波茨坦短芽孢杆菌降解间甲酚和4-氯酚的特性
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摘要
为了高效处理工业含酚废水,研究了波茨坦短芽孢杆菌降解双底物体系过程中间甲酚和4-氯酚的相互作用及苯酚对间甲酚和4-氯酚降解的影响。结果表明,在间甲酚-4-氯酚体系中,4-氯酚会抑制间甲酚的降解,4-氯酚初始质量浓度为40 mg/L时,160 mg/L的间甲酚降解时间延长了8 h;同时间甲酚也抑制4-氯酚的降解,间甲酚初始质量浓度为40 mg/L时,160 mg/L的4-氯酚降解时间延长了4 h。采用Abuhamed动力学方程可以准确描述间甲酚-4-氯酚双底物降解体系中细胞生长的过程,动力学参数I_(1,2)=1.77,I_(2,1)=2.47,决定系数R~2=0.96。拟合参数表明,4-氯酚对间甲酚降解的抑制要强于间甲酚对4-氯酚降解的抑制。酶活测定表明,底物抑制作用增强时苯酚羟化酶和邻苯二酚1,2-双加氧酶的活性降低。添加低质量浓度的苯酚会对间甲酚和4-氯酚的降解产生促进作用,最佳促进质量浓度为200mg/L;添加300 mg/L以上的苯酚会对间甲酚和4-氯酚的降解产生抑制作用,抑制作用随苯酚质量浓度升高而增强。
This paper is aimed to investigate and explore the biodegradation characteristics of m-cresol and 4-chlorophenol in the double-component mixture by using the Brevibacillus borstelensis and their kinetics in hoping to define the effect of phenol on the biodegradation of m-cresol and 4-chlorophenol. To achieve the purpose,we have conducted a batch of experiments and tests in the form of flask cultivation and analysis of phenol,m-cresol and4-chlorophenol via HPLC. The experimental results show that the biodegradation capability of m-cresol tends to be inhibited at presence of 4-chlorophenol. The degradation of 160 mg/L m-cresol can be delayed for 8 h,20 h and 36 h when 40 mg/L,80 mg/L and 120 mg/L 4-chlorophenol were to be added,respectively.Meanwhile,the degradation of 4-chlorophenol would be totally inhibited at presence of m-cresol. Specifically,the delaying time of160 mg/L 4-chlorophenol can be 4 h,16 h and 28 h in the presence of 40 mg/L,80 mg/L and 120 mg/L m-cresol,correspondingly. In addition,we have also adopted a kinetical equation by Abuhamed,which can help to simulate cell growth adequately in the dual-substrate biodegradation system and the interaction parameters can be gained as the result of I_(1,2)= 1. 77 and I_(2,1)= 2. 47 with R~2= 0. 96. The inhibition rate of 4-chlorophenol on m-cresol proves to be stronger than that of m-cresol on the 4-chlorophenol according to the fitting coefficients. What is more,the specific enzyme activities of the phenol hydroxylase and catechol 1,2-dioxygenase in the mixture of 160 mg/L m-cresol and 120 mg/L4-chlorophenol have been found equal to( 0. 115 ± 0. 010) U/mg and( 0. 102 ± 0. 006) U/mg protein,respectively,both of which are lower than those in the single substrate solution of 160 mg/L m-cresol and 120 mg/L 4-chlorophenol. The enzyme assay reveals that,by adding another toxic compound to it,it would be possible to increase the inhibitive power on the enzyme activity,which may lead to lower metabolism of the phenolic compounds.Further exploration helps us to notice that adding 100 mg/L and200 mg/L of phenol may help to speed up obviously the biodegradation of m-cresol and 4-chlorophenol. For instance,in the presence of 200 mg/L phenol,the total degradation time has been found to be 64 h for 160 mg/L m-cresol and 68 h for 120 mg/L 4-chlorophenol,which implies an 8 h and 12 h shorter than that for the dual-substrate mixture,accordingly. But,if increasing the phenol concentration further to 300 mg/L and above,the biodegradation efficiency of m-cresol and 4-chlorophenol may tend to be inhibited and therefore lowered. Moreover,the inhibitive function tends to be increased with the addition of phenolic compounds.
This paper is aimed to investigate and explore the biodegradation characteristics of m-cresol and 4-chlorophenol in the double-component mixture by using the Brevibacillus borstelensis and their kinetics in hoping to define the effect of phenol on the biodegradation of m-cresol and 4-chlorophenol. To achieve the purpose,we have conducted a batch of experiments and tests in the form of flask cultivation and analysis of phenol,m-cresol and4-chlorophenol via HPLC. The experimental results show that the biodegradation capability of m-cresol tends to be inhibited at presence of 4-chlorophenol. The degradation of 160 mg/L m-cresol can be delayed for 8 h,20 h and 36 h when 40 mg/L,80 mg/L and 120 mg/L 4-chlorophenol were to be added,respectively.Meanwhile,the degradation of 4-chlorophenol would be totally inhibited at presence of m-cresol. Specifically,the delaying time of160 mg/L 4-chlorophenol can be 4 h,16 h and 28 h in the presence of 40 mg/L,80 mg/L and 120 mg/L m-cresol,correspondingly. In addition,we have also adopted a kinetical equation by Abuhamed,which can help to simulate cell growth adequately in the dual-substrate biodegradation system and the interaction parameters can be gained as the result of I_(1,2)= 1. 77 and I_(2,1)= 2. 47 with R~2= 0. 96. The inhibition rate of 4-chlorophenol on m-cresol proves to be stronger than that of m-cresol on the 4-chlorophenol according to the fitting coefficients. What is more,the specific enzyme activities of the phenol hydroxylase and catechol 1,2-dioxygenase in the mixture of 160 mg/L m-cresol and 120 mg/L4-chlorophenol have been found equal to( 0. 115 ± 0. 010) U/mg and( 0. 102 ± 0. 006) U/mg protein,respectively,both of which are lower than those in the single substrate solution of 160 mg/L m-cresol and 120 mg/L 4-chlorophenol. The enzyme assay reveals that,by adding another toxic compound to it,it would be possible to increase the inhibitive power on the enzyme activity,which may lead to lower metabolism of the phenolic compounds.Further exploration helps us to notice that adding 100 mg/L and200 mg/L of phenol may help to speed up obviously the biodegradation of m-cresol and 4-chlorophenol. For instance,in the presence of 200 mg/L phenol,the total degradation time has been found to be 64 h for 160 mg/L m-cresol and 68 h for 120 mg/L 4-chlorophenol,which implies an 8 h and 12 h shorter than that for the dual-substrate mixture,accordingly. But,if increasing the phenol concentration further to 300 mg/L and above,the biodegradation efficiency of m-cresol and 4-chlorophenol may tend to be inhibited and therefore lowered. Moreover,the inhibitive function tends to be increased with the addition of phenolic compounds.
引文
[1]FAN Yu(樊瑜),NIE Maiqian(聂麦茜),GE Bizhou(葛碧洲),et al.Screening of high-tolerance-phenol strain and its degrading capacity[J].Journal of Safety and Environment(安全与环境学报),2012,2(4):113-117.
[2]AHAMAD P Y A,KUNHI A A M.Degradation of high concentrations of cresols by Pseudomonas sp.CP4[J].World Journal of Microbiology and Biotechnology,1999,15(2):321-323.
[3]US EPA.National recommended water quality criteria,EPA-822-R-02-047[R].Washington DC:United States Environment Protection Agency,2002.
[4]ZHANG Xinyi(张心意),NIE Maiqian(聂麦茜),ZHAO Jing(赵静),et al.Effect of the co-existent carbon sources on the ring cleavage of phenol by Pseudomonas stutzeri N2[J].Journal of Safety and Environment(安全与环境学报),2015,15(5):239-243.
[5]JIANG Yan(姜岩).Mutation of Candida tropicalis by laser irradiation and its degradation characteristics of phenolic compounds(Candida tropicalis激光育种技术及其降酚特性研究)[D].Tianjin:Tianjin University,2005.
[6]ALEXIEVA Z,YEMENDZHIEV H,ZLATEVA P.Cresols utilization by Trametes versicolor and substrate interactions in the mixture with phenol[J].Biodegradation,2010,21(4):625-635.
[7]LIU Y,LIU J,LI C,et al.Metabolic profiling analysis of the degradation of phenol and 4-chlorophenol by Pseudomonas sp.cbp1-3[J].Biochemical Engineering Journal,2014,90(2):316-323.
[8]GE Qilong(葛启隆),WANG Guoying(王国英),YUE Xiuping(岳秀萍).Phenol degradation by Brevibacillus borstelensis and kinetic analysis[J].Biotechnology Bulletin(生物技术通报),2014,22(3):117-122.
[9]JIA Zilong(贾子龙),WANG Guoying(王国英),YUE Xiuping(岳秀萍),et al.Biodegradation of m-cresol and phenol by Brevibacillus borstelensis[J].Environmental Science and Technology(环境科学与技术),2015,38(11):37-41.
[10]LI Xiaoqian(李啸乾).Characteristic and kinetic study of 4-chlorophenol and phenol biodegradation by Brevibacillus borstelensis(波茨坦短芽孢杆菌降解4-氯酚与苯酚的特性及其动力学研究)[D].Taiyuan:Taiyuan University of Technology,2015.
[11]SALA-TREPAT J M,EVANS W C.The meta cleavage of catechol by Azotobacter species 4-oxalocrotonate pathway[J].European Journal of Biochemistry,1971,20(3):400-413.
[12]GE Qilong(葛启隆),YUE Xiuping(岳秀萍),WANG Guoying(王国英).Isolation and identification of a phenol-degrading strain and optimization for its immobilization using response surface methodology[J].China Environmental Science(中国环境科学),2014,34(2):518-525.
[13]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1):248-254.
[14]ABUHAMED T,BAYRAKTAR E,MEHMETOGLU T,et al.Kinetics model for growth of Pseudomonas putida F1 during benzene,toluene and phenol biodegradation[J].Process Biochemistry,2004,39(8):983-988.
[15]HEIPEIPER H J,KEWELOH H,REHM H J.Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli[J].Applied and Environmental Microbiology,1991,57(4):1213-1217.
[16]LIU Xiaolin(刘小林),WU Zhiquan(伍志权),HUANG Lei(黄磊),et al.Effects of protein denaturants on the activities of polygalacturonase and chemically modified polygalacturonase[J].Journal of Chemical Engineering of Chinese Universities(高校化学工程学报),2012,26(6):1009-1013.
[17]YU Chenyang(于晨阳),MAO Zhen(毛缜).Characteristics and kinetics study of m-cresol biodegradation by Bacillus cereus strain SMC[J].Chemical Industry and Engineering Progress(化工进展),2015,34(5):1453-1458.
[18]SINHA P K,SINHA A,DAS M.Microbial removal of phenol and p-chlorophenol from industrial waste water using Rhodococcus sp.RSP8 and its growth kinetic modeling[J].Journal of Water Resource and Protection,2011,3(8):634-642.
[19]ZHANG Ruijie(张瑞杰).Studies on cresols biotransformation by recombinant E.coli(PH_(IND))(苯酚羟化酶基因工程菌(PH_(IND))转化甲酚类化合物特性研究)[D].Dalian:Dalian University of Technology,2011.
[20]EL-SAYED W S,IBRAHIM M K,OUF S A.Molecular characterization of the alpha subunit of multicomponent phenol hydroxylase from 4-chlorophenol-degrading Pseudomonas sp.strain PT3[J].Journal of Microbiology,2014,52(1):13-19.
[2]AHAMAD P Y A,KUNHI A A M.Degradation of high concentrations of cresols by Pseudomonas sp.CP4[J].World Journal of Microbiology and Biotechnology,1999,15(2):321-323.
[3]US EPA.National recommended water quality criteria,EPA-822-R-02-047[R].Washington DC:United States Environment Protection Agency,2002.
[4]ZHANG Xinyi(张心意),NIE Maiqian(聂麦茜),ZHAO Jing(赵静),et al.Effect of the co-existent carbon sources on the ring cleavage of phenol by Pseudomonas stutzeri N2[J].Journal of Safety and Environment(安全与环境学报),2015,15(5):239-243.
[5]JIANG Yan(姜岩).Mutation of Candida tropicalis by laser irradiation and its degradation characteristics of phenolic compounds(Candida tropicalis激光育种技术及其降酚特性研究)[D].Tianjin:Tianjin University,2005.
[6]ALEXIEVA Z,YEMENDZHIEV H,ZLATEVA P.Cresols utilization by Trametes versicolor and substrate interactions in the mixture with phenol[J].Biodegradation,2010,21(4):625-635.
[7]LIU Y,LIU J,LI C,et al.Metabolic profiling analysis of the degradation of phenol and 4-chlorophenol by Pseudomonas sp.cbp1-3[J].Biochemical Engineering Journal,2014,90(2):316-323.
[8]GE Qilong(葛启隆),WANG Guoying(王国英),YUE Xiuping(岳秀萍).Phenol degradation by Brevibacillus borstelensis and kinetic analysis[J].Biotechnology Bulletin(生物技术通报),2014,22(3):117-122.
[9]JIA Zilong(贾子龙),WANG Guoying(王国英),YUE Xiuping(岳秀萍),et al.Biodegradation of m-cresol and phenol by Brevibacillus borstelensis[J].Environmental Science and Technology(环境科学与技术),2015,38(11):37-41.
[10]LI Xiaoqian(李啸乾).Characteristic and kinetic study of 4-chlorophenol and phenol biodegradation by Brevibacillus borstelensis(波茨坦短芽孢杆菌降解4-氯酚与苯酚的特性及其动力学研究)[D].Taiyuan:Taiyuan University of Technology,2015.
[11]SALA-TREPAT J M,EVANS W C.The meta cleavage of catechol by Azotobacter species 4-oxalocrotonate pathway[J].European Journal of Biochemistry,1971,20(3):400-413.
[12]GE Qilong(葛启隆),YUE Xiuping(岳秀萍),WANG Guoying(王国英).Isolation and identification of a phenol-degrading strain and optimization for its immobilization using response surface methodology[J].China Environmental Science(中国环境科学),2014,34(2):518-525.
[13]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72(1):248-254.
[14]ABUHAMED T,BAYRAKTAR E,MEHMETOGLU T,et al.Kinetics model for growth of Pseudomonas putida F1 during benzene,toluene and phenol biodegradation[J].Process Biochemistry,2004,39(8):983-988.
[15]HEIPEIPER H J,KEWELOH H,REHM H J.Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli[J].Applied and Environmental Microbiology,1991,57(4):1213-1217.
[16]LIU Xiaolin(刘小林),WU Zhiquan(伍志权),HUANG Lei(黄磊),et al.Effects of protein denaturants on the activities of polygalacturonase and chemically modified polygalacturonase[J].Journal of Chemical Engineering of Chinese Universities(高校化学工程学报),2012,26(6):1009-1013.
[17]YU Chenyang(于晨阳),MAO Zhen(毛缜).Characteristics and kinetics study of m-cresol biodegradation by Bacillus cereus strain SMC[J].Chemical Industry and Engineering Progress(化工进展),2015,34(5):1453-1458.
[18]SINHA P K,SINHA A,DAS M.Microbial removal of phenol and p-chlorophenol from industrial waste water using Rhodococcus sp.RSP8 and its growth kinetic modeling[J].Journal of Water Resource and Protection,2011,3(8):634-642.
[19]ZHANG Ruijie(张瑞杰).Studies on cresols biotransformation by recombinant E.coli(PH_(IND))(苯酚羟化酶基因工程菌(PH_(IND))转化甲酚类化合物特性研究)[D].Dalian:Dalian University of Technology,2011.
[20]EL-SAYED W S,IBRAHIM M K,OUF S A.Molecular characterization of the alpha subunit of multicomponent phenol hydroxylase from 4-chlorophenol-degrading Pseudomonas sp.strain PT3[J].Journal of Microbiology,2014,52(1):13-19.