Fermentation Optimization Of Corynebacterium Glutamicim ATCC 14067 Producting L-arginine

As an important semi-essential amino acid,L-arginine is widely used in food,medicine and feed.C.glutamicum is the main strain for the production of L-arginine by biological fermentation,whereas wild ATCC 14067 does not accumulate L-arginine.In the pre-laboratory work,the engineered strain ATCC 14067-XK99E-P_eftu-argB_H268E-argGH?BGH?was obtained by molecular engineering.It was shaken in CGXII medium for 72 h,and L-arginine accumulation was only 0.6 g/L.In this paper,the components of CGXII medium were optimized by genetic algorithm,and the composition ratio of the medium was more suitable for the growth of C.glutamicum and the accumulation of L-arginine.The medium gen₃-14 was obtained after optimization;Although gen₃-14 medium increased the growth stability in the late fermentation period and increased L-arginine accumulation of C.glutamicum,but the growth of the cells in the early fermentation period decreased slightly.In order to further improve the growth of C.glutamicum and increase the accumulation of L-arginine,based on gen₃-14 medium,the effects of adding different concentrations of soy peptone on the growth of C.glutamicum and the accumulation of L-arginine were investigated.And BPgen₃-14 medium which added 2g/L soy peptone to gen₃-14 medium was determined;The bioreactor can regulate process parameters such as pH,temperature and dissolved oxygen.At the bioreactor level,by analyzing the growth and metabolism about L-arginine accumulation of C.glutamicum at different fermentation period under high,medium and low dissolved oxygen levels,a partial dissolved oxygen regulation strategy was determined.Under the condition of partial dissolved oxygen,the accumulation of L-arginine is increased.1.In order to optimize the accumulation of L-arginine,the ratio of CGXII medium components was optimized by genetic algorithm,and gen₃-14 medium with the highest L-arginine accumulation was obtained.The L-arginine accumulation of C.glutamicum BGH was2.37 g/L after fermented on gen₃-14 medium for 72 h in shake flask.Compared with the medium not optimized,L-arginine accumulation increased by 288.52%.By analyzing the growth of C.glutamicum during the optimization process,found that the optimization of the ratio of the components by the genetic algorithm improved the growth of BGH in the late fermentation period.In further metabolic analysis,it was found that the culture medium optimized by the genetic algorithm enhanced the TCA cycle of C.glutamicum BGH and inhibited pyruvate conversing to other miscellaneous acids.2.The gen₃-14 medium obtained by genetic algorithm failed to improve the growth of C.glutamicum in the early fermentation period.In order to further increase the growth of C.glutamicum and the accumulation of L-arginine,the effects of adding different concentrations of soy peptone to gen₃-14 medium on growth and L-arginine accumulation were investigated.It was found that the addition of 2 g/L and 3 g/L of soy peptone significantly increased the growth of C.glutamicum BGH,and the accumulation of L-arginine was the highest with the addition of 2%soy peptone.Thus,a medium BPgen₃-14 which added 2 g/L of soy peptone to gen₃-14 medium was obtained.The L-arginine accumulation of C.glutamicum BGH was 2.87 g/L after fermented for 72 h in BPgen₃-14 medium,which was increased by 28.13%.3.The pH,temperature and dissolved oxygen can be regulated on the bioreactor,and dissolved oxygen is a very important parameter in the industrial production of L-arginine.On the 3L bioreactor,the growth,metabolism and L-arginine accumulation of C.glutamicum at different fermentation stages were analyzed when femented at 5%,20%and 35%dissolved oxygen conditions,and found to be 0-18 h in the early stage of fermentation.It was found that in the early fermentation period of 0-18 h,the accumulation of lactic acid was less and the synthesis rate of L-arginine was faster under the condition of 35%dissolved oxygen;At the late fermentation period of 18-48 h when fermented under 20%dissolved oxygen,the growth of the cells was better and more stable,and the accumulation of alanine is less,and the efficiency of conversion of glutamic acid to L-arginine is higher.Therefore,a partial dissolved oxygen regulation strategy was proposed to control dissolved oxygen at 35%at 0-18 h and control dissolved oxygen at 20%at 18-48 h.The L-arginine accumulation under the condition of partial dissolved oxygen reached 7.02 g/L,which was increased by 10.03%and29.20%compared with 20%and 35%dissolved oxygen.