions by microorganism. Can. Metal. Quart. 24, pp.141-144 (1985)

Vegliò, F., Biase, A., Beolchini, F. and Pagnanelli, F., Heavy metal biosorption in binary system: simulation in single- and two-stage UF/MF membrane reactors. Hydrometallurgy, 66. pp. 107-115 (2002)

Volesky, B. Advance in biosoption of metals：Selection of biomass types. FEMS Microbiol. Rev. 14, pp.291-302 (1994)

Volesky, B. and Holan Z. R. Biosorption of heavy metals. Biotechnol. Prog. 11, pp. 235-250 (1995)

Volesky, B., Detoxification of metal-bearing effluents: Biosorption for the next century. Hydrometallurgy, 59, pp. 203-216 (2001)

Volesky, B., Biosorption process simulation tools. Hydrometallurgy, 71. pp. 179-190 (2003)

Wan, W. S., Endud, C. S. and Mayanar, R., Removal of Cu(II) ion from aqueous solutions onto chitosan and cross linked chitosan beads. Reactive and Functional Polymers, 50, pp. 181-190 (2002)

Wong, P. K., Lam, K. C. and So, C. M. Removal and recovery of Cu(II) from industrial effluent by immobilized cells of Pseudomonas putida II-11. Appl. Microbiol. Biotechnol. 39, pp.127-131 (1993)

Zhan, X.M. and Zhao, X. Mechanism of lead adsorption from aqueous solutions using an adsorbent synthesized from natural condensed tannin. Water Research, 37, pp. 3905–3912 (2003)

行政院環保署，「台灣地區土壤重金屬含量調查總報告(一~ 四冊)」，1990

王一雄，「土壤環境污染與農藥」，國立編譯館，第229-260頁，1997

台南縣環境保護局，「八十九年度台南縣推動土壤污染防治工作計畫期末報告」，2000

屏東縣環境保護局，2002

吳先琪、吳曉芬，「污染土壤之危害分析」，第五屆土壤污染防治研討會論文集，第119-142頁，1997

吳智輝，「Pseudomonas aeruginosa 與Ralstonia taiwanensis 對重金屬與酚之毒理學與生物復育研究」，碩士論文，成功大學化工系，2004

卓英仁，「我國土壤污染現況分析及防治政策之研究」，環保通訊雜誌社，1998

張忠正，「以綠膿桿菌為重金屬生物吸附劑之效果評估」，碩士論文，逢甲大學化工系，1996

張嘉修，「重金屬污染的生物處理技術」，化工，第41卷，第3期, 第52-58頁，1994

張嘉修、羅文鑫，「生物技術在含汞廢水處理上之應用」，工業污染防治，第19卷，第75期, 第1-25頁，2000

曾筠捷，「表現MerP蛋白之基因重組大腸桿菌進行重金屬生物吸附之定性與定量分析」，碩士論文，逢甲大學化工系，2003

詹智遠，「環境微生物處理重金屬之研究」，碩士論文，國立東華大學生物技術研究所碩士班，1990

蘇啟嘉，「以大量表現汞鍵結結合蛋白MerP之基因重組大腸桿菌進行不同重金屬之生物吸附」，碩士論文，逢甲大學化工所，200