Optimizing Fermentation Parameters Of Poly γ-glutamic Acid From Halotolerant Bacillus Licheniformis And Effects Of Environmental Stresses Shock On The Production

The polyγ-glutamic acid（γ-PGA） is a viscous slime biopolymer containing D- and L-glutamate residues.Multifarious applications ofγ-PGA have been developed in food, cosmetics,medicinal industries and agriculture.A low molecular size poly-γ-glutamic acid（γ-PGA） productive strain,halotolerant bacterium WX-02 was isolated from the saline soil of China（Yingcheng）.By physiological,biochemical and 16S rDNA sequence analysis methods,the strain was identified as Bacillus licheniformis.The effect of NaCl concentration onγ-PGA production by WX-02 was investigated in modified E（ME） medium.It was found that theγ-PGA production was salt-inducible,and the highest volumetric yield ofγ-PGA（13.86g/l） was attained with 8%of NaCl.It was also observed that the molecular size ofγ-PGA decreased,when the NaCl concentration increased.This was the first report of isolation of aγ-PGA productive strain,halotolerant B.licheniformis.The flaskes fermentation medium and cultivation conditions were optimized by B. licheniformis WX-02.The optimal fermentation medium contains（g/L）:glucose 70, sodium glutamic acid 70,sodium citrate 8,NH₄Cl 8,MnSO₄·H₂O 0.01,MgSO₄·7H₂O 0.2,K₂HPO₄ 0.8,ZnSO₄ 0.2,CaCl₂·2H₂O 0.2.The optimal cultivation conditions: temperature 37℃,inoculum age 12 h,the inoculum amount 3%,initial pH 7.0.Using the optimal fermentation parameteres,theγ-PGA yield reached 37.05 g/L in flaskes.On the basis of flaskes trials,the cultivation conditions including agitation and aeration rate were optimized in a 3 L bioreactor.Theγ-PGA yield（39.59 g/L） was achieved at agitation rate （800 r/min） and aeration rate（1.5 vvm）,the sodium glutamate apparent conversion rate reached 57%.To improve the sodium glutamate apparent conversion rate,the effect of sodium glutamate concentration on sodium glutamate apparent conversion rate inγ-PGA production was investigated in the medium containing 8%NaCl.The maximum apparent conversion rate（86%） was obtained,when the sodium glutamate was 40 g/L.Effects of different non-nutritional stresses shock methods including KCl-mediated osmotic stress shock,heat shock and pH shock onγ-PGA production were investigated with B. licheniformis WX-02.Under the optimal conditions of osmotic stress shock（supplement 3%of KCl at the beginning of the fermentation）,heat shock（heat shock treatment seed cultures at 50℃for 15 min） and pH shock（adjustment pH8.5 at the beginning of the fermentation）,the sodium glutamate apparent conversion rate increased by 106%,66% and 133%respectively,and theγ-PGA productivity per unit of biomass(Y_P/X)increased by 72%,44%and 110%respectively,as compared with that of the control.By combination of pH shock and heat shock,theγ-PGA yield and sodium glutamate apparent conversion rate reached 29.34 g/L and 73%,respectively,increased by 1.85 times as compared with that of the control.The stress shock methods were simple and effective for enhancement ofγ-PGA production.The increased Y_P/X showed the enhancement ofγ-PGA biosynthesis was a response when B.licheniformis WX-02 was subjected to osmotic stress,heat stress and alkaline pH stress,suggesting theγ-PGA might contribute to protect B.licheniformis from the osmotic stress,heat stress and alkaline pH stress.