一种耐Cr(Ⅵ)微生物筛选新方法
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摘要
六价铬(Cr(Ⅵ))是极具危害的环境污染物,利用微生物治理Cr(Ⅵ)污染是目前生物修复的热点之一,其中耐Cr(Ⅵ)微生物的筛选是关键。针对传统筛选方法培养基与Cr(Ⅵ)易相互作用的问题,文章提出先利用磷酸-Cr(Ⅵ)缓冲液浸泡筛选耐Cr(Ⅵ)微生物,然后在无Cr(Ⅵ)培养基上筛选的新方法(PCB法)。通过3种纯菌株进行筛选效果验证,并在尾矿土和草地土中与常规高温灭菌筛选(HS法)和过滤灭菌筛选法(FS法)进行筛选效果比较。纯菌株筛选结果表明,用含200 mg/L K2Cr2O7磷酸缓冲液(pH 7.4)浸泡微生物细胞2 h能有效筛选耐Cr(Ⅵ)菌株。土壤筛选实验表明,运用PCB法在尾矿土中获取菌落数为7.5×10~4CFU/g,与HS法相当,显著高于FS法;而在草地土中获取菌落数为1.8×10~4CFU/g,与FS法相当,显著低于HS法。HS法Cr(Ⅵ)在含200 mg/L K_2Cr_2O_7的牛肉膏蛋白胨培养基中回收率仅为53.8%,这是HS法获取菌落数高的原因。FS法筛选过程中微生物与Cr(Ⅵ)接触时间长,超过了微生物细胞生长对Cr(Ⅵ)的耐受极限,可能是FS法获得菌落数少的原因。总体而言,PCB法有效避免了上述缺陷,具有快速,准确,菌落生长好的特点。
Hexavalent chromium(Cr(Ⅵ)) is considered as a harmful contaminant in environment, its microbial remediation is one of the research hot spots, and microbial screening method is the key point. Considering the interaction between Cr(Ⅵ)and medium, a novel microbial screening method(PCB method) is proposed. Vegetative cells are firstly soaked in phosphateCr(Ⅵ) buffer, then Cr(Ⅵ) resistant cells are cultured in Cr(Ⅵ) free medium. The effects of PCB method were assessed by three strains. The results indicated that PCB method is an effective way to screen the Cr(Ⅵ) resistant microbial cells. PCB method was also evaluated by screening Cr(Ⅵ) resistant microbes from the tailings and grass land soils, and this method was compared with two traditional methods, including heat temperature sterilization screening method(HS) and filter sterilization screening method(FS). The resistant microbial number was 7.5×10~4 CFU/g of the tailing soil when using PCB method, as much as using HS method but significantly higher than FS method. However, the resistant microbial number was 1.8×10~4 CFU/g of the grass land soil when using PCB method, as much as FS method but significantly lower than HS method. In HS method, Cr(Ⅵ) recovery rate was 53.8% in nutrient broth with 200 mg/L K_2Cr_2O_7, which was the reason that the resistant microbial number increased. Prolonged exposure to high Cr(Ⅵ) levels results in growth tolerance limits of the microbes being exceeded, which was the reason that the resistant microbial number in FS method was low. In general, PCB method could screen Cr(Ⅵ) resistant microbes in a fast and accurate way with good quality of colony.
Hexavalent chromium(Cr(Ⅵ)) is considered as a harmful contaminant in environment, its microbial remediation is one of the research hot spots, and microbial screening method is the key point. Considering the interaction between Cr(Ⅵ)and medium, a novel microbial screening method(PCB method) is proposed. Vegetative cells are firstly soaked in phosphateCr(Ⅵ) buffer, then Cr(Ⅵ) resistant cells are cultured in Cr(Ⅵ) free medium. The effects of PCB method were assessed by three strains. The results indicated that PCB method is an effective way to screen the Cr(Ⅵ) resistant microbial cells. PCB method was also evaluated by screening Cr(Ⅵ) resistant microbes from the tailings and grass land soils, and this method was compared with two traditional methods, including heat temperature sterilization screening method(HS) and filter sterilization screening method(FS). The resistant microbial number was 7.5×10~4 CFU/g of the tailing soil when using PCB method, as much as using HS method but significantly higher than FS method. However, the resistant microbial number was 1.8×10~4 CFU/g of the grass land soil when using PCB method, as much as FS method but significantly lower than HS method. In HS method, Cr(Ⅵ) recovery rate was 53.8% in nutrient broth with 200 mg/L K_2Cr_2O_7, which was the reason that the resistant microbial number increased. Prolonged exposure to high Cr(Ⅵ) levels results in growth tolerance limits of the microbes being exceeded, which was the reason that the resistant microbial number in FS method was low. In general, PCB method could screen Cr(Ⅵ) resistant microbes in a fast and accurate way with good quality of colony.
引文
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[19]Alam M Z,Ahmad S.Toxic chromate reduction by resistant and sensitive bacteria isolated from tannery effluent contaminated soil[J].Annals of Microbiology,2012,62(1):113-121.
[20]Baldiris R,Acosta-Tapia N,Montes A,et al.Reduction of hexavalent chromium and detection of chromate reductase(ChrR)in Stenotrophomonas maltophilia[J].Molecules,2018,23(2):406.
[21]郭丽艳,陈晓明,朱捷,等.不同物质对铬价态测定结果的影响[J].安全与环境学报2012,12(1):165-169.
[22]Tanu F Z,Hakim A,Hoque S.Bacterial tolerance and reduction of chromium(Ⅵ)by Bacillus cereus isolate PGBw4[J].American Journal of Environmental Protection,2016,5(2):35-38.
[23]Kumar R,Nongkhlaw M,Acharya C,et al.Growth media composition and heavy metal tolerance behaviour of bacteria characterized from the sub-surface soil of uranium rich ore bearing site of Domiasiat in Meghalaya[J].Indian Journal of Biotechnology,2013,12(1):115-119.
[24]Fulladosa E,Desjardin V,Murat J C,et al.Cr(Ⅵ)reduction into Cr(Ⅲ)as a mechanism to explain the low sensitivity of Vibrio fischeri bioassay to detect chromium pollution[J].Chemosphere,2006,65(4):644-650.
[25]弭宝彬,杨剑,周火强,等.辣椒秸秆对铬Cr(Ⅵ)的吸附行为及机理[J].环境科学与技术,2017,40(S1):90-96.Mi Baobing,Yang Jian,Zhou Huoqiang,et al.Research on the adsorption behavior and mechanism of Cr(Ⅵ)by pepper stalk[J].Environmental Science&Technology,2017,40(S1):90-96.
[26]Yasemin S,Ayten O.Biosorption of chromium(Ⅵ)ions from aqueous solution by the bacterium Bacillus thuringiensis[J].Process Biochemistry,2005,4:1895-1901.
[27]Zheng Z,Li Y,Zhang X,et al.A Bacillus subtilis strain can reduce hexavalent chromium to trivalent and an nfrA gene is involved[J].International Biodeterioration&Biodegradation,2015,97:90-96.
[28]Ramirez Diaz M I,Diaz Perez C,Vargas E,et al.Mechanisms of bacterial resistance to chromium compounds[J].Biometals,2008,21(3):321-332.
[29]Donati E,Oliver C,Curutchet G.Reduction of chromium(Ⅵ)by the indirect action of Thiobacillus thioparus[J].Brazilian Journal of Chemical Engineering,2003,20:69-73.
[2]Hu G,Wang T,Liu J,et al.Serum protein expression profiling and bioinformatics analysis in workers occupationally exposed to chromium(Ⅵ)[J].Toxicol Lett,2017,277:76-83.
[3]Sawicka E,Dlugosz A.The role of 17beta-estradiol metabolites in chromium-induced oxidative stress[J].Adv Clin Exp Med,2017,26(2):215-221.
[4]Wang Z,Wu J,Humphries B,et al.Upregulation of histonelysine methyltransferases plays a causal role in hexavalent chromium-induced cancer stem cell-like property and cell transformation[J].Toxicol Appl Pharmacol,2018,342:22-30.
[5]Srinath T,Khare S,Ramteke P W.Isolation of hexavalent chromium-reducing Cr-tolerant facultative anaerobes from tannery effluent[J].The Journal of General and Applied Microbiology,2001,47(6):307-312.
[6]Ksheminska H P,Honchar T M,Gayda G Z,et al.Extra-cellular chromate-reducing activity of the yeast cultures[J].Central European Journal of Biology,2006,1(1):137-149.
[7]Vankar P S,Bajpai D.Phyto-remediation of chrome-Ⅵof tannery effluent by Trichoderma species[J].Desalination,2008,222(1):255-262.
[8]Morales-Barrera L,Guillén-Jiménez Fd M,Ortiz-Moreno A,et al.Isolation,identification and characterization of a Hypocrea tawa strain with high Cr(Ⅵ)reduction potential[J].Biochemical Engineering Journal,2008,40(2):284-292.
[9]Sharma S,Adholeya A.Detoxification and accumulation of chromium from tannery effluent and spent chrome effluent by Paecilomyces lilacinus fungi[J].International Biodeterioration&Biodegradation,2011,65(2):309-317.
[10]Han X,Wong Y S,Wong M H,et al.Biosorption and bioreduction of Cr(Ⅵ)by a microalgal isolate,Chlorella miniata[J].Journal of Hazardous Materials,2007,146(1):65-72.
[11]Deng L,Zhang Y,Qin J,et al.Biosorption of Cr(Ⅵ)from aqueous solutions by nonliving green algae Cladophora albida[J].Minerals Engineering,2009,22(4):372-377.
[12]Joutey N T,Sayel H,Bahafid W,et al.Mechanisms of hexavalent chromium resistance and removal by microorganisms[J].Reviews of Environmental Contamination and Toxicology,2015(233):45-69.
[13]牛永艳,陈正军,赵帅,等.铬还原菌的分离筛选及其在微生物燃料电池生物阴极中的应用[J].微生物学通报,2017,44(7):1631-1638.Niu Yongyan,Chen Zhengjun,Zhao Shuai,et al.Isolation of chromium reducing bacteria for application in biological cathode of microbial fuel cell[J].Microbiology China,2017,44(7):1631-1638.
[14]Wu G,Xiao X,Feng P,et al.Gut remediation:a potential approach to reducing chromium accumulation using Lactobacillus plantarum TW1-1[J].Sci Rep,2017,7(1):15000.
[15]马锦民,张烂漫,夏君,等.微生物处理含铬(Ⅵ)废水的研究进展[J].江苏化工,2005,33(2):46-50.
[16]胡永娟,李学梅,王海磊,等.抗铬菌株的筛选鉴定及其生物学特性和吸附特性[J].环境工程学,2014,8(6):2585-2591.Hu Yongjuan,Li Xuemei,Wang Hailei,et al.Screening of chromium resistance strains and their biological and adsorption characteristics[J].Chinese Journal of Environmental Engineering,2014,8(6):2585-2591.
[17]黄天培,张巧铃,潘洁茹,等.高效还原铬的苏云金芽胞杆菌菌株筛选[J].应用与环境生物学,2010,16(6):879-882.
[18]李维宏,杨宁,魏晓峰,等.一株Cr(Ⅵ)还原菌的筛选鉴定及其还原特性研究[J].农业环境科学学报,2015,34(11):2133-2139.Li Weihong,Yang Ning,Wei Xiaofeng,et al.Isolation,identification and Cr(Ⅵ)-reducing capacity of a Cr(Ⅵ)-reducing strain Enterobacterhormaechei[J].Journal of Agroenvironment Science,2015,34(11):2133-2139.
[19]Alam M Z,Ahmad S.Toxic chromate reduction by resistant and sensitive bacteria isolated from tannery effluent contaminated soil[J].Annals of Microbiology,2012,62(1):113-121.
[20]Baldiris R,Acosta-Tapia N,Montes A,et al.Reduction of hexavalent chromium and detection of chromate reductase(ChrR)in Stenotrophomonas maltophilia[J].Molecules,2018,23(2):406.
[21]郭丽艳,陈晓明,朱捷,等.不同物质对铬价态测定结果的影响[J].安全与环境学报2012,12(1):165-169.
[22]Tanu F Z,Hakim A,Hoque S.Bacterial tolerance and reduction of chromium(Ⅵ)by Bacillus cereus isolate PGBw4[J].American Journal of Environmental Protection,2016,5(2):35-38.
[23]Kumar R,Nongkhlaw M,Acharya C,et al.Growth media composition and heavy metal tolerance behaviour of bacteria characterized from the sub-surface soil of uranium rich ore bearing site of Domiasiat in Meghalaya[J].Indian Journal of Biotechnology,2013,12(1):115-119.
[24]Fulladosa E,Desjardin V,Murat J C,et al.Cr(Ⅵ)reduction into Cr(Ⅲ)as a mechanism to explain the low sensitivity of Vibrio fischeri bioassay to detect chromium pollution[J].Chemosphere,2006,65(4):644-650.
[25]弭宝彬,杨剑,周火强,等.辣椒秸秆对铬Cr(Ⅵ)的吸附行为及机理[J].环境科学与技术,2017,40(S1):90-96.Mi Baobing,Yang Jian,Zhou Huoqiang,et al.Research on the adsorption behavior and mechanism of Cr(Ⅵ)by pepper stalk[J].Environmental Science&Technology,2017,40(S1):90-96.
[26]Yasemin S,Ayten O.Biosorption of chromium(Ⅵ)ions from aqueous solution by the bacterium Bacillus thuringiensis[J].Process Biochemistry,2005,4:1895-1901.
[27]Zheng Z,Li Y,Zhang X,et al.A Bacillus subtilis strain can reduce hexavalent chromium to trivalent and an nfrA gene is involved[J].International Biodeterioration&Biodegradation,2015,97:90-96.
[28]Ramirez Diaz M I,Diaz Perez C,Vargas E,et al.Mechanisms of bacterial resistance to chromium compounds[J].Biometals,2008,21(3):321-332.
[29]Donati E,Oliver C,Curutchet G.Reduction of chromium(Ⅵ)by the indirect action of Thiobacillus thioparus[J].Brazilian Journal of Chemical Engineering,2003,20:69-73.
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