Study On Microbial Community Diversity And The Nitrogen Metabolism Flux Of Wine Grapes

Grape is one of the main economic crops in the world,the grape growers put a lot of fertilizer in the economic benefits over the years,the amount of nitrogen fertilizer can make the grape lush foliage,accelerate growth,and promote the formation of protein and chlorophyll,increase the accumulation of nutrient.At the same time,nitrogen is an essential nutrient element for wine yeast growth and fermentation.The composition and content of amino acids in grape juice are affected by grape varieties,producing areas,years,climate,maturity and nitrogen fertilizer.The understanding of wine grape microbes from the source provides a theoretical basis for the study of its effect on the quality and safety of wine.The main conclusions are as follows:Different varieties of wine grapes were analyzed based on T-RFLP,it was found that the specific fragment were 72,73.3,74.6,91.5,103.7,114.8,118.1,419.4,421.3,448.7,453.6,455.2,475.2,482.6,593.6bp respectively.Different varieties of grape rhizosphere microbial flora may be Garcinia Aeromonas,which was 74.6bp,the Fol of rhizosphere Simpson index was the biggest,Sau was the smallest,Shannon index and Simpson index remained the same.The specific bacteria T-RFs fragment of phyllosphere were 72,73.3,74.6,91.5,103.7,114.8,118.1,197.3,242,419.4,421.3,448.7,453.6,475.2,482.5,593.6 bp,the largest Simpson index in phyllosphere was Che,the smallest was Mul,which was agreement with the Shannon index.It was speculated that the comprehensive diversity index of the bacterial community structure of grape was closely related to the species and the colonization site.Based on Illumina technology,the 16S rRNA gene V5-V7 region and ITS1 region of 9 grape varieties on the fruit surface were analyzed.In the phylum level,different varieties of wine grape surface bacteria were mainly distributed in 8 known phylum?no particular order?:Proteobacteria,Actinobacteria,Firmicutes,Bacteroidetes Firmicutes Bacteroidetes,Gemmatimonadetes,Acidobacteria,Chloroflexi and Thermi.In the genus level,different varieties of wine grape surface bacteria were mainly distributed in 6genera?no particular order?:Erwinia,Pseudomonas sp.,Bacillus,Propionibacterium,Kaistobacter and Microbispora.Different varieties of wine grape surface fungal diversity are only distributed in 3phylum:Ascomycota,Basidiomycota and Zygomycota.Different varieties of grape surface fungi mainly distributed in 9 genera?no particular order?:Alternaria sp.,Cladosporium,Phoma,Fusarium,Guehomyces,Alternaria,Cryptococcus,Epicoccum and Myrothecium.Based on Illumina HiSeq2500 analysis of grape berries,leaves and rhizosphere soil bacterial and fungal community diversity,the abundance of 10 highest phylum of rhizosphere soil,leaf and grape berry were mainly distribution in Actinomycetes,Proteobacteria,Firmicutes,Bacillus,Gemmatimonadete,Acidobacteria and Nitrospirae,Chloroflexi,Verrucomicrobia and Planctomycetes.In these bacteria phylum,the highest abundance of rhizosphere soil was Actinomycetes?39.96%?,the lowest abundance was Planctomycetes?0.17%?;phyllosphere and rhizosphere was possed the same highest and lowest phylum,while different abundance,that was,Actinomycetes?39.58%?,and Planctomycetes?0.14%?;the highest abundance of berries was Proteobacteria?40.55%?,the 10 highest abundance of genera in rhizosphere,phyllosphere and berries were Blastococcus,Bacillus,Arthrobacter sp.,Gaiella,Pseudomonas,Nocardia sp.,Sphingolipids,Flavobacterium,Microvirg,Kocuria.Compared with the diversity of bacterial communities,the fungal diversity in grape soil,leaf and grape was far lower than that of bacteria at phylum and genera level.All of the fungi were distribution in the three main phylum?except not classified phylum?:Ascomycota,Basidiomycota and Chytridiomycota.Among all the samples,the highest abundances were the Ascomycota.The proportion in soil,leaves and grapes were66.59%,81.80%and 76.64%,respectively.On the level of genera,soil,leaves and leaf mainly distribution in Alternaria,Guehomyces,Cladosporium sp.Cladosporium,Cryptococcus,Phoma,Acremonium,Fusarium,Chaetomium,Hydropisphaera,Mycoarthris.The genus Alternaria was the highest abundance in all samples,the abundance in soil,leaves and grape was 17.08%,22.21%and 21.57%,respectively.The lowest genus of fungi in the soil was Mycoarthris?0.60%?.The lowest genus in leaves and grapes was Hydropisphaera,the relative abundance in leaves was 0.31%,and the relative abundance in grapes was 0.27%.Due to the participation of wine brewing by microorganisms,lacking of methods for determination of physical and chemical indexes of wine brewing which can guide the metabolic process.Therefore,based on stable isotope mass spectrometry to investigate the application prospect in grape fermentation process of analysis of metabolic flow,setting up nitrogen isotope amino acids in the fermentation liquid of inorganic nitrogen source ¹⁵N labeled amino acid substitution ¹⁵N markers for metabolic flux analysis method and determination method for arginine producing urea metabolic flux analysis.Based on the ¹⁵N labeling technique,a methodological foundation for the analysis of microbial metabolic flow by stable isotope tracing technique was established.In this study,in order to confirm the amino acid retention time,UV detector wavelength was set to 205 nm,first to use pure water as mobile phase.The separation of weak polar amino acids,and contains strong polar amino acids in a large initial peak,and then using the same column with 0.5 mM TFA as mobile phase collected.The size of isotopic fractionation in the system was verified by collecting the front peak,the tail peak and whole peak.The front peak?¹⁵N value?n=6?was-2.33‰,the tail peak was6.47‰,and the whole peak was 2.26‰.The front peak led to the loss of the ¹⁵N value,while the tail peak was enriched with¹⁵N.It was suggested that the isotope fractionation effect can lead to the enrichment of lighter isotope in a given substance peak,and then lacked the heavier isotope.Pretreatment with 0,0.01,0.02,0.05,0.1 g/L phenylalanine standard substances,respectively.The determination range of phenylalanine?¹⁵N measured by HPLC/EA-IRMS was between 8.17-8.25‰and the standard error was0.28‰.The theoretical and measured values were very close with the addition of phenylalanine standard material,and the linear correlation coefficient R²=0.9511.In order to further study whether the HPLC/EA-IRMS method can be used for metabolic flux analysis,inorganic nitrogen source using1-10%labeled ¹⁵NH₄Cl culture of Saccharomyces cerevisiae,it can be seen that,with the addition of ¹⁵NH₄Cl,HPLC/EA-IRMS determination of phenylalanine ¹⁵N values increased gradually,correlation coefficient R²=0.9483.It was suggested that the HPLC/EA-IRMS method can be applied to ¹⁵N labeled inorganic nitrogen sources,and was directly used for metabolic analysis of ¹⁵N markers on the required scale.According to the above methods,the arginine metabolic flow was explored,through the combination of inorganic nitrogen sources and labeled arginine,it was found that the amount of arginine converted to urea was 88.57%,the urea amino mainly derived from arginine,suggesting that the microbial fermentation of Saccharomyces cerevisiae was converted to urea 88.57%in arginine metabolism,which showed that this method can be used in marker in nitrogen.