一株铜抗性细菌的分离鉴定及其耐铜机制
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摘要
利用微生物修复铜污染环境是当前研究的热点之一,筛选铜污染环境生物修复的菌种,研究其耐铜机制对铜污染土壤修复具有重要的意义.从铜矿附近的土壤中分离得到一株具有较强铜抗性的细菌,经形态观察、生理生化实验及系统发育分析,鉴定该菌株为不动杆菌属,命名为Acinetobacter sp. MA9.为探索MA9对铜的耐性、富集及其可能机制,研究不同浓度铜处理下菌株的生长和细菌对培养基铜的去除效率,同时分析铜胁迫下MA9胞外聚合物的含量、菌体表面形貌和官能团的变化.结果表明,铜处理细菌36 h后,以培养基铜浓度表征的半数效应浓度(EC_(50))值为251mg/L,细菌对溶液铜的去除量最高达到了溶液铜浓度的68%.与对照相比,铜胁迫使得MA9产生了更多的胞外聚合物,其中多糖增加62%,蛋白增加185%;利用扫描电镜观察发现,与无铜处理的菌株相比,铜处理菌体表面存在大量颗粒物;能谱分析也显示,MA9细胞表面检测到铜,这说明铜可能与细菌代谢物结合产生胞外吸附;傅里叶红外光谱分析结果表明,菌株在铜处理后细胞表面与醛基官能团相关的吸收峰消失,说明铜主要和菌体表面的醛基官能团结合.本研究表明胞外吸附和胞外沉淀是不动杆菌MA9主要的耐铜机制,醛基是参与MA9和铜反应的主要基团;结果可为MA9在铜污染环境修复中的应用提供数据和基础.(图6表2参36)
Microbial remediation of copper(Cu) pollution is one of the key research topics at present and has received a great deal of attention. Screening bacterial strains that can remediate a Cu contaminated environment and uncoupling the underlying Cu resistance mechanisms are of great significance for engineering practices to clean up Cu polluted soils. A bacterial strain with strong Cu resistance was isolated from soil near a Cu mine. This bacterium was identified and named Acinetobacter sp. MA9, based on morphological, physiological, and biochemical experiments, as well as 16 S rRNA gene sequence analysis. To explore the Cu tolerance, enrichment, and the possible resistance mechanism, MA9 growth and its Cu removal efficiency from the culture medium were studied under different Cu treatments. Variations of the extracellular polymeric substances(EPS), surface morphology, and functional groups of MA9 under Cu stress were also analyzed. The results showed that the concentration for 50% of maximal effect EC50 value of the MA9 treated with Cu for 36 h was 251 mg/L,and the highest Cu removal rate was 68% of the Cu concentration in solution. Compared with the control, Cu stress resulted in more EPS production by MA9, in which polysaccharide increased by 62% and protein increased by 185%. From analysis with a scanning electronic microscope, a large number of particles were formed on the surface of the bacterial cells treated with Cu. Energy dispersive spectrometry analysis also showed that Cu was detected on the surface of MA9 cells, suggesting that Cu might be bound extracellularly with bacterial metabolites. Fourier transform infrared spectroscopy demonstrated that the absorption peaks related to aldehyde functional groups disappeared after Cu treatment, indicating that these groups on the cell surface were involved in Cu binding. In conclusion, extracellular adsorption and precipitation may be the main Cu resistance mechanism of Acinetobacter sp. MA9. The aldehyde group may be the major group involved in the reaction of MA9 and Cu.The results provide a foundation for the application of MA9 in the remediation of Cu polluted environments.
Microbial remediation of copper(Cu) pollution is one of the key research topics at present and has received a great deal of attention. Screening bacterial strains that can remediate a Cu contaminated environment and uncoupling the underlying Cu resistance mechanisms are of great significance for engineering practices to clean up Cu polluted soils. A bacterial strain with strong Cu resistance was isolated from soil near a Cu mine. This bacterium was identified and named Acinetobacter sp. MA9, based on morphological, physiological, and biochemical experiments, as well as 16 S rRNA gene sequence analysis. To explore the Cu tolerance, enrichment, and the possible resistance mechanism, MA9 growth and its Cu removal efficiency from the culture medium were studied under different Cu treatments. Variations of the extracellular polymeric substances(EPS), surface morphology, and functional groups of MA9 under Cu stress were also analyzed. The results showed that the concentration for 50% of maximal effect EC50 value of the MA9 treated with Cu for 36 h was 251 mg/L,and the highest Cu removal rate was 68% of the Cu concentration in solution. Compared with the control, Cu stress resulted in more EPS production by MA9, in which polysaccharide increased by 62% and protein increased by 185%. From analysis with a scanning electronic microscope, a large number of particles were formed on the surface of the bacterial cells treated with Cu. Energy dispersive spectrometry analysis also showed that Cu was detected on the surface of MA9 cells, suggesting that Cu might be bound extracellularly with bacterial metabolites. Fourier transform infrared spectroscopy demonstrated that the absorption peaks related to aldehyde functional groups disappeared after Cu treatment, indicating that these groups on the cell surface were involved in Cu binding. In conclusion, extracellular adsorption and precipitation may be the main Cu resistance mechanism of Acinetobacter sp. MA9. The aldehyde group may be the major group involved in the reaction of MA9 and Cu.The results provide a foundation for the application of MA9 in the remediation of Cu polluted environments.
引文
1 Wahyu I,Adolf JNP,Nida S.Juniche AT.The role of heavy metalsresistant bacter ia Acinetobacter sp.in copper phy toremediation using Eichhornia crasippes[(Mart.)Solms][C]//NRLS Conference Proceedings,International Conference on Natural Resources and Life Sciences(2016),KnE Life Sciences,2017:208-220
2 Grigalavi?ien?I,Rutkovien?V,Marozas V.The accumulation of heavy metals Pb,Cu and Cd at roadside forest soil[J].Pol J Environ Stud,2005,14(1):109-115
3 Mack ie K A,Müller T,Zi keli S,Kandeler E.Long-ter m copper application in an organic vineyard modifies spatial distribution of soil microorganisms[J].Soil Biol Biochem,2013,65(65):245-253
4 Lebrun JD,Trinsoutrotgattin I,Vinceslasakpa M,Bailleul C,Brault A,Mougin C,Laval K.Assessing impacts of copper on soil enzyme activities in regard to their natural spatiotemporal variation under longterm different land uses[J].Soil Biol Biochem,2012,49(6):150-156
5 Li J,Zheng YM,Liu YR,Ma YB,Hu HW,He JZ.Initial copper stress strengthens the resistance of soil microorganisms to a subsequent copper stress[J].Microbial Ecol,2014,67(4):931-941
6黄志钧,李大平.重金属铜离子抗性菌株的筛选和吸附性能[J].应用与环境生物学报,2012,18(6):964-970[Huang ZJ,Li DP.Cu2+Biosorption by a highly copper resistant bacterium isolated from soil[J].Chin J Appl Environ Biol,2012,18(6):964-970]
7林海,朱亦珺,董颖博,程煌,霍汉鑫.一株耐酸耐铜细菌的选育及其吸附铜离子的特性[J].环境化学,2013,32(4):599-604[Lin H,Zhu YJ,Dong YB,Cheng H,Huo HX.Breeding of a strain of acid-resistant and copper-tolerant bacteria and characteristics of copper ion adsorption[J].Environ Chem,2013,32(4):599-604]
8 Manohari,Singh J,Nandabalan YK.Copper(II)bioremoval by a rhizosphere bacterium,Stenotrophomonas acidaminiphila,MYS1-process optimization by RSM using Box-Behnken design[J].Int JEnviron Res,2017,11(1):63-70
9 Jiang CY,Sheng XF,Qian M,Wang QY.Isolation and characterization of a heavy metal-resistant Burkholderia sp.from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil[J].Chemosphere,2008,72(2):157-164
10徐磊辉,黄巧云,陈雯莉.环境重金属污染的细菌修复与检测[J].应用与环境生物学报,2004,10(2):256-262[Xu LH,Huang QY,Chen WL.Bacterial bioremediation and bio-detection of heavy metalcontaminated environments[J].Chin J Appl Environ Biol,2004,10(2):256-262]
11 Langley S,Beveridge TJ.Effect of O-side-chain-lipopolysaccharide chemistry on metal binding[J].Appl Environ Microb,1999,65(2):489-498
12 Chen GC,Chen XC,Yang YQ,Anthony GH,Yu XH,and Chen YX.Sorption and distribution of copper in unsaturated Pseudomonas putida CZ1 biofilms as determined by X-ray fluorescence microscopy[J].Appl Environ Microb,2011,77(14):4719-4727
13 Tao L,Zhu ZK,Li FB,Wang SL.Fe(II)/Cu(II)interaction on goethite stimulated by an iron-reducing bacteria Aeromonas Hydrophila HS01under anaerobic conditions[J].Chemosphere,2017,187:43-51
14 Irawati W,Yuwono T,Rusli A.Detection of plasmids and curing a n a lysis i n c op p e r r e sist a nt ba c t e r ia Ac in e tob a c te r s p.I r C1,Acinetobacter sp.IrC2,and Cupriavidus sp.IrC4[J].Biodiversitas,2016,17:296-300
15 Pan XL,Liu J,Song WJ,Zhang DY.Biosorption of Cu(II)to extracellular polymeric substances(EPS)from Synechoeystis sp.:a f luorescence quenching study[J].Front End Sci Eng,2012,6(4):493-497
16 Wang Y,Qin J,Zhou S,Lin X,Ye L,Song C,Yan Y.Identification of the function of extracellular polymeric substances(EPS)in denitrifying phosphorus removal sludge in the presence of copper ion[J].Water Res,2015,73:252-264
17 Bhaskar PV,Bhosle NB.Microbial extracellular polymeric substances in marine biogeochemical processes[J].Curr Sci,2005,88(1):45-53
18 Papakonstanti EA,Stournaras C.Cell responses regulated by early reorganization of actin cytoskeleton[J].FEBS Lett,2008,582(14):2120-2127
19王宏归,黄晨,姜雅,王卓君,周春洪,严秋香.一株耐铜菌的筛选及其对铜去除能力的研究[J].广东化工,2015,42(19):22-22[Wang HG,Huang C,Jiang Ya,Wang ZJ,Zhou CH,Yan QX.Screening of a coppertolerant bacterial strain and study of its copper removing ability[J].Guangdong Chem Ind,2015,42(19):22-22]
20布坎南,吉本斯.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984[Buchanan RE,Gibbons NE.Berger’s Handbook on Bacterial Identification[M].8th ed.Beijing:Science Press,1984]
21姜琼,谢妤.苯酚-硫酸法测定多糖方法的改进[J].江苏农业科学,2013,41(12):316-318[Jiang Q,Xie Y.Improvement of method for determination of polysaccharide by phenol-sulfuric acid method[J].Jiangsu Agric Sci,2013,41(12):316-318]
22 Lowry OH,Rosebrough NJ,Farr AL,Randall RJ.Protein measurement with the folin phenol reagent[J].J Biol Chem,1951,193(1):265-275
23 Fr?lu nd B,Pal mg re n R,Keid i ng K,Nielse n P.Ex t r a ct ion of extracellular polymers from activated sludge using a cation exchange resin[J].Water Res,1996,30(8):1749-1758
24李哲,张欢,张秀芳,周野,吴迪,李明堂.一株碳酸盐矿化菌的分离鉴定及其对Cu的固定作用[J].环境科学学报,2017,37(10):3687-3695[Li Z,Zhang H,Zhang XF,Zhou Ye,Wu Di,Li MT.Isolation and identification of a carbonate mineralizer and its immobilization on copper[J].Acta Sci Circum,2017,37(10):3687-3695]
25 Cosgaya C,Maríalmirall M,Van AA,Fernández OD,Mosqueda N,Telli M,Huys G,Higgins PG,Seifert H,Lievens B,Roca I,Vila J.Acinetobacter dijkshoorniae sp.nov.a new member of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex mainly recovered from clinical samples in different countries[J].Int J Syst Evol Microbiol,2016,66(10):4105-4111
26 Rooney AP,Dunlap CA,Florweiler LB.Acinetobacter lactucae sp.nov.isolated from iceberg lettuce(Asteraceae:Lactuca sativa)[J].Int J Syst Evol Microbiol,2016,66(9):3566-3572
27 Maldonado J,Diestra E,Huang L,Domènech AM,Villagrasa E.Isolation and identification of a bacterium with high tolerance to lead and copper from a marine microbial mat in Spain[J].Ann Microbiol,2010,60(1):113-120
28 Deepika KV,Raghuram M,Kariali E,Bramhachari PV.Biological responses of symbiotic Rhizobium radiobacter strain VBCK1062 to the arsenic contaminated rhizosphere soils of mung bean[J].Ecotox Environ Safe,2016,134:1-10
29 Williams CL,Neu HM,Gilbreath JJ,Michel SLJ,Zurawski DV,Merrell DS,Dudley EG.Copper resistance of the emerging pathogen Acinetobacter baumannii[J].Appl Environ Microb,2016,82:6174-6188
30王海鸥,支蕊,王立曼,任晶,艾妍.一株耐铜不动杆菌UST B-F的富集特性[J].环境化学,2011,30(4):832-837[Wang HO,Zhi R,Wang LM,Ren J,Ai Y.Enrichment characteristics of a strain of copperresistant Acinetobacter:USTB-F[J].Environ Chem,2011,30(4):832-837]
31 Ueshima M,Ginn BR,Haack EA,Jennifer ES,Fein JB.Cd adsorption onto Pseudomonas putida,in the presence and absence of extracellular polymeric substances[J].Geochim Cosmochim Acta,2008,72(24):5885-5895
32 Garnham GW,Codd GA,Gadd GM.Accumulation of cobalt,zinc and manganese by the estuarine green microalga Chlorella salina immobilized in alginate microbeads[J].Environ Sci Technol,1992,26(26):1764-1770
33许朝阳,柏庭春,孟涛,周锋,吕惠,庞一山.铁细菌A生物修复铅铜污染土壤[J].河海大学学报(自然科学版),2015,43(6):569-573[Xu ZY,Bai TC,Meng T,Zhou F,Lv H,Pang YS.Bioremediation of soil contaminated by Pb2+or Cu2+using iron bacteria A[J].J Hohai Univ(Nat Sci Ed),2015,43(6):569-573]
34陈永华,向捷,吴晓芙,冯冲凌,袁斯文.三株耐铅锌菌的分离、鉴定及其吸附能力[J].生态学杂志,2015,34(9):2665-2672[Chen YH,Xiang Jie,Wu XF,Feng CL,Yuan SW.Isolation,identification and biosorption ability of three Pb2+and Zn2+tolerant bacterial strains[J].JAppl Ecol,2015,34(9):2665-2672]
35 Sayyadi S,Ahmady-Asbchin S,Kamali K,Tavakoli N.Thermodynamic,equilibrium and kinetic studies on biosorption of Pb+2,from aqueous solution by Bacillus pumilus sp.AS1 isolated from soil at abandoned lead mine[J].J Taiwan Inst Chem E,2017,80:701-708
36 Choińska-Pulit A,Sobolczyk-Bednarek J,?aba W.Optimization of copper,lead and cadmium biosorption onto newly isolated bacterium using a Box-Behnken design[J].Ecotox Environ Safe,2018,149:275-283
2 Grigalavi?ien?I,Rutkovien?V,Marozas V.The accumulation of heavy metals Pb,Cu and Cd at roadside forest soil[J].Pol J Environ Stud,2005,14(1):109-115
3 Mack ie K A,Müller T,Zi keli S,Kandeler E.Long-ter m copper application in an organic vineyard modifies spatial distribution of soil microorganisms[J].Soil Biol Biochem,2013,65(65):245-253
4 Lebrun JD,Trinsoutrotgattin I,Vinceslasakpa M,Bailleul C,Brault A,Mougin C,Laval K.Assessing impacts of copper on soil enzyme activities in regard to their natural spatiotemporal variation under longterm different land uses[J].Soil Biol Biochem,2012,49(6):150-156
5 Li J,Zheng YM,Liu YR,Ma YB,Hu HW,He JZ.Initial copper stress strengthens the resistance of soil microorganisms to a subsequent copper stress[J].Microbial Ecol,2014,67(4):931-941
6黄志钧,李大平.重金属铜离子抗性菌株的筛选和吸附性能[J].应用与环境生物学报,2012,18(6):964-970[Huang ZJ,Li DP.Cu2+Biosorption by a highly copper resistant bacterium isolated from soil[J].Chin J Appl Environ Biol,2012,18(6):964-970]
7林海,朱亦珺,董颖博,程煌,霍汉鑫.一株耐酸耐铜细菌的选育及其吸附铜离子的特性[J].环境化学,2013,32(4):599-604[Lin H,Zhu YJ,Dong YB,Cheng H,Huo HX.Breeding of a strain of acid-resistant and copper-tolerant bacteria and characteristics of copper ion adsorption[J].Environ Chem,2013,32(4):599-604]
8 Manohari,Singh J,Nandabalan YK.Copper(II)bioremoval by a rhizosphere bacterium,Stenotrophomonas acidaminiphila,MYS1-process optimization by RSM using Box-Behnken design[J].Int JEnviron Res,2017,11(1):63-70
9 Jiang CY,Sheng XF,Qian M,Wang QY.Isolation and characterization of a heavy metal-resistant Burkholderia sp.from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil[J].Chemosphere,2008,72(2):157-164
10徐磊辉,黄巧云,陈雯莉.环境重金属污染的细菌修复与检测[J].应用与环境生物学报,2004,10(2):256-262[Xu LH,Huang QY,Chen WL.Bacterial bioremediation and bio-detection of heavy metalcontaminated environments[J].Chin J Appl Environ Biol,2004,10(2):256-262]
11 Langley S,Beveridge TJ.Effect of O-side-chain-lipopolysaccharide chemistry on metal binding[J].Appl Environ Microb,1999,65(2):489-498
12 Chen GC,Chen XC,Yang YQ,Anthony GH,Yu XH,and Chen YX.Sorption and distribution of copper in unsaturated Pseudomonas putida CZ1 biofilms as determined by X-ray fluorescence microscopy[J].Appl Environ Microb,2011,77(14):4719-4727
13 Tao L,Zhu ZK,Li FB,Wang SL.Fe(II)/Cu(II)interaction on goethite stimulated by an iron-reducing bacteria Aeromonas Hydrophila HS01under anaerobic conditions[J].Chemosphere,2017,187:43-51
14 Irawati W,Yuwono T,Rusli A.Detection of plasmids and curing a n a lysis i n c op p e r r e sist a nt ba c t e r ia Ac in e tob a c te r s p.I r C1,Acinetobacter sp.IrC2,and Cupriavidus sp.IrC4[J].Biodiversitas,2016,17:296-300
15 Pan XL,Liu J,Song WJ,Zhang DY.Biosorption of Cu(II)to extracellular polymeric substances(EPS)from Synechoeystis sp.:a f luorescence quenching study[J].Front End Sci Eng,2012,6(4):493-497
16 Wang Y,Qin J,Zhou S,Lin X,Ye L,Song C,Yan Y.Identification of the function of extracellular polymeric substances(EPS)in denitrifying phosphorus removal sludge in the presence of copper ion[J].Water Res,2015,73:252-264
17 Bhaskar PV,Bhosle NB.Microbial extracellular polymeric substances in marine biogeochemical processes[J].Curr Sci,2005,88(1):45-53
18 Papakonstanti EA,Stournaras C.Cell responses regulated by early reorganization of actin cytoskeleton[J].FEBS Lett,2008,582(14):2120-2127
19王宏归,黄晨,姜雅,王卓君,周春洪,严秋香.一株耐铜菌的筛选及其对铜去除能力的研究[J].广东化工,2015,42(19):22-22[Wang HG,Huang C,Jiang Ya,Wang ZJ,Zhou CH,Yan QX.Screening of a coppertolerant bacterial strain and study of its copper removing ability[J].Guangdong Chem Ind,2015,42(19):22-22]
20布坎南,吉本斯.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984[Buchanan RE,Gibbons NE.Berger’s Handbook on Bacterial Identification[M].8th ed.Beijing:Science Press,1984]
21姜琼,谢妤.苯酚-硫酸法测定多糖方法的改进[J].江苏农业科学,2013,41(12):316-318[Jiang Q,Xie Y.Improvement of method for determination of polysaccharide by phenol-sulfuric acid method[J].Jiangsu Agric Sci,2013,41(12):316-318]
22 Lowry OH,Rosebrough NJ,Farr AL,Randall RJ.Protein measurement with the folin phenol reagent[J].J Biol Chem,1951,193(1):265-275
23 Fr?lu nd B,Pal mg re n R,Keid i ng K,Nielse n P.Ex t r a ct ion of extracellular polymers from activated sludge using a cation exchange resin[J].Water Res,1996,30(8):1749-1758
24李哲,张欢,张秀芳,周野,吴迪,李明堂.一株碳酸盐矿化菌的分离鉴定及其对Cu的固定作用[J].环境科学学报,2017,37(10):3687-3695[Li Z,Zhang H,Zhang XF,Zhou Ye,Wu Di,Li MT.Isolation and identification of a carbonate mineralizer and its immobilization on copper[J].Acta Sci Circum,2017,37(10):3687-3695]
25 Cosgaya C,Maríalmirall M,Van AA,Fernández OD,Mosqueda N,Telli M,Huys G,Higgins PG,Seifert H,Lievens B,Roca I,Vila J.Acinetobacter dijkshoorniae sp.nov.a new member of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex mainly recovered from clinical samples in different countries[J].Int J Syst Evol Microbiol,2016,66(10):4105-4111
26 Rooney AP,Dunlap CA,Florweiler LB.Acinetobacter lactucae sp.nov.isolated from iceberg lettuce(Asteraceae:Lactuca sativa)[J].Int J Syst Evol Microbiol,2016,66(9):3566-3572
27 Maldonado J,Diestra E,Huang L,Domènech AM,Villagrasa E.Isolation and identification of a bacterium with high tolerance to lead and copper from a marine microbial mat in Spain[J].Ann Microbiol,2010,60(1):113-120
28 Deepika KV,Raghuram M,Kariali E,Bramhachari PV.Biological responses of symbiotic Rhizobium radiobacter strain VBCK1062 to the arsenic contaminated rhizosphere soils of mung bean[J].Ecotox Environ Safe,2016,134:1-10
29 Williams CL,Neu HM,Gilbreath JJ,Michel SLJ,Zurawski DV,Merrell DS,Dudley EG.Copper resistance of the emerging pathogen Acinetobacter baumannii[J].Appl Environ Microb,2016,82:6174-6188
30王海鸥,支蕊,王立曼,任晶,艾妍.一株耐铜不动杆菌UST B-F的富集特性[J].环境化学,2011,30(4):832-837[Wang HO,Zhi R,Wang LM,Ren J,Ai Y.Enrichment characteristics of a strain of copperresistant Acinetobacter:USTB-F[J].Environ Chem,2011,30(4):832-837]
31 Ueshima M,Ginn BR,Haack EA,Jennifer ES,Fein JB.Cd adsorption onto Pseudomonas putida,in the presence and absence of extracellular polymeric substances[J].Geochim Cosmochim Acta,2008,72(24):5885-5895
32 Garnham GW,Codd GA,Gadd GM.Accumulation of cobalt,zinc and manganese by the estuarine green microalga Chlorella salina immobilized in alginate microbeads[J].Environ Sci Technol,1992,26(26):1764-1770
33许朝阳,柏庭春,孟涛,周锋,吕惠,庞一山.铁细菌A生物修复铅铜污染土壤[J].河海大学学报(自然科学版),2015,43(6):569-573[Xu ZY,Bai TC,Meng T,Zhou F,Lv H,Pang YS.Bioremediation of soil contaminated by Pb2+or Cu2+using iron bacteria A[J].J Hohai Univ(Nat Sci Ed),2015,43(6):569-573]
34陈永华,向捷,吴晓芙,冯冲凌,袁斯文.三株耐铅锌菌的分离、鉴定及其吸附能力[J].生态学杂志,2015,34(9):2665-2672[Chen YH,Xiang Jie,Wu XF,Feng CL,Yuan SW.Isolation,identification and biosorption ability of three Pb2+and Zn2+tolerant bacterial strains[J].JAppl Ecol,2015,34(9):2665-2672]
35 Sayyadi S,Ahmady-Asbchin S,Kamali K,Tavakoli N.Thermodynamic,equilibrium and kinetic studies on biosorption of Pb+2,from aqueous solution by Bacillus pumilus sp.AS1 isolated from soil at abandoned lead mine[J].J Taiwan Inst Chem E,2017,80:701-708
36 Choińska-Pulit A,Sobolczyk-Bednarek J,?aba W.Optimization of copper,lead and cadmium biosorption onto newly isolated bacterium using a Box-Behnken design[J].Ecotox Environ Safe,2018,149:275-283
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