Researched On The Production Of Poly ?-Glutamic Acid By Bacillus Subtilis And 300L Pilot Scale Fermentation

In this work,with y-PGA as the target molecule,a production process was studied which includes the optimization of culture the medium and cultivation conditions in shake-flask scale and jar fermentor scale,and the development of separation and purification of y-PGA from fermentation broth.The optimization of Bacillus subtilis HCUL-B-115 culture conditions and the scale-up of fermentation process were investigated.The optimal medium composition in shake-flask scale were determined as followings:corn saccharification liquid 60.00g/L,peptone 5g/L,L-glutamate 60.00g/L?NaCl 12.00g/L,MgSO4 1.00g/L,CaCl2 0.42g/L,KH2PO4 4.00g/L,MnSO4 0.08g/L;flask content 50ml/250ml,rotation speed 150rpm,temperatuer 37?,and fermentation time 72h.The final y-PGA productivity of 58.12g/L.A high productive poly y-glutamic acid strain Bacillus subtilis HCUL B-115 was used in this study.Flask-shaking fed batch fermentation was carried out in the study by feedding glucose,peptone,monosodium glutamate and complete fermentation medium.the experiment result showed that the suitable initial concentration of glucose,peptone,monosodium glutamate respectively was 20.00g/L,4g/L,and 30.00g/L.To supplement complete fermentation medium every 12 hours from 24th hour of fermentation process,the production yield of y-PGA obtained 73.39gL by flask-shaking fermentation for 72 hours.The fermentation of?-PGA from Bacillus subtilis HCUL-B-115 was investigated by batch culture and fed-batch culture in 5 liter fermenter.The results showed that the y-PGA yield enhanced from 11.30g/L to 30.86g/L with 250r/min at batch culture.In the course of fed-batch culture,when glucose concentration in broth was below 20.00g/L,glucose was fed into the broth.The y-PGA yield reached 52.20g/L.Fermentative production of y-PGA from glucose by Bacillus subtilis HCUL-B-115 has been conducted up to the scale of 300L.The experimental results showed that the final mass concentration,molar yield of ?-PGA to glucosewere 42.03g/L and 70.05%in 300L reactor.By using ethanol precipitative pretreatment,the product y-PGA with pure had been harvested.After the different concentrations of ethanol extraction conditions,different concentration of ?-PGA under the conditions of the experiment,to determine the absolute alcohol for?-PGA fermentation liquor extraction is most obvious,fermentation and ethanol proportion at 1:6,extraction of high 67.47g/L.At the end of pilot scale fermentation,if in order to ensure the y-PGA extraction volume,will be the proportion of isolates of y-PGA 600L,consumption of ethanol,greatly improve the production costs.It is suggested that respond to the fermentation liquid concentration,using high purity ethanol solution with 1:3 scale purification of y-PGA.Product identification:The product was detected by IR and UY,We could see that the polymer was polyamide and its largest ultraviolet absorption wavelength is located in 206 nm place;The molecular weight of y-PGA was 700kDa and 960kDa determined by SDS-PAGE and GFC;Observed the freeze dried samples of y-PGA by electron microscopy,photographs showing the freeze-drying without disrupting the y-PGA internal structures,so the drying method is feasible.