Studies On Soil Microbial Diversity And Application In Shiyang River Basin And Wushaoling

The soil is the most appropriate growth environment for microorganisms.The regions of Shiyang river basin and Wushaoling contain various typical ecosystems in temperate climate zones such as Liushitan,alpine meadow,coniferous forest,broadleaf forest,grassland and desert.The diversity of environments has produced a variety of soil microbial populations and they were good regions for microbial resource investigation.So in this master thesis,the soil microbial diversities in typical ecosystems were researched by using traditional and high-throughput sequencing technologies on Shiyang river basin and Wushaoling.At the same time,the application values of the isolated culturable microorganisms were studied.A wild strain with inhibitory effect on staphylococcus aureus was successfully isolated and carried out studies on isolation and identification of the strain,mutagenic breeding and optimization of fermentation medium for breeding high-yield strain.The main results of the study were as follows:1 The studies of the soil microbial diversities on the regions of Shiyang river basin and WushaolingThe soil microbial diversities in typical ecosystems were researched by using traditional and high-throughput sequencing technologies on Shiyang river basin and Wushaoling.The main results of the study were as follows:?1?The number of culturable microorganisms in the soil of sampling sites basically conformed the rule that the number of bacteria was greater than that of actinomycetes than that of fungi.?2?The soil microbial contents,species and diversities were highly correlated with aboveground vegetation types and water conditions.In general,the microbial contents,species and diversities basically conformed to the rule of forest ecosystem>meadow ecosystem>grassland ecosystem>desert ecosystem and which were affected by the micro environment and had great variation.?3?There were 136 species of bacteria and 8 species of fungi in all the samples,the dominant microbial population types in various places were relatively consistent.The bacteria were mainly Actinobacteria,Proteobacteria and Acidobacteria,followed by Bacterioidetes,Chloroflexi and Gemmatimonadetes.The fungi were mainly Unclassified,followed by Ascomycota and Basidiomycota??4?There was a transitional microbial community structure between desert ecosystems and forest ecosystem in the grassland ecosystem.2 Studies on the application value of culturable microorganismsWe isolated culturable microorganisms from different sampling sites by method of the traditional separation,and the representative culturable fungi?F4-8,F1-1 and F13-10?in three typical ecosystems of the Shiyang River Basin were screened and studied which of the tolerance of heavy ion beam and ultraviolet light,and antibacterial experiment of Staphylococcus aureus.It was found that compared with the strain F4-8spores,F1-1 spores and F13-10 spores had higher survival rate under ¹²C⁶⁺heavy ion beam and ultraviolet irradiation environment,but the radiation tolerance of these two strains of fungi was far lower than the currently known radiation-resistant fungi.Antibacterial experiments showed that F1-1 can significantly inhibit the growth of Staphylococcus aureus than the other two fungi.After identification,F1-1 was Penicillium chrysogenum and its antibacterial product was penicillin G.After comprehensively comparing the characteristics of these three fungi,it was found that F1-1 has more application potential because of its antibacterial effect.3 The mutagenic screening and optimization of fermentation medium for Penicillium chrysogenum F1-1In order to select high-yield penicillin producing strains,this study induced the wild Penicillium chrysogenum strain F1-1 by the heavy ion beam irradiation and optimized the fermentation medium of high-yield strains.After irradiation,we conducted a preliminary screening,at the same time we counted the survival rete,positive and negative mutation rates.The results showed that the positive mutation rate was the highest when the irradiation dose was 150 Gy,which was 8.82%.The negative mutation rate was lower than other irradiation doses,and the survival rate was 24.09%,which was more conducive to screening high-yielding mutants.Finally,fifteen kind of positive mutant strains were selected from 168 strains,and then these 15 strains were shake-fermented and obtained two high-yield penicillin mutant strains C12 and D28.The two strains were fermented in shake flasks for six days,their penicillin production were 413.02 U/mL and 414.56 U/mL.Compared with wild strains,the penicillin production increased by 11.36%and 11.78%.By adjusting the carbon source and adding precursors to optimize the fermentation medium,it was found that the most suitable carbon source for strain D28 was sucrose?11%?.At the same time,the addition of precursors could effectively increase the yield of penicillin?486.60 U/mL,which increased 17.38%?.In this work,we studied the application value of the isolated target strain using heavy ion irradiation technology basised on the field investigation and sampling was studied.The obtained target strain has been identified as Penicillium chrysogenum and it was a penicillin-producing strain with application value.Although this strain did not have any production advantages compared with the current mature penicillin-producing strain,it has abundanted the antimicrobial resources.Therefore,it has been reported to the Chinese Industrial Microbial Culture Collection Management Center for deposit?deposit number MT341466-MT341467?.