| ?-Poly(glutamic acid)(??-PGA) is a water-soluble macromolecular peptide that consists of D-and L-glutamic acids and is polymerized by??-glutamyl bonds.??-PGA is produced by a variety of Bacillus species and is freely excreted into the culture broth.It holds thickeners,humectants,substained release materials,form films and fibers.The polymer has been expected to be a new industrial biodegradable resources,which will solve some of the world-wide environmental problems,abandoning of waste plastic.Moreover,its use in the fields of food,cosmetics,or medicine has been proposed,and biodegradable fibers and hydrogels have already been made from ?-PGA.The polymer firmly stands as a potential candidate for replacing currently used nonbiodegradable water-soluble polymers. In this study,a high productive mutant of ?-PGA was obtained from Bacillus licheniformis WBL-3 with mutagenesis。The mutritional and environmental conditions were investigated。The effects of dissolved oxygen on the batch production of ?-PGA in a 5 L stirred fermentor by Bacillus licheniformis C9was studied。 Metabolic mechanism of carbon source of the production strain was studied。Main results are as follows:A high productive mutant of ?-PGA was obtained from Bacillus licheniformis WBL-3 with mutagenesis by NTG and 60Co。The productivity of ?-PGA was increased from 9.44g/L to 19.76g/L,the mutant showed genetic stability。The effects of shake flask conditions of Bacillus licheniformis C9 on the ?-PGA were investigated。According to the single-factor experiments,the L9(34) orthogonal experiments were carried out to optimize the concentrations of the nutrients。Aiming at achiving high ?-PGA concentration,the optimal mass concentrations of the nutrients were gained as following:citric acid 12.0g/L,glycerol 80.0g/L,L-glutamic acid 23.0g/L,ammonium chloride 7.0g/L. According to the single-factor experiments,the L25(56) orthogonal experiments were carried out to optimize the concentrations of the nutrients.The optimal mass concentrations of the nutrients were gained as following:FeCl3?6H2O 0.06g/L,CaCl2?2H2O 0.40g/L,(NH4)6Mo7O24?4H2O 0.20g/L,MgSO4 0.60g/L,K2HPO4 0.60g/L,MnSO4?H2O 0.104g/L.The environmental conditions of ?-PGA production were also investigated and an optimal combination was developed:an initial pH of 7.0,a medium content of 50 mL medium in 500mL flask,and an inoculum size of 5%,temperature 37℃.The mode of pH control is important factor for batch ?-PGA fermentation.At suitable culture conditions of temperature 37℃,aeration 1.17vvm.When Bacillus licheniformis C9 was cultivated without pH control,cell growth and ?-PGA production were inhibited to a certain extent due to the fact that pH decreased dramatically.At fermentation time 48h,DCW reached 6.22g/L,???h ?-PGA concentration reached 26.82g/L.At pH6.5 and fermentation time 40h,DCW reached 7.48g/L,???h ?-PGA concentration reached 29.16g/L.Batch fermentation of ?-PGA in 5.0L bioreactor was carried out.The effect of dissolved oxygen(DO)concentration on the batch production of ?-PGA in a 5L stirred fermentor by Bacillus licheniformis C9 was studied.It was found that DCW and ?-PGA production were greatly influenced by dissolved oxygen.When add 2%dodecane,??-PGA concentration higher than agitation rate 600. At fermentation time 48h,??-PGA concentration reached 38.06%,which increased 30.5% comparison to agitation rate 600.We undertook a set of experiments in which certain carbon sources were removed from medium in order to examine the role of each carbon source in terms of growth and ?-PGA production.It is presumed that L-glutamic acid is produced from citric acid via isocitric acid and ??keto glutaric acid in the TCA cycle.The presence of ethanol,acetic acid,pyruvic acid is indicative with HPLC.Glycerol was transforms to ATP via EMP cycle,which provides a great deal of energies for microorganism.
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