一株李氏禾内生细菌去除Cr(VI)的特性
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摘要
李氏禾(Leersia hexandra)是中国境内发现的第一种铬超积累植物,该文对李氏禾内生菌及其除铬性能进行了研究。采用添加Cr(VI)的牛肉膏蛋白胨固体平板培养方法,从李氏禾根部分离筛选获得一株具有较强Cr(VI)抗性的内生细菌G04,分子生物学鉴定结果表明该菌株属于阴沟肠杆菌(Enterobacter cloacae)。采用摇瓶培养方法,以Cr(VI)去除率、总Cr(铬)的去除率以及菌体生物量为指标,考察了pH、温度、底物浓度、装液量、接种量、摇床转速以及反应时间等因素对Cr(VI)去除率、总铬去除率和菌株生长的影响。结果表明:在牛肉膏蛋白胨液体培养基中,菌株E. cloacae G04去除Cr(VI)的较优反应条件为初始pH5. 0、温度37℃、Cr(VI)浓度为100 mg·L~(-1)、装液量80 mL(250 mL三角瓶)、接种量15%、摇床转速为120r·min~(-1)、反应时间48 h。在此条件下,菌株E. cloacae G04对Cr(VI)和总铬的去除率分别为84%和8%。根据Cr(VI)去除率和总铬去除率的结果推测该菌株去除Cr(VI)的机制可能是以还原为主、吸附为辅。这表明李氏禾内生细菌E. cloacae G04菌株具有较好的应用潜力,既有可能直接用于土壤、水环境铬污染的修复,也有可能作为促植物修复铬污染的后备菌株,另外可为深入研究李氏禾的铬积累作用机制提供参考。
Leersia hexandra is a chromium hyper-accumulative plant which was firstly found in China. The endophytic bacteria of L. hexandra and their removal capacity for Cr( VI) were studied. A Cr( VI)-resistant endophytic bacterium G04 was isolated from the roots of L. hexandra by solid plate culture method using the media of beef extract peptone containing Cr( VI). Biological identification results showed the strain belonged to Enterobacter cloacae. Effects of culture conditions,such as initial p H,temperature,Cr( VI) concentration,liquid volume,inoculation amount,shaking speed and culture time,on the removal rate of Cr( VI),removal rate of total Cr and the growth of the strain were studied in detail by shaking flask culture. The results showed that the optimal conditions for the removal of Cr( VI) by E. cloacae G04 were initial p H of 5.0,culture temperature of 37 ℃,substrate concentration of 100 mg·L~(-1),liquid volume of 80 m L in250 m L conical flask,inoculum size of 15%,shaking speed of 100 r·min~(-1) and culture time of 48 h. Under these conditions,the removal rate of Cr( VI) and total chromium were about 84% and 8%,respectively. The results of this study indicate that the endophytic bacteria Enterobacter cloacae G04 has better application potential for removing chromium. It may be used directly for remediation of soil and water environment contaminated of chromium,and also may be used as alternative strain for promoting plant remediation of chromium pollution. Furthermore,the result has an important reference value for illuminating the mechanism of chromium hyper-accumulation of L. hexandra.
Leersia hexandra is a chromium hyper-accumulative plant which was firstly found in China. The endophytic bacteria of L. hexandra and their removal capacity for Cr( VI) were studied. A Cr( VI)-resistant endophytic bacterium G04 was isolated from the roots of L. hexandra by solid plate culture method using the media of beef extract peptone containing Cr( VI). Biological identification results showed the strain belonged to Enterobacter cloacae. Effects of culture conditions,such as initial p H,temperature,Cr( VI) concentration,liquid volume,inoculation amount,shaking speed and culture time,on the removal rate of Cr( VI),removal rate of total Cr and the growth of the strain were studied in detail by shaking flask culture. The results showed that the optimal conditions for the removal of Cr( VI) by E. cloacae G04 were initial p H of 5.0,culture temperature of 37 ℃,substrate concentration of 100 mg·L~(-1),liquid volume of 80 m L in250 m L conical flask,inoculum size of 15%,shaking speed of 100 r·min~(-1) and culture time of 48 h. Under these conditions,the removal rate of Cr( VI) and total chromium were about 84% and 8%,respectively. The results of this study indicate that the endophytic bacteria Enterobacter cloacae G04 has better application potential for removing chromium. It may be used directly for remediation of soil and water environment contaminated of chromium,and also may be used as alternative strain for promoting plant remediation of chromium pollution. Furthermore,the result has an important reference value for illuminating the mechanism of chromium hyper-accumulation of L. hexandra.
引文
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COSTA M,1997.Toxicity and carcinogenicity of Cr(VI)in animal models and humans[J].Crit Rev Toxicol,27(5):431-442.
DHAL B,THATOI H,DAS N,et al.,2010.Reduction of hexavalent chromium by Bacillus sp.isolated from chromite mine soils and characterization of reduced product[J].JChem Technol Biotechnol,85(11):1471-1479.
DONG RZ,2012.Application of endophytes of arsenic hyperaccumulator in the resistance and adsorption of arsenic and lead in the aqueous solution[D].Nanchang:Nanchang Hangkong University.[董睿智,2012.砷超累积植物内生菌对重金属砷、铅抗性及吸附性能的研究[D].南昌:南昌航空大学.]
GE S,ZHOU M,DONG X,et al.,2013.Distinct and effective biotransformation of hexavalent chromium by a novel isolate under aerobic growth followed by facultative anaerobic incubation[J].Appl Microbiol Biotechnol,97(5):2131-2137.
HU C,2008.Study on mechanism of Cr accumulation and detoxification in Leersia hexandra Swartz[D].Guilin:Guilin University of Technology.[胡澄,2008.李氏禾对铬的富集解毒机制研究[D].桂林:桂林工学院.]
LIN KM,GUAN XF,MA LN,et al.,2008.The biological characteristics of organophosphorus pesticide-degrading bacterium,Enterobacter cloacae[J].Chin Agric Sci Bull,24(9):382-386.[林抗美,官雪芳,马丽娜,等,2008.有机磷农药降解菌-阴沟肠杆菌的生物学特性[J].中国农学通报,24(9):382-386.]
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LU WX,2015.Identification and characterization of manganeseresistant endophytic bacteria from manganese hyperaccumulator Phytolacca acinosa[D].Quanzhou:Fujian Normal University.[卢文显,2015.超富集植物商陆抗锰内生菌的筛选鉴定及其特性的研究[D].泉州:福建师范大学.]
LU YY,ZHANG XH,LIU J,et al.,2013.Mechanism of Cr(VI)uptake by hyperaccumulator Leersia hexandra Swartz[J].JAgro Environ Sci,32(11):2140-2144.[卢媛媛,张学洪,刘杰,等,2013.超富集植物李氏禾根系对Cr(Ⅵ)吸收机制的研究[J].农业环境科学学报,32(11):2140-2144.]
MALIK RN,HUSAIN SZ,NAZIR I,2010.Heavy metal contamination and accumulation in soil and wild plant species from industrial area of Islamabad,Pakistan[J].Pakistan JBot,42(1):291-301.
MA XZ,FEI BJ,JIN N,et al.,2009.Characteristic of reduce Cr(VI)by Desulfovibrio SRB7[J].Microbiol China,32(9):1324-1328.[马小珍,费保进,金楠,等,2009.脱硫弧菌SRB7对重金属铬Cr(VI)的还原特性[J].微生物学通报,32(9):1324-1328.]
OPPERMAN DJ,VAN HE,2007.Aerobic Cr(VI)reduction by Thermus scotoductus strain SA-01[J].J Appl Microbiol,103(5):1907-1913.
POOPAL AC,LAXMAN RS,2008.Hexavalent chromate reduction by immobilized Streptomyces griseus[J].Biotechnol Lett,30(6):1005-1010.
RAHMAN A,NAHAR N,OLSSON B,et al.,2016.Complete genome sequence of Enterobacter cloacae B2-DHA,a chromium-resistant bacterium[J].Genome Announc,4(3):e00483-516.
RAMASAMY S,ARUMUGAM A,CHANDRAN P,2017.Optimization of Enterobacter cloacae,(KU923381)for diesel oil degradation using response surface methodology(RSM)[J].J Microbiol,55(2):104-111.
SHANKER AK,CERVANTES C,LOZA-TAVERA H,et al.,2005.Chromium toxicity in plants[J].Environ Int,31(5):739-753.
SHI R,WU XF,LI Y,et al.,2015.Plant species applied in phytoremediation of heavy metal contaminated soils[J].JCentr S Univ For Technol,(4):139-146.[石润,吴晓芙,李芸,等,2015.应用于重金属污染土壤植物修复中的植物种类[J].中南林业科技大学学报,(4):139-146.]
WANG PC,MORI T,KOMORI K,et al.,1989.Isolation and characterization of an Enterobacter cloacae strain that reduces hexavalent chromium under anaerobic conditions[J].Appl Environ Microbiol,55(7):1665-1669.
WANG XJ,LIU J,2012.Study on experimental conditions of determination the total chromium in water by flame atomic absorption spectrophotometry[J].J Med Pest Contr,(4):462-464.[王新建,刘健,2012.原子吸收火焰光度法测定水中总铬实验条件的探讨[J].医学动物防制,(4):462-464.]
XU C,HE S,LIU Y,et al.,2017.Bioadsorption and biostabilization of cadmium by Enterobacter cloacae TU[J].Chemosphere,173:622.
XU YP,ZANG RC,CHEN WL,et al.,2001.Promoting plant growth and IAA of Enterobacter cloacae B8 fermentation liquid[J].J Zhejiang Univ(Agric Life Sci),27(3):282-284.[徐幼平,臧荣春,陈卫良,等,2001.阴沟肠杆菌B8发酵液对植物的促生作用和IAA分析[J].浙江大学学报(农业与生命科学版),27(3):282-284.]
XU WH,2007.Research on characteristic and mechanisms of microbial Cr(VI)reduction[D].Changsha:Hunan University.[徐卫华,2007.微生物还原Cr(Ⅵ)的特性与机理研究[D].长沙:湖南大学.]
YANG HL,SUN XL,SONG W,et al.,2001.Studies on the rice endophytic bacteria Enterobacter cloacae MR12's identification and it's effects of nitrogen fixation and biological control to plant disease[J].Acta Phytopathol Sin,31(1):92-93.[杨海莲,孙晓璐,宋未,等,2001.水稻内生阴沟肠杆菌MR12的鉴定及其固氮和防病作用研究[J].植物病理学报,31(1):92-93.]
ZHANG J,2011.Screening,identification and characteristics of a strain with high Cr(VI)degradation capacity[D].Xiangtan:Xiangtan University.[张剑,2011.高效降Cr(Ⅵ)菌的筛选、鉴定及其降解特性[D].湘潭:湘潭大学]
ZHANG JK,WANG ZH,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].Appl Biochem Biotechnol,180(7):1328-1344.
ZHANG XH,LIU J,HUANG HT,et al.,2007.Chromium accumulation by the hyperaccumulator plant Leersia hexandra Swartz[J].Chemosphere,67(6):1138-1143.
BROOKS RR,CHAMBERS MF,NICKS LJ,et al.,1998.Phytomining[J].Trends Plant Sci,3(9):359-362.
CAO Z,LUO SL,ZENG GM,et al.,2009.Removal of Cd2+by an endophytic bacteria SDE06 obtained from Solanum nigrum L.[J].Microbiol Chin,36(3):328-333.[曹喆,罗胜联,曾光明,等,2009.一株龙葵内生细菌SDE06去除Cd2+的实验[J].微生物学通报,36(3):328-333.]
CHEN J,WANG DQ,ZHANG XH,et al.,2008.Study on the potential of Leersia hexandra Swartz for remediation of heavy metal(Cr,Cu,Ni)polluted water[J].J Agro Environ Sci,27(4):1514-1518.[陈俊,王敦球,张学洪,等,2008.李氏禾修复重金属(Cr,Cu,Ni)污染水体的潜力研究[J].农业环境科学,27(4):1514-1518.]
CHEN Z,ZOU L,ZHANG H,et al.,2014.Thioredoxin is involved in hexavalent chromium reduction in Streptomyces violaceoruber strain LZ-26-1 isolated from the Lanzhou reaches of the Yellow River[J].Int Biodeterior Biodegrad,94:146-151.
CHIBUIKE GU,OBIORA SC,2014.Heavy metal polluted soils:Effect on plants and bioremediation methods[J].Appl Environ Soil Sci,2014:1-12.
COSTA M,1997.Toxicity and carcinogenicity of Cr(VI)in animal models and humans[J].Crit Rev Toxicol,27(5):431-442.
DHAL B,THATOI H,DAS N,et al.,2010.Reduction of hexavalent chromium by Bacillus sp.isolated from chromite mine soils and characterization of reduced product[J].JChem Technol Biotechnol,85(11):1471-1479.
DONG RZ,2012.Application of endophytes of arsenic hyperaccumulator in the resistance and adsorption of arsenic and lead in the aqueous solution[D].Nanchang:Nanchang Hangkong University.[董睿智,2012.砷超累积植物内生菌对重金属砷、铅抗性及吸附性能的研究[D].南昌:南昌航空大学.]
GE S,ZHOU M,DONG X,et al.,2013.Distinct and effective biotransformation of hexavalent chromium by a novel isolate under aerobic growth followed by facultative anaerobic incubation[J].Appl Microbiol Biotechnol,97(5):2131-2137.
HU C,2008.Study on mechanism of Cr accumulation and detoxification in Leersia hexandra Swartz[D].Guilin:Guilin University of Technology.[胡澄,2008.李氏禾对铬的富集解毒机制研究[D].桂林:桂林工学院.]
LIN KM,GUAN XF,MA LN,et al.,2008.The biological characteristics of organophosphorus pesticide-degrading bacterium,Enterobacter cloacae[J].Chin Agric Sci Bull,24(9):382-386.[林抗美,官雪芳,马丽娜,等,2008.有机磷农药降解菌-阴沟肠杆菌的生物学特性[J].中国农学通报,24(9):382-386.]
LONG XX,CHEN XM,HUANG HZ,et al.,2013.Feasibility of enhanced phytoextraction of Zn contaminated soil with Zn mobilizing and plant growth promoting endophyticbacteria[J].Trans Nonferr Metal Soc Chin,(8):2389-2396.[龙新宪,陈雪梅,黄焕忠,等,2013.促植物生长内生细菌强化植物修复锌污染土壤[J].中国有色金属学报,(8):2389-2396.]
LU WX,2015.Identification and characterization of manganeseresistant endophytic bacteria from manganese hyperaccumulator Phytolacca acinosa[D].Quanzhou:Fujian Normal University.[卢文显,2015.超富集植物商陆抗锰内生菌的筛选鉴定及其特性的研究[D].泉州:福建师范大学.]
LU YY,ZHANG XH,LIU J,et al.,2013.Mechanism of Cr(VI)uptake by hyperaccumulator Leersia hexandra Swartz[J].JAgro Environ Sci,32(11):2140-2144.[卢媛媛,张学洪,刘杰,等,2013.超富集植物李氏禾根系对Cr(Ⅵ)吸收机制的研究[J].农业环境科学学报,32(11):2140-2144.]
MALIK RN,HUSAIN SZ,NAZIR I,2010.Heavy metal contamination and accumulation in soil and wild plant species from industrial area of Islamabad,Pakistan[J].Pakistan JBot,42(1):291-301.
MA XZ,FEI BJ,JIN N,et al.,2009.Characteristic of reduce Cr(VI)by Desulfovibrio SRB7[J].Microbiol China,32(9):1324-1328.[马小珍,费保进,金楠,等,2009.脱硫弧菌SRB7对重金属铬Cr(VI)的还原特性[J].微生物学通报,32(9):1324-1328.]
OPPERMAN DJ,VAN HE,2007.Aerobic Cr(VI)reduction by Thermus scotoductus strain SA-01[J].J Appl Microbiol,103(5):1907-1913.
POOPAL AC,LAXMAN RS,2008.Hexavalent chromate reduction by immobilized Streptomyces griseus[J].Biotechnol Lett,30(6):1005-1010.
RAHMAN A,NAHAR N,OLSSON B,et al.,2016.Complete genome sequence of Enterobacter cloacae B2-DHA,a chromium-resistant bacterium[J].Genome Announc,4(3):e00483-516.
RAMASAMY S,ARUMUGAM A,CHANDRAN P,2017.Optimization of Enterobacter cloacae,(KU923381)for diesel oil degradation using response surface methodology(RSM)[J].J Microbiol,55(2):104-111.
SHANKER AK,CERVANTES C,LOZA-TAVERA H,et al.,2005.Chromium toxicity in plants[J].Environ Int,31(5):739-753.
SHI R,WU XF,LI Y,et al.,2015.Plant species applied in phytoremediation of heavy metal contaminated soils[J].JCentr S Univ For Technol,(4):139-146.[石润,吴晓芙,李芸,等,2015.应用于重金属污染土壤植物修复中的植物种类[J].中南林业科技大学学报,(4):139-146.]
WANG PC,MORI T,KOMORI K,et al.,1989.Isolation and characterization of an Enterobacter cloacae strain that reduces hexavalent chromium under anaerobic conditions[J].Appl Environ Microbiol,55(7):1665-1669.
WANG XJ,LIU J,2012.Study on experimental conditions of determination the total chromium in water by flame atomic absorption spectrophotometry[J].J Med Pest Contr,(4):462-464.[王新建,刘健,2012.原子吸收火焰光度法测定水中总铬实验条件的探讨[J].医学动物防制,(4):462-464.]
XU C,HE S,LIU Y,et al.,2017.Bioadsorption and biostabilization of cadmium by Enterobacter cloacae TU[J].Chemosphere,173:622.
XU YP,ZANG RC,CHEN WL,et al.,2001.Promoting plant growth and IAA of Enterobacter cloacae B8 fermentation liquid[J].J Zhejiang Univ(Agric Life Sci),27(3):282-284.[徐幼平,臧荣春,陈卫良,等,2001.阴沟肠杆菌B8发酵液对植物的促生作用和IAA分析[J].浙江大学学报(农业与生命科学版),27(3):282-284.]
XU WH,2007.Research on characteristic and mechanisms of microbial Cr(VI)reduction[D].Changsha:Hunan University.[徐卫华,2007.微生物还原Cr(Ⅵ)的特性与机理研究[D].长沙:湖南大学.]
YANG HL,SUN XL,SONG W,et al.,2001.Studies on the rice endophytic bacteria Enterobacter cloacae MR12's identification and it's effects of nitrogen fixation and biological control to plant disease[J].Acta Phytopathol Sin,31(1):92-93.[杨海莲,孙晓璐,宋未,等,2001.水稻内生阴沟肠杆菌MR12的鉴定及其固氮和防病作用研究[J].植物病理学报,31(1):92-93.]
ZHANG J,2011.Screening,identification and characteristics of a strain with high Cr(VI)degradation capacity[D].Xiangtan:Xiangtan University.[张剑,2011.高效降Cr(Ⅵ)菌的筛选、鉴定及其降解特性[D].湘潭:湘潭大学]
ZHANG JK,WANG ZH,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].Appl Biochem Biotechnol,180(7):1328-1344.
ZHANG XH,LIU J,HUANG HT,et al.,2007.Chromium accumulation by the hyperaccumulator plant Leersia hexandra Swartz[J].Chemosphere,67(6):1138-1143.