| L- lactic acid is a monomer used for poly lactic acid synthesis and poly lactic acid is one kind of biomedical polymer materials.Poly lactic acid, by the reason of good biological compatibility and biodegradability, is widely concerned in the field of biomedicine and environmental protection.With the rapid development of poly lactic acid’s study, interests in the production of lactic acid have been ever increasing. The nutritional requirement of Rhizopus oryzae is simple and its output is in high optical purity of greater than 99% that is easy to purify from the medium in fermentation. Hence, Rhizopus oryzae has become the main strain in preparation of L- lactic acid with high optical purity. However, the fermentation of L- lactic acid by Rhizopus oryzae is faced with a problem of high substrate cost in industrialization. Biomass renewable resources- natural cellulose(tobacco stem, etc.), due to the low cost and rich resource, is considered to be effective carbon source in lactic acid production. While, intertwined combinations among pectin, cellulose, hemicellulose and lignin can prevent microorganism using cellulose to produce lactic acid, increase cost s of enzyme preparation, weaken costs saving in raw material.Rhizopus oryzae can produce pectinases to degrade pectin and there is a growing market demand of pectinases for that pectinases are widely used in food, textile, medicine and so on.Rhizopus oryzae, a safe strain which owns the ability to degrade pectin and is rich in commercial value, was immobilized on the matrix which is developed by our laboratory to achieve semi-continuous fermentation of pectinases and degradation of pectin in tobacco stem in this research. Firstly, the pure pectin was selected as the fermentation substrate. The comparison of enzymes production between immobilized cells and free cells was made. Semi- continuous fermentation of pectinases was achieved after optimization of culture medium and growth conditions and some properties of the enzyme were under research. Then, the tobacco stem was selected as the s ubstrate. Semi-continuous fermentation of pectinases was achieved after optimization of culture medium and growth conditions. K inetic behaviors of immobilized cells growth on different culture medium above were simulated by Logistic equation in one fermentaion. Finally, the crude enzyme liquid by fermentation was applied to degradation of tobacco stem pectin and the immobilized Rhizopus oryzae was used to degrade tobacco stem.First of all, the study of pure pectin was carried out.The fermentation period decreased by 33% and production improved by 115% when cells were immobilized compared with free cells. Optimization of culture medium and growth conditions was investigated. The optimized results were composed of pectin 2.5%,(NH4)2SO4 1.5%, Zn SO40.6 mmol/L,Tween80 0.15%, K2HPO4 0.4%,KH2PO4 0.4%, rotatory speed 190 r/min, liquid volume 50 m L/250 m L, temperature 30oC, p H 5.0, spore concentration 0.75×106/m L. After 24 hours, the PEC and Endo-PG activity were 973.47U/m L and 165.08U/m L separately. K inetic behavior of immobilized cells growth was in accord with Logistic equation.The highest PEC and Endo-PG activity were 1 253.95U/m L and 181.94U/m L in 5th batch, about 29.8% and 18.3% higher than 1st batch.Secondly, optimization of culture medium made from tobacco stem and growth conditions was investigated. The optimized results were composed of tobacco stem 10%,(NH4)2SO4 1.5%, Zn SO40.6 mmol/L,Tween80 0.1%, K2HPO4 0.2%,KH2PO4 0.2%,rotatory speed 170 r/min, liquid volume 50 m L/250 m L, temperature 30oC, initial p H 5.0, initial spore concentration 0.50×106/m L. After 48 hours, the PEC activity and Endo-PG activity were 361.27U/m L and 49.22U/m L separately.The highest PEC activity and Endo-PG activity were 430.83U/m L and 51.73U/m L in the 2 nd batch and PEC activity was about 22.8% higher than the 1st batch.Finally, the crude enzyme liquid and the immobilized Rhizopus oryzae was used to degrade tobacco stem. The results showed that: when PEC activity from pure pectin fermentation was 600U/m L and liquid-solid ratio was 20, the pectin degradation rate was 55.6%, the relative contents of cellulose increased by 130% and both are higher than PEC activity from tobaccostem leach liquorfermentation.In the study of tobacco stem fermentation, culture medium was confirmed as the following:tobacco stem 2.0%, glucose 0.6%,(NH4)2SO40.3%, Zn SO40.6 mmol/L,Tween80 0.1%, K2HPO4 0.2%,KH2PO4 0.2%. Under the same growth conditions as tobacco stem leach liquor fermentation and after 24 hours, the PEC activity and Endo-PG activity were 342.10U/m L and 52.67U/m L separately. The degradation study of tobacco stem by immobilized Rhizopus oryzae showed after 96 hours pectin degradation rate was 74.1%, hemicellulose degradation rate was 54.5%, cellulose degradation rate was 13.3%.In conclusion, this study had realized semi-continuous fermentation of pectinases and degradation of pectin in tobacco stem by immobilized Rhizopus oryzae. |
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