Study On Lactic Acid Fermentation Technology By Rhizopus Oryzae And Lactobacillus Rhamnosus

Lactic acid and its derivatives have wide applications in food, pharmaceutical, leather and textile industry or as resources for base chemicals production. Especially, preparation of biodegradable polymers like polylactic acid (PLA) substituting synthetic plastic derived from petroleum feedstock, medical sutures, and green solvents has been caused a continuous increase in its demand.About 90% of the lactic acid produced world wide every year is made by lactic acid fermentation and the rest is produced synthetically. Only two categories of microorganism are generally used for lactic acid fermentation, one is bacteria, mostly is Lactic acid bacteria in unareation, another is mold such as Rhizopus oryzae mostly in aeration. The nutritional requirements of Rhizopus oryzae are simple, need only a small amount of inorganic nitrogen and other inorganic salts. Its most advantegy is that it can use starch as carbon source due to its abitility to secrete amylase. However the conversion ratio is of little low as 75% theoretically. Lactic acid bacteirum such as Lactobacillus rhamnosus is heterotrophic. It requires rich nutrients and can not use starch as carbon resource directly, however it has very high conversion ratio of 100% therortically to lactic using glucose as substrate. Take into account the fermentation characteristics of both Rhizopus oryzae and Lactic acid bacteria listed above, a novel fermentation process of co-immobilized Rhizopus oryzae and Lactic acid bacterium with starch directly as substrate materials is proposed in this work. Lactic acid bacteria such as Lactobacillus rhamnosus are immobilized in the internal of microcapsules, whereas Rhizopus oryzae are immobilized in the out layer of microcapsules. It is hoped that under the condition of aeration, Rhizopus oryzae in the outer layer of microcapsules saccharifies starch and produce lactic acid aerobicly, in the mean time it provides glucose transferring into the internal of microcapsules for lactic acid by immobilized Lactic acid bacteria unaerobicly. This process not only has the bilateral fermentation (SSF) characteristics but also has a superposition effect for lactic acid production, which may settle the problems of low carrier utilization rate, low productivity and low conversion rate in L-lactic acid fermentation by immobilized Rhizopus oryzae with starch directly.In this paper the fermentation conditions, culture medium and fermentation kinetics in suspension fermentation for both Rhizopus oryzae AS3.819 and Lactobacillus rhamnosus AS1.549 are studied firstly, and then lactic acid fermentation by co-immobilized Rhizopus oryzae and Lactobacillus rhamnosus in microcapsules using starch was researched preliminarily. The conclusions were obtained as follows.1. The shake-flask suspension fermentation conditions of L-lactic acid production by Rhizopus oryzae AS3.819 are investigated. The optimal composition of culture medium is glucose 80g/L, (NH4)2SO4 2g/L, KH2PO4 0.3g/L, ZnSO4?7H2O 0.05g/L and MgSO4 0.3g/L. The suitable fermentation temperature is 34℃skaker rotation speed is 160rpm, seed age is 18 hours and the optimal inoculum size is 10%. Under such fermentation conditions, the final concentration of the L-lactic acid arrives 61.5g/L and the maximal yield based on initial glucose concentration is 76.9%.2. Kinetics model of L-actic acid production by Rhizopus oryzae AS3.819 using glucose was setup with consideration of product inhibition on cell growth, and kinetic parameters are determinated and optimized using the experimental results at optimal conditions optimized. Simulated results indicated that the formation of lactic acid by Rhizopus oryzae under this condition is a combined kinetic mechanism, mainly dominated by cell growth rate. Optimization kinetic parameters have been successfully used to predict the fermentation processes with initial glucose concentration of both 72g/L,74g/L respectively.3. Fermentation conditions and culture medium optimization in shake-flask suspension fermentation by Lactobacillus rhamnosus 1.549 were carried out. The optimal composition of substrate is peptone 15g/L, yeast 4g/L, glucose 50g/L, Diammonium hydrogen citrate 1g/L, K2HPO4 2g/L, sodium acetate 2g/L, MgSO4 0.3g/L, MnSO4 ? H2O 0.03g/L, FeSO4 ? 7H2O 0.03g/L. The most suitable fermentation conditions are 34℃, seed age of 8 hours and 5% for inoculum size. Under above fermentation conditions, the final concentration of the L-lactic acid is around 45g/L and the maximal yield based on initial glucose concentration is 90%.4. Kinetics model of L-actic acid production by Lactobacillus rhamnosus 1.549 using glucose substrate was setup, and kinetic parameters are determinated and optimized with different initial glucose concentration of 55g/L,80g/L and 110g/L. Simulated results indicated that the formation of lactic acid by Lactobacillus rhamnosus is laso partially growth associated. The inhibition of both high concentration glucose and lactic acid accumulated on fermentation does not acted on substrate consumption and production formation itself directly, but on the cell growth.5. The lactic acid fermentation conditions by co-immobilized Rhizopus oryzae and Lactobacillus rhamnosus using starch directly is investigated rudimentarily The optimal immobilization conditions is the concentration of sodium alginate 1%, The optimal composition of substrate is starch 60g/L, yeast 6g/L, KH2PO4 0.5g/L,ZnSO4?7H2O 0.02g/L,MgSO4 0.4g/L .Under such conditions the final concentration of the L-lactic acid is around 50g/L and the maximal yield based on initial glucose concentration is 86.7% by co-immobilization. Furthermore, in the same fermentation conditions and culture medium composition, mixed fermentations by both Rhizopus oryzae and Lactobacillus rhamnosus in co-immobilized or Rhizopus oryzae suspension plusing immobilized Lactobacillus rhamnosus demonstrate a little modification with lastic acid increasing 8-10%.