Mutation Breeding Of Adenosine And Optimal Control Of Fermentation Process

Adenosine is an important endogenous purine nucleoside within the body.It has a wide range of physiological and pharmacological effects.The production methods of adenosine mainly include chemical synthesis,RNA degradation and microbial fermentation.The production of adenosine by microbial fermentation has the advantages of low production cost,high yield,and no pollution to the environment.In this study,microbial fermentation was used to prepare adenosine.First,it imposes compound mutation to B.subtilis B-0 with Atmospheric and Room Temperature Plasma(ARTP)and 5-bromouracil(5-BU).After six rounds of mutations,it acquires adenosine strains with high yield B.subtilis B-6.Then by comparing different bioreactors,the results showed that the baffled flask was more suitable for the fermentation production of adenosine,and the fermentation conditions of adenosine were studied.Finally,a kinetic model of adenosine batch fermentation with Bacillus subtilis was established in baffled flasks.(1)Mutation breeding of high adenosine-producing strains.The compound mutagenesis of B.subtilis B-0 was carried out with ARTP and 5-BU,and the adenosine high-yield strain was selected quickly by the culture method of sulfaguanidine(SG)resistance primary screening and 48-well plates quantitative screening.The results showed that 48-well plates had marginal pore effect in Bacillus subtilis fermentation process,and 2 mL aseptic water filling could eliminate the marginal pore effect.Futhermore,a genetic stable mutant strain B.subtilis B-6 with 35%higher yield of adenosine than that of B.subtilis B-0 was finally obtained by 6 rounds of compound mutagenesis.Briefly,the combination of ARTP and 5-BU mutagenesis combined with 48-well plates can significantly improve the breeding efficiency and accuracy of adenosine high-yielding strains,and also can provide a theoretical reference for the selection of other nucleoside strains.(2)Comparison of adenosine fermentation bioreactors and fermentation conditions studies.Determine the most suitable bioreactors for adenosine fermentation,and effects of rotation speed,seed age,inoculation amount and initial pH on the production of adenosine by B.subtilis B-6 were studied.The results showed that under the same culture conditions,the baffled flask had better oxygen dissolution performance,and was more suitable for the production of adenosine by fermentation with B.subtilis B-6.The optimistic condition for adenosine fermentation in baffled flasks was 8%of inoculating quantity,inoculating age of 12 hours,initial pH of 7.5 and rotation rate of 200-240 r/min.The production volume of adenosine under this ideal condition will reach as high as 15.37 g/L,15.61%higher than normal conditions and 35.65%higher than that of unbaffled shake flasks.(3)A kinetic model of adenosine production by batch fermentation with Bacillus subtilis.Culture B.subtilis B-6 by baffled flasks,taking 6-8h sampling analysis on each.The study intends to establish a regression equation that includes cell growth,substrate consumption,and adenosine synthesis on the basis of Logistic model and Luedeking-Piret equation,using 1stOpt software to solve the model parameters.The result showed that the dynamic model cohered well with the experimental values of fermentation.And the model reflects the dynamic process of adenosine production by batch fermentation with Bacillus subtilis.The established three mathematical models for adenosine fermentation are as following:Kinetic model of cell growth:(?)Kinetic model of adenosine synthesis:P(t)=0.603X(t)+0.294+0.0091n(0.928+0.072e0.157t)Kinetic model of substrate consumption:S(t)=86.542-4.779X(t)-0.4751n(0.928+0.072e0.157t)...