一株用于石油污染修复的产漆酶菌种产酶条件优化及性能评价
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摘要
选取血红密孔菌Pycnoporus coccineus为产漆酶菌种,通过开展单因素产漆酶条件优化研究,明确最佳液体发酵工艺参数,制备出粗酶制剂并进行原油降解性能评价。结果表明:1)漆酶最佳液体发酵工艺参数为初始pH值5～7、初始温度30℃、摇床转速200r/min,接种量1.5%、碳源为羧甲基纤维素钠20.00g/L、氮源为硝酸钠5.00g/L、磷源磷酸氢二钾为2.00g/L、Cu2+浓度为1.00mmol/L、诱导剂为蔥2.00×10-4 mmol/L;复合制剂(粗酶制剂+菌剂)TPHs降解率高于单一制剂(仅投加菌液),环境耐受性、微生物底物作用能力及原油降解速率均显著提升。
Pycnoporuscoccineus was selected as the laccase producing strain.By optimizing the conditions of single factor laccase production,the optimal liquid fermentation process parameters were determined,and the crude enzyme was prepared and the degradation performance of crude oil was evaluated.The results show that:1)the optimum liquid fermentation process parameters for laccase are initial pH was between 5 and 7;the initial temperature was 30℃;the shaking speed was 200r/min;the inoculum was 1.5%(V/V);the alternative carbon source was carboxymethyl cellulose sodium(20.00g/L);the alternative nitrogen source was NaNO_3(5.00g/L);the alternative phosphorus source was KH_2PO_3(2.00g/L);the Cu~(2+)concentration was 1.00mmol/L;add inducer was shallot(2.00×10~(-4)mmol/L);2)the degradation rate of TPHs in the composite preparation(crude enzyme+bacterial agent)was higher than the single preparation(bacterial agent),and the environmental tolerance,the microbial substrate action ability and the crude oil degradation rate were all significantly improved.
Pycnoporuscoccineus was selected as the laccase producing strain.By optimizing the conditions of single factor laccase production,the optimal liquid fermentation process parameters were determined,and the crude enzyme was prepared and the degradation performance of crude oil was evaluated.The results show that:1)the optimum liquid fermentation process parameters for laccase are initial pH was between 5 and 7;the initial temperature was 30℃;the shaking speed was 200r/min;the inoculum was 1.5%(V/V);the alternative carbon source was carboxymethyl cellulose sodium(20.00g/L);the alternative nitrogen source was NaNO_3(5.00g/L);the alternative phosphorus source was KH_2PO_3(2.00g/L);the Cu~(2+)concentration was 1.00mmol/L;add inducer was shallot(2.00×10~(-4)mmol/L);2)the degradation rate of TPHs in the composite preparation(crude enzyme+bacterial agent)was higher than the single preparation(bacterial agent),and the environmental tolerance,the microbial substrate action ability and the crude oil degradation rate were all significantly improved.
引文
[1]周宇,马刚平,张向伟,等.多环芳烃类污染土壤强化生物修复技术研究及应用[J].环境工程,2017,35(12):184~188.
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[3]Zheng Zhongming,Jeffrey Philip Obbard.Effect of non-ionic surfactants on elimination of polycyclic aromatic hydrocarbons(PAHs)in soil-slurry by Phanerochaete chrysosporium[J].Journal of Chemical Technology&Biotechnology,2001,76(4):423~429.
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[10]Biao Fan,Yuechun Zhao,Ganhui Mo,et al.Co-remediation of DDT-contaminated soil using white rot fungi and laccase extract from white rot fungi[J].Journal of Soils and Sediments,2013,13(7):1232~1245.
[11]Gemma Eibes,A.Arca-Ramos,Gumersindo Feijoo,et al.Enzymatic technologies for remediation of hydrophobic organic pollutants in soil[J].Applied Microbiology and Biotechnology,2015,99(21):1~15.
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[13]宋佳宇,刘思敏,刘玉龙.固定化高效石油降解生物制剂制备及效果评价[J].油气田环境保护,2018.28(01):21~24.
[14]Eugene L.Madsen.Determining in situ biodegradation[J].Environmental Science and Technology,1991,25(10):1662~1673.
[15]史文婷.重油污染海岸线石油降解酶制剂-生物菌剂联合修复研究[D].北京:中国石油大学(北京),2016.
[16]Madhavi Vernekar,Smita S Lele.Laccase:properties and applications[J].Bioresources,2009,4(4):1694~1717.
[17]Karthikeyan,K,Singh Kuldeep.Bioremediation of polluted soils:An overview[D].Haryana(India):Haryana Agricultural University Hisar,2002.
[18]张雪莹,赵敏.发酵罐法培养疣孢漆斑菌产漆酶培养条件的研究[J].精细与专用化学品,2017,25(12):38~41.
[19]赵凤霞,张莉.白腐菌Trametes pubescens MB89产漆酶发酵条件的优化及其部分酶学性质的研究[J].西北农业学报,2009,18(4):175~180.
[20]张瑞玲,李鑫钢,黄国强,等.固体微生物菌剂现场修复油田污染土壤的应用研究初探[J].上海环境科学,2009(3):97~100.
[21]K Praveen,Buddolla Viswanath,KY Usha,et al.Lignolytic Enzymes of a Mushroom Stereum ostrea Isolated from Wood Logs[J].Enzyme Research,2011,2011(2-3):749518.
[22]王蕾,张永,田乔鹏,等.IMBH-2固态发酵产漆酶条件的优化及酶学性质的初步研究[J].科学技术与工程,2018,18(3):1671~1851.
[23]李环明.白腐真菌产漆酶培养基的优化及对嘧菌酯的降解[D].佳木斯:佳木斯大学,2017.
[24]Sergio Luiz Moreira,Adriane M F Milagres,Solange I Mussatto.Reactive dyes and textile effluent decolorization by a mediator system of salt-tolerant laccase from Peniophora cinerea[J].Separation and Purification Technology,2014,135(1):183~189.
[25]Feng Wang,Jian-Hua Hu,Chen Guo,et al.Enhanced laccase production by Trametes versicolor using corn steep liquor as both nitrogen source and inducer[J].Bioresource Technology,2014,166(1):602~605.
[26]Maria Teresa Cambria,Santa Ragusa,Vittorio Calabrese,et al.Enhanced laccase production in white-rot fungus Rigidoporus lignosus by the addition of selected phenolic and aromatic compounds[J].Applied biochemistry and biotechnology,2011,163(3):415~422.
[27]Miriam Lorenzo,Diego Moldes,Maria Angeles Sanromán.Effect of heavy metals on the production of several laccase isoenzymes by Trametes versicolor and on their ability to decolourise dyes[J].Chemosphere,2006,63(6):912~917.
[2]余欣欣,李建洲,陈立秀,等.微生物和酶在降解环境有机污染物中的应用[J].环境工程,2012,30(S2):97~100.
[3]Zheng Zhongming,Jeffrey Philip Obbard.Effect of non-ionic surfactants on elimination of polycyclic aromatic hydrocarbons(PAHs)in soil-slurry by Phanerochaete chrysosporium[J].Journal of Chemical Technology&Biotechnology,2001,76(4):423~429.
[4]张丽.铜离子诱导白腐菌高产漆酶及其用于降解染料的研究[D].广州:华南理工大学,2013.
[5]刘家扬,焦国宝,有小娟,等.真菌漆酶的性质、生产及应用研究进展[J].生物技术通报,2016,32(4):24~33.
[6]C RüttimannJohnson,R T Lamar.Polymerization of pentachlorophenol and ferulic acid by fungal extracellular lignin-degrading enzymes[J].Applied and Environmental Microbiology,1996,62(10):3890.
[7]A Krastanov.Removal of phenols from mixtures by co-immobilized laccase/tyrosinase and Polyclar adsorption[J].Journal of Industrial Microbiology and Biotechnology,2000,24(6):383~388.
[8]SusanaRodríguez Couto,JoséLuisToca Herrera.Industrial and biotechnological applications of laccases:a review[J].Biotechnology Advances,2006,24(5):500~513.
[9]司静,李伟,崔宝凯,等.真菌漆酶性质、分子生物学及其应用研究进展[J].生物技术通报,2011(2):48~55.
[10]Biao Fan,Yuechun Zhao,Ganhui Mo,et al.Co-remediation of DDT-contaminated soil using white rot fungi and laccase extract from white rot fungi[J].Journal of Soils and Sediments,2013,13(7):1232~1245.
[11]Gemma Eibes,A.Arca-Ramos,Gumersindo Feijoo,et al.Enzymatic technologies for remediation of hydrophobic organic pollutants in soil[J].Applied Microbiology and Biotechnology,2015,99(21):1~15.
[12]郑红婷,张秀霞,钟哲森,等.漆酶修复石油污染土壤优化实验[J].现代化工,2017.37(04):109~112.
[13]宋佳宇,刘思敏,刘玉龙.固定化高效石油降解生物制剂制备及效果评价[J].油气田环境保护,2018.28(01):21~24.
[14]Eugene L.Madsen.Determining in situ biodegradation[J].Environmental Science and Technology,1991,25(10):1662~1673.
[15]史文婷.重油污染海岸线石油降解酶制剂-生物菌剂联合修复研究[D].北京:中国石油大学(北京),2016.
[16]Madhavi Vernekar,Smita S Lele.Laccase:properties and applications[J].Bioresources,2009,4(4):1694~1717.
[17]Karthikeyan,K,Singh Kuldeep.Bioremediation of polluted soils:An overview[D].Haryana(India):Haryana Agricultural University Hisar,2002.
[18]张雪莹,赵敏.发酵罐法培养疣孢漆斑菌产漆酶培养条件的研究[J].精细与专用化学品,2017,25(12):38~41.
[19]赵凤霞,张莉.白腐菌Trametes pubescens MB89产漆酶发酵条件的优化及其部分酶学性质的研究[J].西北农业学报,2009,18(4):175~180.
[20]张瑞玲,李鑫钢,黄国强,等.固体微生物菌剂现场修复油田污染土壤的应用研究初探[J].上海环境科学,2009(3):97~100.
[21]K Praveen,Buddolla Viswanath,KY Usha,et al.Lignolytic Enzymes of a Mushroom Stereum ostrea Isolated from Wood Logs[J].Enzyme Research,2011,2011(2-3):749518.
[22]王蕾,张永,田乔鹏,等.IMBH-2固态发酵产漆酶条件的优化及酶学性质的初步研究[J].科学技术与工程,2018,18(3):1671~1851.
[23]李环明.白腐真菌产漆酶培养基的优化及对嘧菌酯的降解[D].佳木斯:佳木斯大学,2017.
[24]Sergio Luiz Moreira,Adriane M F Milagres,Solange I Mussatto.Reactive dyes and textile effluent decolorization by a mediator system of salt-tolerant laccase from Peniophora cinerea[J].Separation and Purification Technology,2014,135(1):183~189.
[25]Feng Wang,Jian-Hua Hu,Chen Guo,et al.Enhanced laccase production by Trametes versicolor using corn steep liquor as both nitrogen source and inducer[J].Bioresource Technology,2014,166(1):602~605.
[26]Maria Teresa Cambria,Santa Ragusa,Vittorio Calabrese,et al.Enhanced laccase production in white-rot fungus Rigidoporus lignosus by the addition of selected phenolic and aromatic compounds[J].Applied biochemistry and biotechnology,2011,163(3):415~422.
[27]Miriam Lorenzo,Diego Moldes,Maria Angeles Sanromán.Effect of heavy metals on the production of several laccase isoenzymes by Trametes versicolor and on their ability to decolourise dyes[J].Chemosphere,2006,63(6):912~917.