High-cell-density Cultivation Of A Salt-tolerant Yeast Zygosaccharomyces Rouxii

Salt-tolerant yeast Zygosaccharomyces rouxii is a key strain to produce flavor in soy sauce brewing. The technology of direct inoculation yeasts in brine fermentation phase has applied for many years. However, the fermentation activity and stability of yeasts in the process of soy sauce fermentation were not satisfying. Active dry yeast(ADY) has the characteristics of high activity, good stability, etc. So, there has a huge potential for market applications in developping an appropriate ADY for soy sauce brewing. This paper studies the process of high-cell-density cultivaton of Z. rouxii, based on the production of ADY. The results are as follows:(1) Response surface methodology(RSM) was used to optimize fermentation medium for Z. rouxii CCTCC M 2013310. Plackett-Burman(PB)design was used to evaluate the influence of related factors in medium. The results of PB design indicated that contents of glucose, corn steep liquor and KH2PO4 significantly affected yield of biomass. The optimum medium was obtained with central composite design and response surface analysis method as follows: glucose 12.03%, corn steep liquor 2.24%, yeast extract 1%, KH2PO4 0.26%, Glycerol 3%, VB1 0.001%. The maximum cell mass in optimum medium was 28.28 g/L, which increased 4.5 times than that with YEPD 5.12 g/L。(2) In experiments of optimization of the shaking flask fermentation condition, the culture conditions such as p H value, culture temperature and seed volume were investigated. The results shown that p H value at 5.0 was the best choice for Z. rouxii. In the late experiments of constant p H value in 10 L stirred tank reactor the same result was observed. Culture temperature and seed volume at 28℃ and 5% respectively were the optimal. The result also showed that in the conditions of agitation speed at 500 r/min and aeration rate at 1 vvm the DCW was the highest.(3) An exponential feeding strategy in 10 L stirred tank reactor was applied where the specific growth rate was fixed by limitation of the fed carbon source. The set point of specific growth rate of exponential feeding for fed-batch culture was chosen to be 0.03~0.07 h-1. The optimal set point of specific growth rate(μset) of exponential feeding started after glucose depletion for fed-batch culture was found to be 0.05 h-1. Maximum biomass concentration of 72.91 g/L dry cell weight was reached. Furthermore, a not bad biomass concentration was obtained in the culture of exponential feeding started after biomass reach the highest in batch culture with μset=0.05 h-1. According to all the feeding experiments, the specific growth rate of 0.05 h-1 with feeding start after glucose depletion can satisfy the high density fermentation of Z. rouxii.