以前饋控制策略建立批次饋料醱酵 的酵母菌生長動力模式

Establishing a feed-forward control strategy of fed-batch fermentation for the kinetic model of yeast growth

游越(Yehn Yu)

指導教授 ： 古源光 ； 郭嘉信

國立屏東科技大學/農學院/食品科學系所/碩士(2012年)

https://doi.org/10.6346/NPUST.2012.00048

摘要

本研究是利用前饋控制的策略，以批次饋料的方式，建立微生物的生長動力學模式。隨著烘培酵母菌(Saccharomyces cerevisiae)的生長，計算出饋料的間隔時間，藉以維持培養基中的碳源濃度，使烘焙酵母菌保持在對數生長期狀況，配合微調校正，有效達到高密度細胞培養的目標。為了避免酵母菌株及培養基成份的個別差異，造成微生物生長動力學模式探討的問題，本研究利用實驗設計法的成分替換及因子分析法，篩選出適合酵母菌生長的碳源和氮源，並利用最陡上升法，並找出適合酵母菌生長的碳氮比。結果顯示，蔗糖和大豆腖分別為最佳的碳源及氮源(p <0.05)；最陡上升法的結果顯示，將蔗糖濃度及碳氮比分別調整為2 g/L及15：1時，YX/S可達到51.35%。以此培養基配方進行酵母菌的批次醱酵後，對數生長期大約落於0~3 h，其YX/S及比生長速率(μ)分別為74.29%及0.26 h-1。根據此數據計算酵母菌在對數生長期消耗碳源的速率，並間歇饋入相對的碳源消耗量，藉此將批次饋料醱酵期間培養基的糖濃度控制在1~2 g/L的範圍內。初步的結果顯示，此前饋控制模組雖然可在18.10小時內產生5 g/L的酵母菌濃度，但其中仍有未被考量的因子存在，導致培養基中的糖濃度有持續升高的現象。為了修正預測值的偏差，首先，計算出菌體生長的遲滯期為0.5 h，並將其加入饋料時間的排程當中；接著，計算出糖消耗量之預測值與實驗值間的相關性，並在前饋控制模組計算饋料時間時，加入此相關性的參數，藉以校正培養基中糖濃度持續升高的狀況。為了使模組能夠更準確的預測總糖量的變化，再根據總糖消耗量的變化趨勢進行模組的細部微調。經過校正後的結果顯示，此糖量變化的前饋控制模組實際上為一2次方程式，於批次饋料醱酵的期間能將培養基中的糖濃度穩定控制0.85~1.90 g/L的範圍內，使酵母菌以0.16 h-1的比生長速率維持在對數生長期中，並於醱酵18.95 h後，菌體量達到初始細胞濃度的28.33倍。本研究的前饋控制模組能夠透過簡單的計算及校正模式，穩定地控制培養基中的糖濃度，此模組可運用在醱酵槽自動化控制系統上，協助探討高濃度微生物生產之用。

關鍵字

酵母菌 ； 碳源濃度 ； 前饋控制模組 ； 比生長速率

並列摘要

The object of this study is to establish a kinetic model for Saccharomyces cerevisia growth in a fed-batch fermentation to achieve a high cell density using feed-forward strategy. The feeding time course was adjusted based on the prediction of yeast growth to keep the concentration of carbon course in the fermentation medium within a certain rang, so that the growth of S. cerevidiae maintained at its log phase. Component and factorial analyses were also conducted to identify a set of carbon and nitrogen sources for this study. Steepest ascent was used to identify an appropriate C/N ratio. The results indicated that sucrose and soytone were the most effective carbon and nitrogen sources, respectively, for biomass production in this case (p <0.05). When the medium contains 2 g/L of sucrose and has a C/N ratio of 15:1, the yield of cell mass based on the sucrose consumption (YX/S) reaches 51.35%. Under these conditions, the growth of yeast can reach the log phase within 0~3 h post-incubation. The YX/S and specific growth rate (μ) during this log phase were 74.29% and 0.26 h-1, respectively. The concentration of carbon source in the fermentation medium was controlled in a range of 1~2 g/L through adjustment of feeding frequency. Unfortunately, the primary result showed that the sugar concentration in the
medium accumulated up to 5 g/L after 18.10 h post-incubation due to unknown influence factors. To adjusted the deviation of the predicted outcome, a holding time of 0.5 h lag phase was applied and an extra delay time was also calculated and added based on the increasing sugar concentration for each feeding interval. For more accurate prediction on sugar consumption, fine tune the prediction model according to the track of experimental sugar demand. The result shows that the prediction model was approximately a quadratic equation, and the variation of substrate concentration seemed to be steadily controlled in a range of the prediction model from 0.85 g/L to 1.90 g/L with a stable growth of yeast in the specific growth rate of 0.16 h-1, and harvest 28.33 folds of initial biomass after 18.95 h post-incubation. In conclusion, the design of this feed-forward model is capable of controlling yeast growth effectively, and may provide a potential approach to develop an automatic process for high cell mass production using yeast.

並列關鍵字

yeast ； sugar concentration ； feed-forward control model ； specific growth rate

參考文獻

杜姿瑩、黃進發、李河水、華傑。2000。酵母在食品工業之應用。食品工業發展研究所。新竹市。台灣。

Bae CS, Yang DS, Lee J, Park YH. 1999. Improved process for production of recombinant yeast-derived monomeric human G-CSF. Applied Microbiology and Biotechnology 52: 338-44.

Botz DW. 2000. Experimental design for fermentation media development: statistical design or global random search? Journal of Bioscience and Bioengineer 90 (5): 473-83.

Chou CC, Cheng SJ. 2000. Recovery of low-temperature stressed E.coli O157:H7 and its susceptibility to crystal violet, bile salt, sodium chloride and ethanol. International Journal of Food Microbiology 61: 127-36.

Ferreira BS, Calado CRC, Keulen FV, Fonseca LP, Cabral JMS, Fonseca MMRD. 2003. Recombinant Saccharomyces cerevisiae strain triggers acetate production to fuel biosynthetic pathways. Journal of Biotechnology 109: 159-67.