Separation Of Growth-promoting Bacteria Sm-1 And Purification And Identification Of Siderophore

In the tobacco rhizosphere,there lives a wide variety of microorganisms,these microbes played an important role in changing the soil fertility level and nutrition structure,among which the beneficial microbes,formed many physiological mechanism to promote the growth and development of each other after a long period time of coevolution with tobacco.This study screened a growth-promoting bacteria named Sm-1 that poccess a strong ability of secreting siderophore from K326 tobacco rhizosphere,and Sm-1 strain was proved to be Herbaspirillum spp.through 16 S rDNA identification.High performance liquid chromatography(HPLC)method was used for the purification and identification of the siderophore,and Mass Spectrometry(MS)and Nuclear Magnetic Resonance(NMR)spectroscopy for the characterization.In order to obtain higher output of siderophore,Plackett-Burman experiment was conducted to optimize the fementative conditions of Sm-1.The main results of this paper was as follows:1.6 strains significantly different in exterior appearance were isolated from the K326 tobacco rhizosphere using the method of dilution butter on plates,according to the result of the CAS blue plate assay,Sm-1 was intended to be the one secreting the most siderophore.And 16 S rDNA identification of Sm-1 showed that the 16 S rDNA sequence of Sm-1 strain and Herbaspirillum spp.achieved a similarity of 99%,that is to say Sm-1 strain was identified to be Herbaspirillum spp.2.SM medium that insufficient of iron was chosed for the medium induced Sm-1 strain to secrete a large amount of siderophore for the 50 L bulk fermentation.Siderophore in the fermentation liquor of Sm-1 was separated and purified based on high performance liquid chromatography(HPLC),pure substance Sm-1-1-2-1,Sm-1-1-2-2 and Sm-1-1-3-1 were obtained,with a purity of 98%,respectively.Using mass spectrometry(MS)and nuclear magnetic resonance(NMR)spectroscopy to characterize the three substances,the results show that the Sm-1-1-2-1,Sm-1-1-2-2 and Sm-1-1-2-1 the molecular weight of 162.0461,444.1596 and 444.1592 respectively.3.SM culture medium for comparison,Plackett-Burman optimization experimental design of 17 factors,N=20 of Sm-1 was layed out,to screening the private factors that affecting the siderophore production of Sm-1 strain,the results showed that calcium and sulfate performed a most significantly effect on the production of siderophore of Sm-1 strain,vitamin B1 is the main resistance factor,C and N sources and other constituents have no significant influence for the production of the siderophore secreted by Sm-1 strain,the optimized fermentation culture medium of Sm-1 strain consist of calcium sulfate 0.3 g,vitamin B1 1.5 g,aspartic acid 1.5 g,potassium hydrogen phosphate 1.5 g,succinic acid 1.5 g and magnesium sulfate 0.3 g.The fermentation liquor of the Sm-1 strain cultivated in the optimized medium formed a much bigger orange halo(3.6 cm)on the CAS blue agar plate than that of SM medium(3.0 cm),it demonstrated that Sm-1 strain cultivated in the optimized medium can produce more siderophore.In conclusion,Sm-1 strain has a strong ability to secrete siderophore,the 16 S rDNA identification proved that Sm-1 strain was one of the Herbaspirillum huttiense.Iron deficiency culture medium can induce Sm-1 strain secrete siderophore,through the fermentation medium composition optimization can help increase the production of as much siderophore as possible of Sm-1 strain at a low cost.Siderophore secreted by Sm-1 strain separated and purified using high performance liquid chromatography(HPLC)method,which monomer,is beneficial to the future research related to the interaction mechanism between plant and rhizospheric microorganism,and the chelation mechanism between siderophore and heavy metals.The achievement of this paper provides theoretical basis and technical support to the exploration of siderophore secreted by rhizosphere bacteria of tobacco in promoting tobacco growth and the biological control of soil heavy metal pollution,also has extensive application in improving yield and quality of tobacco and soil ecological safety.