一株铬还原菌的分离鉴定及铬还原特性研究
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摘要
铬污染是目前最为普遍的重金属污染物之一,一定浓度的Cr(Ⅵ)会威胁动植物健康。用微生物修复技术降解高毒性的Cr(Ⅵ)可为环保高效的铬污染治理开辟新途径。针对从湖南某铬盐厂污染区土壤中分离筛选出的铬还原菌G12进行菌种鉴定及铬还原特性研究,明确该菌株的最适生长条件,考察其在不同环境条件下的铬还原效果,为原位微生物修复技术实际工程应用提供理论依据。通过菌株形态特征和16S r RNA基因序列分析,确定该菌株为革兰氏阳性芽孢杆菌,鉴定为短小芽孢杆菌(Bacillus pumilus)。研究发现B.pumilus G12的最佳生长温度和p H分别为30℃和9.0;且菌株铬还原能力随着初始Cr(Ⅵ)浓度的升高而下降;在50、100、200、400和600 mg·L~(-1)初始Cr(Ⅵ)浓度条件下其铬还原率分别为66.2%,35.7%,26.1%,16.0%和6.0%。在改变环境过程中,该菌株以甘油为外加碳源电子供体时对Cr(Ⅵ)的还原率可达100%,在60 h可将50mg·L~(-1)Cr(Ⅵ)还原为零;菌株G12培养可耐受较高盐浓度,在10 g·L~(-1) Na Cl盐浓度下菌株的还原能力最佳,Cr(Ⅵ)的去除率为70%;将菌株分别培养在含不同重金属离子的培养液中,菌株G12还原能力均受到抑制。对菌株G12的铬还原能力的初步研究结果表明,菌株G12在铬污染修复中具有良好的应用潜力。
Chromium contamination is one of the most common heavy metal pollutants, and a certain concentration of hexavalent chromium [Cr(Ⅵ)] will be a threat to the health of plants and animals. The use of microbial remediation technology of high toxicity degradation of Cr(Ⅵ) may be the environment-friendly and efficient management of chromium pollution. In this paper, the chromium-reducing bacteria screened from the contaminated area of a chromium salt plant in Hunan Province were used to identify the species and study the chromium reduction characteristics. Chromium reduction was also investigated under different environmental conditions, and the optimum growth conditions of the strain were determined. In order to remove off Cr(Ⅵ) from the chromium-contaminated sites, the study provides a theoretical basis for the practical engineering application of in-situ microbial remediation technology. In this study, an alkaline heterotrophic chromium reduction strain G12 was screened out. Through the morphological characteristics of the strains and 16 S rRNA gene sequence analysis, the strain was determined as Gram-positive Bacillus and identified as Bacillus pumilus. The optimum growth temperature and p H of Bacillus pumilus strain G12 were found to be 30 ℃ and 9.0. And the hexavalent chromium reduction ability of the strain decreased with the increase of initial Cr(Ⅵ) concentration, the reduction rates were 66.2%, 35.7%, 26.1%, 16.0% and 6.0% at initial Cr(Ⅵ) concentrations of 50, 100, 200, 400 and 600 mg·L~(-1), respectively. In the process of changing the environment, the reduction rate of this strain was 100% when using glycerol as carbon donor and electron donor, and 50 mg·L~(-1) of Cr(Ⅵ) could be reduced to zero at 60 h. Strain G12 could tolerate the higher salt concentration, the best reduction ability and the removal rate of Cr(Ⅵ) was 70% under the 10 g·L~(-1) Na Cl salt concentration. However, the strain was cultured in culture medium containing different heavy metal ions. The results of preliminary studies on the reduction ability of chromium in strain G12 show that strain G12 has good potential in chromium pollution repair.
Chromium contamination is one of the most common heavy metal pollutants, and a certain concentration of hexavalent chromium [Cr(Ⅵ)] will be a threat to the health of plants and animals. The use of microbial remediation technology of high toxicity degradation of Cr(Ⅵ) may be the environment-friendly and efficient management of chromium pollution. In this paper, the chromium-reducing bacteria screened from the contaminated area of a chromium salt plant in Hunan Province were used to identify the species and study the chromium reduction characteristics. Chromium reduction was also investigated under different environmental conditions, and the optimum growth conditions of the strain were determined. In order to remove off Cr(Ⅵ) from the chromium-contaminated sites, the study provides a theoretical basis for the practical engineering application of in-situ microbial remediation technology. In this study, an alkaline heterotrophic chromium reduction strain G12 was screened out. Through the morphological characteristics of the strains and 16 S rRNA gene sequence analysis, the strain was determined as Gram-positive Bacillus and identified as Bacillus pumilus. The optimum growth temperature and p H of Bacillus pumilus strain G12 were found to be 30 ℃ and 9.0. And the hexavalent chromium reduction ability of the strain decreased with the increase of initial Cr(Ⅵ) concentration, the reduction rates were 66.2%, 35.7%, 26.1%, 16.0% and 6.0% at initial Cr(Ⅵ) concentrations of 50, 100, 200, 400 and 600 mg·L~(-1), respectively. In the process of changing the environment, the reduction rate of this strain was 100% when using glycerol as carbon donor and electron donor, and 50 mg·L~(-1) of Cr(Ⅵ) could be reduced to zero at 60 h. Strain G12 could tolerate the higher salt concentration, the best reduction ability and the removal rate of Cr(Ⅵ) was 70% under the 10 g·L~(-1) Na Cl salt concentration. However, the strain was cultured in culture medium containing different heavy metal ions. The results of preliminary studies on the reduction ability of chromium in strain G12 show that strain G12 has good potential in chromium pollution repair.
引文
ALAM M Z,AHMAD S.2011.Chromium Removal through Biosorption and Bioaccumulation by Bacteria from Tannery Effluents Contaminated Soil[J].Clean-Soil Air Water,39(3):226-237.
ALOTHMAN Z A,ALI R,NAUSHAD M.2012.Hexavalent chromium removal from aqueous medium by activated carbon prepared from peanut shell:Adsorption kinetics,equilibrium and thermodynamic studies[J].Chemical Engineering Journal,184(2):238-247.
ALVARADO L,TORRES I R,CHEN A.2013.Integration of ion exchange and electrodeionization as a new approach for the continuous treatment of hexavalent chromium wastewater[J].Separation&Purification Technology,105(105):55-62.
CHEN J,CHEN R,HONG M.2015.Influence of p H on hexavalent chromium reduction by Fe(II)and sulfide compounds[J].Water Science&Technology A Journal of the International Association on Water Pollution Research,72(1):22-8.
CIACCI C,BARMO C,GALLO G,et al.2012.Effects of sublethal,environmentally relevant concentrations of hexavalent chromium in the gills of Mytilus galloprovincialis[J].Aquatic Toxicology,120-121:109-118.
DEVECI H,KAR Y.2013.Adsorption of hexavalent chromium from aqueous solutions by bio-chars obtained during biomass pyrolysis[J].Journal of Industrial&Engineering Chemistry,19(1):190-196.
DHAL B,ABHILASH,PANDEY B D.2013.Process optimization for bio-beneficiation of a chromite concentrate by a Cr(Ⅵ)reducing native microbe(Bacillus sp.)[J].International Journal of Mineral Processing,123(36):129-136.
DHARNAIK A S,GHOSH P K.2014.Hexavalent chromium[Cr(Ⅵ)]removal by the electrochemical ion-exchange process[J].EnⅥronmental Technology,35(18):2272-2279.
GE S M,GE S C,ZHOU M H,et al.2015.Bioremediation of hexavalent chromate using permeabilized Brevibacterium sp.and Stenotrophomonas sp.Cells[J].Journal of Environmental Management,157:54-59.
GRAHAM A M,BOUWER E J.2010.Rates of Hexavalent Chromium Reduction in Anoxic Estuarine Sediments:p H Effects and the Role of Acid Volatile Sulfides[J].Environmental Science&Technology,44(1):136-142.
GUTIéRREZ A M,PE?A CABRIALES J J,VEGA M M.2010.Isolation and characterization of hexavalent chromium-reducing rhizospheric bacteria from a wetland[J].International Journal of Phytoremediation,12(4):317-334.
HAMDI M,?BRAHIM P.2013.Batch study and kinetics of hexavalent chromium removal from aqueous solutions by anion exchange resin(Dowex 21 KCl)[J].Desalination&Water Treatment,51(10-12):2116-2120.
HE Z,LI S,WANG L,et al.2014.Characterization of Five Chromium-Removing Bacteria Isolated from Chromium-Contaminated Soil[J].Water Air&Soil Pollution,225(3):1-10.
JUNG C,HEO J Y,HAN H J,et al.2013.Hexavalent chromium removal by various adsorbents:Powdered activated carbon,chitosan,and single/multi-walled carbon nanotubes[J].Separation&Purification Technology,106(6):63-71.
KHORRAMABADI G S,SOLTANI R D C,REZAEE A,et al.2012.Utilisation of immobilised activated sludge for the biosorption of chromium(Ⅵ)[J].Canadian Journal of Chemical Engineering,90(6):1539-1546.
LIN X,LUO M,LI W,et al.2011.Reduction of hexavalent chromium by pannonibacter phragmitetus,LSSE-09 stimulated with external electron donors under alkaline conditions[J].Journal of Hazardous Materials,185(2-3):1169-1176.
NARAYANI M,VIDYA SHETTY K.2013.Chromium-resistant bacteria and their environmental condition for hexavalent chromium removal:Areview[J].Critical Reviews in Environmental Science&Technology,43(9):955-1009.
POORNIMA K,KARTHIK L,SWADHINI S P,et al.2010.Degradation of chromium by using a novel strains of Pseudomonas species[J].Journal of Microbial&Biochemical Technology,(4):95-99.
QIU L,MAO Y,GONG A,et al.2016.Inhibition effect of Bdellovibrio bacteriovorus on the corrosion of X70 pipeline steel induced by sulfate-reducing bacteria[J].Anti-Corrosion Methods and Materials,63(4):269-274.
SHIH Y J,CHEN C W,HSIA K F,et al.2015.Granulation for extended-release of nanoscale zero-valent iron exemplified by hexavalent chromium reduction in aqueous solution[J].Separation&Purification Technology,56(Part 3):1073-1081.
TERAHARA T,XU X,KOBAYASHI T,et al.2015.Isolation and Characterization of Cr(Ⅵ)-Reducing Actinomycetes from Estuarine Sediments[J].Applied Biochemistry&Biotechnology,175(7):3297-309.
THACKER U,SHOUCHE P Y,MADAMWAR D.2006.Hexavalent chromium reduction by Providencia sp[J].Process Biochemistry,41(6):1332-1337.
VITI C,PACE A,GIOVANNETTI L.2003.Characterization of Cr(Ⅵ)-resistant bacteria isolated from chromium-contaminated soil by tannery activity[J].Current Microbiology,46(1):1-5.
WANG J,PAN K,HE Q,et al.2013.Polyacrylonitrile/polypyrrole core/shell nanofiber mat for the removal of hexavalent chromium from aqueous solution[J].Journal of Hazardous Materials,244-245(9):121-129.
XAFENIAS N,ZHANG Y,BANKS C J.2015.Evaluating hexavalent chromium reduction and electricity production in microbial fuel cells with alkaline cathodes[J].International Journal of Environmental Science&Technology,12(8):2435-2446.
ZAHOOR A,REHMAN A.2009.Isolation of Cr(Ⅵ)reducing bacteria from industrial effluents and their potential use in bioremediation of chromium containing wastewater[J].Journal of Environmental Sciences,21(6):814-820.
ZHANG J K,WANG Z H,YE Y.2016.Heavy Metal Resistances and Chromium Removal of a Novel Cr(Ⅵ)-Reducing Pseudomonad Strain Isolated from Circulating Cooling Water of Iron and Steel Plant[J].Applied Biochemistry&Biotechnology,180(7):1328-1344.
ZHAO Q,LIU H,YING Z,et al.2010.Engineering of protease-resistant phytase from Penicillium sp.:High thermal stability,low optimal temperature and p H[J].Journal of Bioscience&Bioengineering,110(6):638-645.
ZHONG Y L,QIU X,CHEN D Y,et al.2016.Fabrication of Unique Magnetic Bionanocomposite for Highly Efficient Removal of Hexavalent Chromium from Water[J].Scientific Reports,6:31090.
陈育翔.2008.二苯碳酰二肼分光光度法测定电镀废水中六价铬的改进研究[J].化学工程与装备,(6):121-123.
郝宇.2015.湿地土壤重金属含量与理化性质相关性研究及生物修复策略[D].齐齐哈尔:齐齐哈尔大学生命科学与农林学院:24-36.
杨文玲,王继雯,慕琦,等.2013.耐Cr(Ⅵ)菌株的筛选及条件优化[J].河南科学,31(8):1175-1179.
ALOTHMAN Z A,ALI R,NAUSHAD M.2012.Hexavalent chromium removal from aqueous medium by activated carbon prepared from peanut shell:Adsorption kinetics,equilibrium and thermodynamic studies[J].Chemical Engineering Journal,184(2):238-247.
ALVARADO L,TORRES I R,CHEN A.2013.Integration of ion exchange and electrodeionization as a new approach for the continuous treatment of hexavalent chromium wastewater[J].Separation&Purification Technology,105(105):55-62.
CHEN J,CHEN R,HONG M.2015.Influence of p H on hexavalent chromium reduction by Fe(II)and sulfide compounds[J].Water Science&Technology A Journal of the International Association on Water Pollution Research,72(1):22-8.
CIACCI C,BARMO C,GALLO G,et al.2012.Effects of sublethal,environmentally relevant concentrations of hexavalent chromium in the gills of Mytilus galloprovincialis[J].Aquatic Toxicology,120-121:109-118.
DEVECI H,KAR Y.2013.Adsorption of hexavalent chromium from aqueous solutions by bio-chars obtained during biomass pyrolysis[J].Journal of Industrial&Engineering Chemistry,19(1):190-196.
DHAL B,ABHILASH,PANDEY B D.2013.Process optimization for bio-beneficiation of a chromite concentrate by a Cr(Ⅵ)reducing native microbe(Bacillus sp.)[J].International Journal of Mineral Processing,123(36):129-136.
DHARNAIK A S,GHOSH P K.2014.Hexavalent chromium[Cr(Ⅵ)]removal by the electrochemical ion-exchange process[J].EnⅥronmental Technology,35(18):2272-2279.
GE S M,GE S C,ZHOU M H,et al.2015.Bioremediation of hexavalent chromate using permeabilized Brevibacterium sp.and Stenotrophomonas sp.Cells[J].Journal of Environmental Management,157:54-59.
GRAHAM A M,BOUWER E J.2010.Rates of Hexavalent Chromium Reduction in Anoxic Estuarine Sediments:p H Effects and the Role of Acid Volatile Sulfides[J].Environmental Science&Technology,44(1):136-142.
GUTIéRREZ A M,PE?A CABRIALES J J,VEGA M M.2010.Isolation and characterization of hexavalent chromium-reducing rhizospheric bacteria from a wetland[J].International Journal of Phytoremediation,12(4):317-334.
HAMDI M,?BRAHIM P.2013.Batch study and kinetics of hexavalent chromium removal from aqueous solutions by anion exchange resin(Dowex 21 KCl)[J].Desalination&Water Treatment,51(10-12):2116-2120.
HE Z,LI S,WANG L,et al.2014.Characterization of Five Chromium-Removing Bacteria Isolated from Chromium-Contaminated Soil[J].Water Air&Soil Pollution,225(3):1-10.
JUNG C,HEO J Y,HAN H J,et al.2013.Hexavalent chromium removal by various adsorbents:Powdered activated carbon,chitosan,and single/multi-walled carbon nanotubes[J].Separation&Purification Technology,106(6):63-71.
KHORRAMABADI G S,SOLTANI R D C,REZAEE A,et al.2012.Utilisation of immobilised activated sludge for the biosorption of chromium(Ⅵ)[J].Canadian Journal of Chemical Engineering,90(6):1539-1546.
LIN X,LUO M,LI W,et al.2011.Reduction of hexavalent chromium by pannonibacter phragmitetus,LSSE-09 stimulated with external electron donors under alkaline conditions[J].Journal of Hazardous Materials,185(2-3):1169-1176.
NARAYANI M,VIDYA SHETTY K.2013.Chromium-resistant bacteria and their environmental condition for hexavalent chromium removal:Areview[J].Critical Reviews in Environmental Science&Technology,43(9):955-1009.
POORNIMA K,KARTHIK L,SWADHINI S P,et al.2010.Degradation of chromium by using a novel strains of Pseudomonas species[J].Journal of Microbial&Biochemical Technology,(4):95-99.
QIU L,MAO Y,GONG A,et al.2016.Inhibition effect of Bdellovibrio bacteriovorus on the corrosion of X70 pipeline steel induced by sulfate-reducing bacteria[J].Anti-Corrosion Methods and Materials,63(4):269-274.
SHIH Y J,CHEN C W,HSIA K F,et al.2015.Granulation for extended-release of nanoscale zero-valent iron exemplified by hexavalent chromium reduction in aqueous solution[J].Separation&Purification Technology,56(Part 3):1073-1081.
TERAHARA T,XU X,KOBAYASHI T,et al.2015.Isolation and Characterization of Cr(Ⅵ)-Reducing Actinomycetes from Estuarine Sediments[J].Applied Biochemistry&Biotechnology,175(7):3297-309.
THACKER U,SHOUCHE P Y,MADAMWAR D.2006.Hexavalent chromium reduction by Providencia sp[J].Process Biochemistry,41(6):1332-1337.
VITI C,PACE A,GIOVANNETTI L.2003.Characterization of Cr(Ⅵ)-resistant bacteria isolated from chromium-contaminated soil by tannery activity[J].Current Microbiology,46(1):1-5.
WANG J,PAN K,HE Q,et al.2013.Polyacrylonitrile/polypyrrole core/shell nanofiber mat for the removal of hexavalent chromium from aqueous solution[J].Journal of Hazardous Materials,244-245(9):121-129.
XAFENIAS N,ZHANG Y,BANKS C J.2015.Evaluating hexavalent chromium reduction and electricity production in microbial fuel cells with alkaline cathodes[J].International Journal of Environmental Science&Technology,12(8):2435-2446.
ZAHOOR A,REHMAN A.2009.Isolation of Cr(Ⅵ)reducing bacteria from industrial effluents and their potential use in bioremediation of chromium containing wastewater[J].Journal of Environmental Sciences,21(6):814-820.
ZHANG J K,WANG Z H,YE Y.2016.Heavy Metal Resistances and Chromium Removal of a Novel Cr(Ⅵ)-Reducing Pseudomonad Strain Isolated from Circulating Cooling Water of Iron and Steel Plant[J].Applied Biochemistry&Biotechnology,180(7):1328-1344.
ZHAO Q,LIU H,YING Z,et al.2010.Engineering of protease-resistant phytase from Penicillium sp.:High thermal stability,low optimal temperature and p H[J].Journal of Bioscience&Bioengineering,110(6):638-645.
ZHONG Y L,QIU X,CHEN D Y,et al.2016.Fabrication of Unique Magnetic Bionanocomposite for Highly Efficient Removal of Hexavalent Chromium from Water[J].Scientific Reports,6:31090.
陈育翔.2008.二苯碳酰二肼分光光度法测定电镀废水中六价铬的改进研究[J].化学工程与装备,(6):121-123.
郝宇.2015.湿地土壤重金属含量与理化性质相关性研究及生物修复策略[D].齐齐哈尔:齐齐哈尔大学生命科学与农林学院:24-36.
杨文玲,王继雯,慕琦,等.2013.耐Cr(Ⅵ)菌株的筛选及条件优化[J].河南科学,31(8):1175-1179.