Optimization Of Fermentation Conditions Of Physcion In Aspergillus Chevalieri BYST01

Physcion is a plant-derived fungicide developed independently in China,which has a good control effect on crop powdery mildew,downy mildew,grey mould,and anthracnose.However,the low yield of physcion in plant rhubarb leads to high production costs.Chemical synthesis is an important means to obtain the target compound on a large scale,but the existing chemical synthesis routes of physcion have disadvantages such as low yield,complex synthesis routes,and high production costs.How to improve the yield and reduce the production cost of physcion is a prominent problem that needs to be solved for this bio-sourced pesticide.The advantages of obtaining physcion from microbial ferments are very obvious:shorter growth period,easy strain selection,simple production conditions,and easy mass production.A strain of Aspergillus chevalieri BYST01,a high-yielding fungus of physcion,was isolated from the gut of the black-winged soil termite in the laboratory.For this reason,this thesis focuses on the optimization of the solid and liquid fermentation process conditions of BYST01 to lay the foundation for increasing its yield and reducing production costs.The main results obtained are as follows:(1)The components of BYST01’s solid fermentation medium were optimized by using physcion produced from agricultural waste.The experimental results showed that when sugar beet residue,wheat bran,rice bran,corn stalk,and bagasse were added as carbon sources on the basis of PDB medium,it was found that the yield of physcion was the highest when sugar beet residue was used as the carbon source,which was 20.42 mg/kg.On this basis,rapeseed cake,tea cake,sake lees,peanut cake,and soybean cake were added as nitrogen sources,and it was found that the content of physcion was highest when rapeseed cake was used as the nitrogen source,which was 29.25 mg/kg.It was further found that when the mass ratio of beet residue to rapeseed cake was 3:1,the physcion content was the highest,reaching 38.12 mg/kg dry weight.The results show that physcion can be produced by fermentation of BYST01 from agricultural waste.(2)Single-factor experiments were used to optimize the composition of BYST01’s liquid fermentation medium and culture conditions.The experimental results showed that when glucose,maltose,xylose,sucrose,and fructose were added as carbon sources,glucose as a carbon source was the most beneficial to the production of physcion BYST01.However,the addition of glycine,urea,ammonium chloride,ammonium sulfate,and yeast extract as nitrogen sources,and six inorganic salt ions,sodium acetate,calcium chloride,manganese chloride,sodium chloride,potassium chloride,and ferric chloride,could inhibit the production of physcion.The optimal liquid fermentation medium formula is as follows:add30 g/L glucose.The influence of culture conditions on fermentation was significant,especially p H and temperature on the yield of physcion.The optimum fermentation conditions were as follows:temperature 28℃,p H 6.5,shaking speed 180 r/min,fermentation time 11d.After the optimization of medium composition and culture conditions,the yield of physcion in BYST01 fermentation broth could reach 74 mg/L.(3)The Plackett-Burman method was used to select three factors that had significant effects on the yield of physcion,such as temperature,rotational speed,and p H.The response surface(RSM)Box-Behnken method was used to optimize the fermentation conditions.A,B,and C were used to define the three significant key factors of temperature,speed and p H,and the physcion yield was used as the response value,and Y was used to define,the regression equation obtained was:Y=79.04-11.67A-6.23B+5.2C+2.33AB-8.76AC-7.48BC-28.09A~2-33.86B~2-29.33C~2.Then optimal fermentation conditions were as follows:p H 6.6,temperature 28℃,shaking speed 177 r/min,and the yield of physcion in BYST01’s liquid fermentation reached 82 mg/L.Therefore,after the combination of single-factor and response surface methods,the yield of physcion produced by BYST01 was 2.93 times that before optimization,and the optimization effect was obvious.(4)We conducted a fermentation scale-up experiment on a 5 L fermentation tank by further using the obtained optimal formula of the medium and fermentation conditions.The results showed that the yield of physcion increased slowly before five days,and the highest yield was 85.2 mg/L at eight days,which was equivalentto the yield of shaking flask liquid fermentation at 11 days.(5)The isothermal plasma mutagenesis(ARTP)of BYST01 was performed for 500-700sec.The results showed that the fatality rate was low(23.2%)when the mutagenesis time was 500 sec,and high(97.2%)when the mutagenesis time was 700 sec.However,the yield of physcion produced by the mutated strain was similar to that of the original strain.