解淀粉芽孢杆菌L-H15产促生物质分析及发酵工艺优化
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摘要
植物根际促生菌可以分解转化土壤中的有机质和矿物质,预防和控制农作物病害,减少农药和化肥的使用,将其应用于农业被认为是一种提高农作物产量的新型方法。实验室分离筛选到一株优良的解淀粉芽孢杆菌L-H15,通过前期L-H15全基因组测序的提示,考察L-H15能够产生的促生物质,并优化其发酵工艺,开发出高活性的固态菌剂。结果表明:解淀粉芽孢杆菌L-H15能够产生相对表达量为65.53%的铁载体;其产1-氨基环丙烷-1-羧酸脱氨酶的能力也较强,比活力测定结果为0.401 U/mg;采用酶联免疫吸附法检测到该菌株亦能产生植物激素吲哚乙酸和细胞分裂素,产细胞分裂素的能力尤为显著,可达到2.157 7×10~(-10) mol/L;运用气相色谱-质谱检测出L-H15产生的挥发性物质中含有乙酰甲基原醇。这些因素均可有效刺激植物的生长发育。采用Box-Behnken响应面试验优化解淀粉芽孢杆菌L-H15的发酵工艺,L-H15的最佳发酵条件为:接种量8%、装液量67.8%、发酵时间11.5 h、发酵温度32℃、转速196 r/min。此时,解淀粉芽孢杆菌L-H15活菌数最高可达到1.21×10~9 CFU/mL。综合评定,解淀粉芽孢杆菌L-H15是一株优良的植物根际促生菌,具有广阔的开发成生物肥料的前景。
Plant growth promoting rhizobacteria(PGPR)can decompose and transform organic substances and minerals in soil,prevent and control plant diseases and reduce the use of pesticides and fertilizers.Thus,PGPR are considered to be used as a new strategy to improve agricultural production.Bacillus amyloliquefaciens L-H15 is an excellent plant growth promoting rhizobacterium.Based on the results of previous studies of its full-length genomic sequence,we investigated the plant growth promoting properties of B.amyloliquefaciens L-H15 and optimized its culture conditions in order to develop a highly active solid starter culture.Our results indicated that B.amyloliquefaciens L-H15 produced a high level of siderophore(65.53%)and strong activity of 1-aminocyclopropane-1-carboxylate(ACC)deaminase showing a specific activity of 0.401 U/mg.This strain could also produce indole-3-acetic(IAA)and cytokinins(CTK).The ability to produce CTK was particularly significant,which was as high as 2.157 7×10~(-10) mol/L.Acetyl methyl alcohol,which can promote plant growth,was also detected in the volatiles generated by L-H15.These factors were able to effectively stimulate plant growth.We used response surface methodology based on Box-Behnken design to optimize the culture conditions of B.amyloliquefaciens L-H15 as follows:an inoculum size of 8%,a proportion of medium in a 500-mL volumetric flask of 67.8%,a culture temperature of 32℃,a culture time of 11.5 h,and a rotational speed of 196 r/min.Under these conditions,the viable cell count of B.amyloliquefaciens L-H15 was 1.21×10~9 CFU/mL.In conclusion,B.amyloliquefaciens L-H15 is an excellent PGPR,which has a broad prospect of development and utilization as a biofertilizer.
Plant growth promoting rhizobacteria(PGPR)can decompose and transform organic substances and minerals in soil,prevent and control plant diseases and reduce the use of pesticides and fertilizers.Thus,PGPR are considered to be used as a new strategy to improve agricultural production.Bacillus amyloliquefaciens L-H15 is an excellent plant growth promoting rhizobacterium.Based on the results of previous studies of its full-length genomic sequence,we investigated the plant growth promoting properties of B.amyloliquefaciens L-H15 and optimized its culture conditions in order to develop a highly active solid starter culture.Our results indicated that B.amyloliquefaciens L-H15 produced a high level of siderophore(65.53%)and strong activity of 1-aminocyclopropane-1-carboxylate(ACC)deaminase showing a specific activity of 0.401 U/mg.This strain could also produce indole-3-acetic(IAA)and cytokinins(CTK).The ability to produce CTK was particularly significant,which was as high as 2.157 7×10~(-10) mol/L.Acetyl methyl alcohol,which can promote plant growth,was also detected in the volatiles generated by L-H15.These factors were able to effectively stimulate plant growth.We used response surface methodology based on Box-Behnken design to optimize the culture conditions of B.amyloliquefaciens L-H15 as follows:an inoculum size of 8%,a proportion of medium in a 500-mL volumetric flask of 67.8%,a culture temperature of 32℃,a culture time of 11.5 h,and a rotational speed of 196 r/min.Under these conditions,the viable cell count of B.amyloliquefaciens L-H15 was 1.21×10~9 CFU/mL.In conclusion,B.amyloliquefaciens L-H15 is an excellent PGPR,which has a broad prospect of development and utilization as a biofertilizer.
引文
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[12]WANG Z,LIU Z,YU F,et al.Siderophore-modified Fenton-like system for the degradation of propranolol in aqueous solutions at near neutral p H values[J].Chemical Engineering Journal,2013,229(8):177-182.DOI:10.1016/j.cej.2013.05.118.
[13]蒙渊.产铁载体和ACC脱氨酶的氢氧化细菌筛选及促生作用研究[D].西安:西北大学,2011.
[14]孙红启.黑曲霉An76铁载体研究[D].济南:山东大学,2005.
[15]席琳乔,李德峰,王静芳,等.棉花根际促生菌固氮和分泌生长激素能力的测定[J].干旱区研究,2008,5(5):690-694.
[16]ORTíZCASTRO R,CONTRERASCORNEJO H A,MACíASRODRíGUEZ L.The role of microbial signals in plant growth and development[J].Plant Signaling&Behavior,2009,4(8):701-712.
[17]MIYAKAWA T,FUJITA Y,YAMAGUCHISHINOZAKI K,et al.Structure and function of abscisic acid receptors[J].Trends in Plant Science,2013,18(5):259-266.
[18]ZHANG J,LIU J,MENG L,et al.Isolation and characterization of plant growth-promoting rhizobacteria from wheat roots by wheat germ agglutinin labeled with fluorescein isothiocyanate[J].Journal of Microbiology,2012,50(2):191-198.DOI:10.1007/s12275-012-1472-3.
[19]DAMMES E V,FOUQUAERT E,LANNOO N,et al.Novel concepts about the role of lectins in the plant cell[J].Advances in Experimental Medicine&Biology,2011,705:271-294.
[20]王海滨,王平,陈永华.脱氨酶的作用机理和转基因的应用[J].生物技术通报,2009,4(2):40-43.
[21]RIBEIRO C M.Isolation,selection and characterization of rootassociated growth promoting bacteria in Brazil Pine(Araucaria angustifolia)[J].Microbiological Research,2012,167(2):69-78.DOI:10.1016/j.micres.2011.03.003.
[22]马嘉敏.苜蓿根际促生菌的分离及促生作用研究[D].哈尔滨:哈尔滨师范大学,2013.
[23]龚凤娟.具有ACC脱氨酶活性的内生细菌及根际细菌的分离鉴定及其植物促生作用[D].吉首:吉首大学,2012.
[24]周仲强.植物根际促生细菌功能菌株的筛选[D].长春:吉林大学,2007.
[25]GUERRERO-RODRíGUEZ J D,REVELL D K,BELLOTTI W D.Mineral composition of Lucerne(Medicago sativa)and white melilot(Melilotus albus)is affected by Na Cl salinity of the irrigation water[J].Fuel&Energy Abstracts,2011,170(12):97-104.DOI:10.1016/j.anifeedsci.2011.07.011.
[26]杨柳,郑华张,邑帆,等.凝结芽孢杆菌芽孢高产固体发酵的研究[J].家畜生态学报,2013,34(7):49-53.DOI:10.3969/j.issn.1673-1182.2013.07.011.
[2]Abd HALIM M R,SINNIAH U R,TORABI M,et al.Influence of salinity on the germination of Iranian alfalfa ecotypes[J].African Journal of Agricultural Research,2011,6(19):4624-4630.
[3]SCHWYN B,NEILANDS J B.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160(1):47-56.DOI:10.1016/0003-2697(87)90612-9.
[4]ADAMS D,YANG S F.Ethylene biosynthesis:identification of1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene[J].Proceeding of National Academy Science of the USA,1979,76(1):170-174.DOI:10.1073/pnas.76.1.170.
[5]RYU C M,FARAG M A,HU C H.Bacterial volatiles promote growth in Arabidopsis[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(8):4927-4932.
[6]KUKLINSKY-SOBRAL J,ARAúJO W L,MENDES R,et al.Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion[J].Environmental Microbiology,2005,6(12):1244-1251.DOI:10.1111/j.1462-2920.2004.00658.x.
[7]FARIA D C,DIAS A F,MELO I S,et al.Endophytic bacteria isolated from orchid and their potential to promote plant growth[J].World Journal of Microbiology&Biotechnology,2013,29(2):217-221.
[8]BOUTARD-HUNT C,SMART C D,THALER J,et al.Impact of plant growth-promoting rhizobacteria and natural enemies on myzus persicae infestations in pepper[J].Journal of Economic Entomology,2009,102(6):2183-2191.
[9]VACHERON J,DESBROSSES G,BOUFFAUD M L,et al.Plant growth-promoting rhizobacteria and root system functioning[J].Frontiers in Plant Science,2013,4(9):356-357.DOI:10.3389/fpls.2013.00356.
[10]VOLOVA T G,KISELEV E G,SHISHATSKAYA E I,et al.Cell growth and accumulation of polyhydroxyalkanoates CO2 and H2 of a hydrogen-oxidizing bacterium,Cupriavidus eutrophus B-10646[J].Bioresource Technology,2013,146(10):215-222.
[11]FISHER K,NEWTON W E.Nitrogenase proteins from Gluconacetobacter diazotrophicus,a sugarcane-colonizing bacterium[J].Biochimica Et Biophysica Acta,2005,1750(2):154-165.DOI:10.1016/j.bbapap.2005.04.010.
[12]WANG Z,LIU Z,YU F,et al.Siderophore-modified Fenton-like system for the degradation of propranolol in aqueous solutions at near neutral p H values[J].Chemical Engineering Journal,2013,229(8):177-182.DOI:10.1016/j.cej.2013.05.118.
[13]蒙渊.产铁载体和ACC脱氨酶的氢氧化细菌筛选及促生作用研究[D].西安:西北大学,2011.
[14]孙红启.黑曲霉An76铁载体研究[D].济南:山东大学,2005.
[15]席琳乔,李德峰,王静芳,等.棉花根际促生菌固氮和分泌生长激素能力的测定[J].干旱区研究,2008,5(5):690-694.
[16]ORTíZCASTRO R,CONTRERASCORNEJO H A,MACíASRODRíGUEZ L.The role of microbial signals in plant growth and development[J].Plant Signaling&Behavior,2009,4(8):701-712.
[17]MIYAKAWA T,FUJITA Y,YAMAGUCHISHINOZAKI K,et al.Structure and function of abscisic acid receptors[J].Trends in Plant Science,2013,18(5):259-266.
[18]ZHANG J,LIU J,MENG L,et al.Isolation and characterization of plant growth-promoting rhizobacteria from wheat roots by wheat germ agglutinin labeled with fluorescein isothiocyanate[J].Journal of Microbiology,2012,50(2):191-198.DOI:10.1007/s12275-012-1472-3.
[19]DAMMES E V,FOUQUAERT E,LANNOO N,et al.Novel concepts about the role of lectins in the plant cell[J].Advances in Experimental Medicine&Biology,2011,705:271-294.
[20]王海滨,王平,陈永华.脱氨酶的作用机理和转基因的应用[J].生物技术通报,2009,4(2):40-43.
[21]RIBEIRO C M.Isolation,selection and characterization of rootassociated growth promoting bacteria in Brazil Pine(Araucaria angustifolia)[J].Microbiological Research,2012,167(2):69-78.DOI:10.1016/j.micres.2011.03.003.
[22]马嘉敏.苜蓿根际促生菌的分离及促生作用研究[D].哈尔滨:哈尔滨师范大学,2013.
[23]龚凤娟.具有ACC脱氨酶活性的内生细菌及根际细菌的分离鉴定及其植物促生作用[D].吉首:吉首大学,2012.
[24]周仲强.植物根际促生细菌功能菌株的筛选[D].长春:吉林大学,2007.
[25]GUERRERO-RODRíGUEZ J D,REVELL D K,BELLOTTI W D.Mineral composition of Lucerne(Medicago sativa)and white melilot(Melilotus albus)is affected by Na Cl salinity of the irrigation water[J].Fuel&Energy Abstracts,2011,170(12):97-104.DOI:10.1016/j.anifeedsci.2011.07.011.
[26]杨柳,郑华张,邑帆,等.凝结芽孢杆菌芽孢高产固体发酵的研究[J].家畜生态学报,2013,34(7):49-53.DOI:10.3969/j.issn.1673-1182.2013.07.011.