Studies On Mutagenesis Of Serratia Marcescens ZSG Producing Prodigiosin And Cultivation Process Optimization In 5L Fermentor

Prodigiosin is known as a potential anticancer drug.The synthetic method of prodigiosin mainly includes the chemical synthesis and microbial fermentation.First of all,the optimum conditions of high-yield prodigiosin strain breeding by DES-UV compound mutagenesis were studied.Then the culture medium formula were optimized by single factor and orthogonal experiments.At last,The fermentation conditions of the mutagenic strain in 5 L fermentor were optimized by single factor experiments,which may provides the reference for the industrialized production of prodigiosin.All the studies and results were as follows:Taking Serratia marcescens ZSG which was separated in own laboratory as original strain,the strain was mutated by DES-UV compound mutagenesis and screened according to prodigiosin production.The results showed that the mutant strain ZSG7 was obtained which had a higher yield of prodigiosin after the original strain was mutated by DES(1.0%,V/V)20 min and exposed in ultraviolet 20 s.In comparison of fermentation results in the shaking flask,the prodigiosin yield of ZSG7 was 0.104 g/L which was 1.63 times compared to original strain yield(0.064 g/L).In comparison of fermentation results in 5 L fermentor,the prodigiosin yield of ZSG7 was 0.1723 g/L which was 3.69 times compared to original strain yield(0.0467 g/L).Through five genetic stability tests of the mutant strain ZSG7,prodigiosin production is between 0.102 and 0.109 g/L,showing that the mutant strain ZSG7 has good genetic stability.Taking the mutant strain ZSG7 as production strain,carbon source,nitrogen source,inorganic salt and the surfactant of its culture medium ingredients were optimized by single factor experiments and the orthogonal experiment.The optimal culture medium formula was as follows: oleic acid 1.0%,peptone 1.5%,KCl 0.25%,Tween-80 1.0%,NaCl 0.5%.With the optimal culture medium in 250 mL flask,the prodigiosin production of the fermentation was 0.2079 g/L which was increased by 1 time compared to the unoptimized.The temperature,dissolved oxygen coupling speed,constant pH and the fed-batch process of the fermentation of Serratia marcescens ZSG7 in 5 L fermenter were optimized.The results were as follows: the loading volume 3 L in 5 L fermenter,fermentation time 78 h,fermentation temperature 29 ?,the initial speed 200 r/min,dissolved oxygen(DO)10% in 0-12 h,DO 30% in 12-24 h,DO 60% after 24 h,the constant pH7,the fed-batch components oleic acid 0.5%,peptone 0.5% and Tween-80 0.5%.DO value which the DO probe monitored real-time,was kept constant with adjusting speed automatically by the software of the fermentation system.In the process of fermentation,it was to guarantee the speed between 200 and 500 r/min by adjusting ventilation appropriately.The pH value which pH probe monitored real-time,was kept constant by the software of the fermentation system.The fed-batch medium was started to be supplemented through flow feeding by the peristaltic pump in the fermentation system at 42 h,which took 12 hours.By the optimization of the cultivation process,dry cell weight(DCW)had soared.The biggest DCWs before and after the fed-batch process were 1.49 g/L and 2.19 g/L,respectively 1.39 times and 2.05 times compared to the DCW 1.07 g/L of ZSG7 before optimization in 5 L fermentor.And prodigiosin production had soared.The biggest prodigiosin productions before and after the fed-batch process were 0.3822 g/L and 0.5643 g/L,respectively 1.78 times and 2.63 times compared to the prodigiosin production 0.2149 g/L of ZSG7 before optimization in 5 L fermentor.Kinetic models about bacteria growth and PG formation of the batch fermentation and the fed-batch fermentation were built based on the Logistic equation and Luedeking-Piret equation.With the fitting model parameters from Origin 8.0,the models could provide reasonable description for the processes of the batch fermentation and the fed-batch fermentation.