Efficient Fermentation Of Rhamnolipids For Preparing Rhamnose Via Hydrolysis

As a sweetener,rhamnose is widely used and can be prepared by hydrolysis of rhamnolipids.Rhamnolipids are biosurfactants produced by Pseudomonas aeruginosa,which are of low toxicity,environment-friendliness and biodegradation.At present,the production of rhamnolipids has been industrialized,but still have problems such as low yield,abnormal fermentation,and difficult foam control.This thesis intends to improve the fermentation efficiency for reducing the production cost of rhamnolipids via optimizing the cultivation process.Also,the maximal hydrolysis of rhamnolipids were developed for producing rhamnose.Firstly,the negative effect of fermentation tank material on the fermentation process was evaluated by adding stainless steel into shake flasks and the corresponding strategy was proposed.As found,the use of a low phosphate level in the culture medium could reduce the side effect of high iron ion dose on rhamnolipids production.Different stainless steel materials,such as carbon steel,304 and 316L,had different effects on fermentation,with a lowest impact of 316 L.Such impact of stainless steel on fermentation was significantly attenuated at the low phosphate level compared with that at the high phosphate level.The reduced medium loading in ordinary shake flasks could fascinate the production of rhamnolipids accompanied by the high oil conversion rate.The addition of nitrogen further increase the conversion rate of soybean oil at 4 days from 0.508 to 0.615,enhancing the rhamnolipids productivity to 58g/L at 7 days.Secondly,the control of severe foaming included modification of the medium composition for regulating pH of the fermentation broth and optimization of the mechanical defoaming equipment.For reducing the stability of foam,pH of fermentation broth was controlled at a neutral value by the addition of yeast extract in the medium.Moreover,phosphoric acid was fed during the fermentation process for further regulating pH.On the other hand,the slit defoaming device was mechanistically studied and its structural design was optimized by numerical simulation.The optimized parameter was set at following:a slit width of 1mm with the spacing of 10mm,and a layout of horizontal position.The improvement of industrial fermentation was largely on overcoming the negative impact of tank material.Baffled shaking flasks supplied with 304 stainless steel in culture medium were used to simulate the industrial fermentation system.Using the baffled shaking flasks whereas the phosphate concentration was set at 0.5g/L in the medium,the rhamnolipid productivity was enhanced with an oil conversion rate of about 0.53.This fermentation condition was applied to an industrial fermentation at a scale of 20T,by reducing the negative impact of iron ion,feeding with nitrogen and oil during the fermentation and foam controlling with a slit defoamer.The rhamnolipids concentration reached 46g/L at 4 days with the oil conversion rate of 0.554 and the volumetric productivity of 0.11g·L-1·h-1.Lastly,rhamnolipids were hydrolyzed to prepare rhamnose.The response surface methodology(RSM)was used to optimize the hydrolysis reaction combined with BoxBehnken central combination design and the response surface methodology.The optimal condition of hydrolysis were obtained from the model:sulfuric acid concentration of 2.3%,hydrolysis temperature of 137℃ and hydrolysis time of 2.8h.Under the optimal conditions,the actual yield of rhamnose is 81.6%,which is close to the predicted value of 82.35%.In addition,it was found that if the temperature was too high or the reaction time was too long,rhamnose could convert to by-products such as methylfurfural,resulting a reduction of the yield of the rhamnose.Finally,the process for preparing rhamnose by hydrolysis of rhamnolipids was scaled up to an industrial scale,reaching a hydrolysis yield of 72.6%and the rhamnose yield of 30.4%.In summary,this study largely improved the industrial fermentation of rhamnolipids and developed the preparation of rhamnose by hydrolizing rhamnolipids at an industrial scale.It will lay the foundation for the large-scale production of rhamnose via rhamnolipids fermentation.