Study On Population Of Marine Actinomycetes And Antibacterial Strains From Sediment Of Northern Yellow Sea

The bacterial population difference of the different functional categories of the North Yellow Sea were studied with 16S rDNA V3 variable region by denaturing gradient gel electrophoresis?DGGE?technology.The culturable actinomycetes in the samples were separated by combination culture,and the richness and difference of actinomycetes in marine samples were revealed.In order to develop and utilize marine actinomycetes further,three strains with different antibacterial functions were screened and identified,and their volatile metabolites,solid culture metabolites and liquid fermentation broth were researched to provide reference for the development and utilization of actinomycetes resources from different angles.The main results are as follows.1.The differences of bacterial populations and major environmental factors among different functional zones of the north Yellow Sea were clarified.Marine sediment samples were collected from 8 function zone,and the populations of bacteria in different samples and dominant species were determined and analyzed by PCR-DGGE.DGGE bands number were ranging from 18 to 26,and the main representative groups of these samples were revealed basically.Cluster analysis showed that all samples were clustered into three groups.The group I was Y1collected from saltern of Dandong,and Y2,Y4,Y5,Y6 which collected from the Dandong reserve areas and Dalian mariculture zones samples were clustered into group II,Y3,Y7and Y8 which all were from Dalian Xiaoyao Bay and bathing placewere clustered into one group III.Dominant DGGE bands were sequenced,the results showed that the dominant bacteria in all samples were Escherichia albertii and Methylophaga sp.of?-Proteobacteria.Amycolatopsis sp.,Phaeocystidibacter sp.and Salinimicrobium gaetbulicola were the dominant bacteria in Y2 which come from reserve areas of Dandong.Salinimicrobium sp.and Nitrosospira multiformis which were affiliated with?-Proteobacteria were detected as predominant species in Y4,Y5 andY6 sample of mariculture zones.There has no unique dominant species were detected in bathing beach sediment samples.The bacterial population diversity index include Richness index?S?,Shannon-Wiener index?H'?and Pielou index?E_h?were analyzed and showed that there was a little difference in the abundance of main bacterial populations in different function zone,and the Shannon index is between 2.41 and 3.10,and the evenness is between 0.835 and 0.985.RDA analysis of microbial structure and environmental factors showed that pH,total carbon and total nitrogen were important driving factor to bacterial community composition and structure diversity,and that the content of humic acid has little effect on the diversity of bacterial community.2.The main groups of actinomycetes in north Yellow Sea were identified and 3 excellent active strains were screened.145 marine actinomycetes were isolated from eight sites marine sediments with 8 media.114Streptomyces accounting for 78.6%and rare genus 31 strains accounted for 21.4%.Rare actinomycetes include:Lentzea,Marinobacter,Microbacterium,Micromonospora,Nesterenkonia,Nocardiopsis,nonomuraea,Pseudonocardia and Saccharothrix and so on.The results of Actinomycetes isolated and environmental factor determined showed the isolation rate of Actinomycetes dramatically differed among mariculture zones.The influencing factors may be the higher pH,type of aquatic products and antibacterial agents.The lowest detection rate of saltworks?Y1?indicated the extreme special environment has close influence on the species of actinomycetes.The detected rate of rare actinomyces showed media M1,GH SC and ISP5 was the optimum separation method of marine actinomycetes.The antibacterial activity of the volatile gas metabolites,solid culture products and liquid fermentation products were determined respectively.3 strains of actinomycetes HW80,HW85 and GQ-17 with excellent bacteriostatic effect were screened by determining the inhibition activity against common target strains.Then,classification,identification and metabolites of these strains will be study further.3.The strain HW80 was identified and its volatile metabolite components were defined.Taxonomic identification based on 16S rDNA sequence analysis,morphology,physiological and biochemical characterization were conducted.The result showed that strain HW80 was identified as Nocardiopsis dassonvillei HW80 because of the highest relatedness consistency with 99.93%similarity,and consistent cultural morphological characteristics,physiological and biochemical reactions phenomenon between HW80 and Nocardiopsis dassonvillei subsp.dassonvillei DSM 43111^T.The result of tested showed that volatile metabolites of HW80 have strong antifungal ability.It has inhibitory effect on all 9 tested plant pathogens strains except Exserohilum turcicum.Volatile metabolites cause the tip of filamentous mycelium to expand,thus affecting normal growth of mycelia.The composition analysis results of volatile metabolites from HW80 was carried out by HS-SPME-GC-MS showed that the volatile metabolites produced by HW80 contain 33 compounds,including 9 alcohols,9alkanes,6 alkenes,3 ketones and 2 acids.The main 6 compounds,including 3-nonyl alcohol?21.25%?,3-methyl-4-carbonyl pentanoic acid?16.99%?,1,4-dithiane-1-oxygen?10.81%?,3-methyl-p-mint-8-ene?7.65%?,methane sulfonic acid?5.63%?,and dimethyl ether?5.36%?.4.The classification status of strain HW85 and the antibacterial composition of the metabolites in solid culture were clarified.HW85 was identified as S.vinaceusdrappus HW85 according to the similarity of 16S rDNA sequence between HW85 and S.vinaceusdrappus NRRL 2363^T was 99.93%and the more similar morphological features and physiological and biochemical reaction.The HW85 has broad spectrum antibacterial activity to pathogenic fungi using the bacterial cake.It was cultured on modified fresh water medium?soluble starch 20g,KNO₃ 1g,K₂HPO₄ 0.5g,MgSO₄ 4.11g,Agar 15g,1L deionized water,pH 7.2⁷.4?.Active metabolites were extracted from solid culture by ethyl acetate.The studied on physical and chemical properties showed active components was not sensitive to temperature,acid,ultraviolet ray and protease.The light yellow crude product was extracted by TLC and silica gel column chromatography separation.High performance liquid chromatography?HPLC?was adopted with 254nm as the detection wavelength,with 85%-80%-85%methanol/aqueous solution as the mobile phase,and C18 reverse phase column was used for separation,thin layer chromatography for preparation and semi-preparation liquid phase for collection and concentration,then the white substances with a purity of 97.89%was obtained.UPLC-Q-TOF-MS,UV,IR,¹H NMR and ¹³C NMR were used for structural analysis,and the molecular weight was determined to be 913.55.Comparisons of literature and related spectrograms confirmed the white powder molecular formula was C₅₁H₇₉NO₁₃3 and unsaturation was13.The chemical name was:?3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS?-9,10,12,13,14,21,22,23,24,25,26,34a-16 Hydrogen-9,27-Two hydroxy-3-[?1R?-2-[?1 S,3R,4R?-hydroxyl-methoxy–cyclohexyl-]-1-methyl ethyl]-10,21-two methoxy-6,8,12,14,20,26-six methyl-23,27--3h-pyridine and[2,1-c][1,4]oxygen Miscellaneous nitrogen Miscellaneous 31-cyclohexene-1,5,one,?4H,6H,31H?-amyl ketone.It's reported triene macrocyclic lactone active material Rapamycin in literature,but this is the first time isolated from Streptomyces aeruginosa.5.The classification status of the strain GQ-17 and its effect on preventing disease and promoting growth of the metabolites in fermentation broth were clarified.Sequencing and comparison of 16S rDNA of GQ-17 and construction of phylogenetic tree with similar sequences showed the closest genetic relationship due to the similarity of 99.93%between strain GQ-17and the type stain Streptomyces rimosus subsp.rimosus JCM4667^T that produced oxytetracycline and rimocidin.Analysis of morphology,mycelium characteristics and physiological and biochemical reaction were performed,the results showed that all feature of GQ-17 were highly consistent with the type strain except for the resistance to acid and alkali?pH 4-12?and high temperature resistance?45??.GQ-17 was identified as Streptomyces rimosus GQ-17 ultimately.The active substance of GQ-17 fermentation liquid has a spectral inhibition effect on plant pathogenic fungi as the production stain of rimocidin and oxytetracycline.In this study,active metabolin ST-2 include GQ-17 was developed by the cultivation of mixed S.diastaticus YH-91 and S.pactum YH-23,and the fermentation conditions were optimized,and the effect of growth-promoting and anti-fungi were verified.The best fermentation medium and the optimal inoculation condition were determined by single-factor experiment and orthogonal experiment respectively.The best fermentation medium for ST-2 was screened out,which contains ingredients as follows:corn flour 8 g/L,soybean meal 10 g/L,sodium chloride 2.5 g/L,calcium carbonate 2.0 g/L,and nature pH.The optimum fermentation conditions was:the combination ratio of GQ-17,YH-91 and YH-23 was 3:3:2?V/V?,and the inoculation ratio was 3%into 50mL liquid medium in 250mL triangular flask,shaking at 31?for 5 d with 150 r/min.And the relative control effect was65.89%.In addition,ST-2 had obvious promoting effect because the stem length and diameter of plant were significant increased at 29.75%and 12.69%after using of the combination preparation than control respectively.