Study On Fermentation Production Of γ-Polyglutamic Acid By Bacillus Subtilis HD11

γ-Polyglutamic acid (γ-PGA), an extracellular macromolecular peptide mainly produced by somebacteria in Bacillus, which consists of D-and L-glutamic acids through γ-glutamyl bonds. γ-PGA is apromising environmental friendly material with outstanding water solubility, biocompatibility anddegradability. For its excellent characters, γ-PGA has potential applications in a wide range of medicine,agriculture and food fields. The paper concentrates on the identification of strain producing γ-PGA, theoptimization of fermentation process and the solid fermentation conditions. Main results are as follows:(1) The strain of Bacillus sp. HD11producing γ-PGA was isolated and preserved by laboratory. Thestrain was identified as Bacillus subtilis through electron microscopy scanning and16S rRNA sequencinganalysis, and was nominated as Bacillus subtilis HD11.(2) A high productive strain F12of γ-PGA was obtained from Bacillus subtilis HD11by diethyl sulfateand nitrosoguanidine mutation. The yield of γ-PGA attained20.27g/L increasing by39.2%, and the mutantshowed genetic stability during10generation.(3) The liquid fermentation process of the strain F12screened by mutagenicity were investigated. Theexperimental results showed that the initial concentration and the supplemental methods of glucose andmonosodium glutamate could significantly influence the yield of γ-PGA. The optimal initial concentrationof glucose was15g/L. During the fermentation process, the glucose was added at l2h and24h respectivelywith the same amount and total amount was60g/L, in which the yield of γ-PGA attained26.32g/L. Theoptimal initial concentration of monosodium glutamate was30g/L, and its supplement occured at24h with40g/L total amount, in which the yield of γ-PGA attained28.4g/L. It was shown that the yield of γ-PGAcould reaches to30.57g/L.with supplement of glucose at12h and24h and monosodium glutamate at24h.Besides, the yield of γ-PGA could be improved to31.02g/L by adding30g/L glycerol at l6h. The yield ofγ-PGAcould reach to34.25g/L by adding glucose, monosodium glutamate and glycerol.(4) The fermentation conditions of γ-PGA with F12was optimized by adding of amino acids andorganic acids (L-glutamic acid, L-phenylalanine, L-glutamine, L-asparagine, L-valine, propylene acid,oxalic acid, and fumaric acid). Results showed that the synthesis of γ-PGA was improved by supplementingwith L-glutamic acid (4.0g/L), L-glutamine (3.0g/L) and L-valine (3.0g/L) individually. The final yield rate of γ-PGA reached43.68g/L,43.88g/L and34.68g/L respectively.(5) The solid state fermentation (SSF) of the strain F12was carried out to study the production ofγ-PGA, including the single factor test of culture medium matrix, carrier, ratio of material to water,inoculum, culture time, plus carbon and nitrogen, respectively. The optimal condition of the γ-PGAproduction was obtained in the mixed substrates of soybean powder and wheat bran, with ratio of materialto water1:1, inoculum8%, and incubated for36h. Supplemented with glucose (20g/kg), monosodiumglutamate (20g/kg) and urea (20g/kg) individually, the yield rate of γ-PGA reached90g/kg,101g/kg and98g/kg respectively.