## 3 Results and Discussion
### Optimization of Culture Conditions by Single Factor Design
The yield of 3-Met from strain YHM-G increased rapidly at a pH value between 3 to 4 and was optimal at a pH value between 4.0 and 5.5 (Figure 2d). The yield decreased with increasing pH when the pH was \(\geq\)6 (Figure 2d), which was consistent with the study of [PERSON] et al. [2] but not with that of [PERSON] et al. [5]. The result showed that an initial of pH 4.0-5.5 would result in a final pH between 3.34-3.74, while when the initial pH was set at 3.0-3.5 and 6.0-7.0 and the final pH was 2.78-3.15 and 4.2-6.2, respectively. In addition, the result also illustrated that the final fermentation biomass was positively correlated with the initial pH (Figure 2d). Apparently, the optimal pH for 3-Met production was slightly lower than the optimal pH for the growth of strain YHM-G. Thus, the reason for these results was that enzyme activity in the Ehrlich pathway for 3-Met production was probably optimal under slightly acidic conditions due to its long-term survival in the slightly acidic environment of _Baijiu_ making [2; 5]. Temperature is an important factor affecting the growth of microorganisms and the production of metabolites. If the temperature is too low, microbial growth will be greatly compromised; an excessive temperature may lead to cell death or enzyme inactivation, discouraging the accumulation of the end-products [8; 64; 65]. Because of the growth temperature range of strain YHM-G, the optimum temperature for 3-Met production was measured from 20 to 40 \({}^{\circ}\)C. The 3-Met production increased as the temperature of incubation increased from 20 to 32 \({}^{\circ}\)C (Figure 2e), which was consistent with the study by [PERSON] et al. [2]. As previously reported, the relationship between temperature and 3-Met production was not linear [2]. The increases in 3-Met formation were large, at 300% and 150%, for an increase in temperature of 4 \({}^{\circ}\)C, from 20 to 24 \({}^{\circ}\)C, and from 24 to 28 \({}^{\circ}\)C (Figure 2e), respectively, which was not consistent with a previous study [2], mainly because of the different strains. The highest yield of 3-Met occurred at 32 \({}^{\circ}\)C (Figure 2e), similar to _K. lactis_ KL71 [2], which was consistent with their optimum growth temperature [2; 5]. At this temperature, growth was faster and the enzymes for 3-Met biosynthesis may have exhibited a higher activity [2]. A reduction in the growth rate and activity of enzymes related to 3-Met synthesis led to a decrease in 3-Met production at high temperatures [8; 66]. Although yeasts are facultative anaerobic microorganisms, their growth and most biological metabolic activities benefit from aerobic conditions [5; 43]. The metabolic pathways for different metabolites differ and the oxygen concentration required also varies [25; 43]. In shaking flask fermentation, the shaking speed and loading volume are the two parameters that most directly affect oxygen concentration during the fermentation process. They both regulate the production of metabolites by affecting oxygen concentration [43]. Therefore, the effect of shaking speed and loading volume on 3-Met synthesis were investigated. The results showed that 3-Met produced by strain YHM-G first increased then decreased with increasing shaking speed or loading volume (Figure 2f). With increasing shaking speed or decreasing loading volume, the oxygen concentration in the fermentation system rapidly increased growth and invigorated the metabolism, significantly increasing the yield of 3-Met [5]. When the shaking speed was 135 rpm or the loading volume was 75 mL/250 mL, the yield of 3-Met reached its highest value, at almost 10 times that achieved in an unaerated fermentation (shaking speed = 0 rpm), which was consistent with a previous study (Figure 2f) [2]. With a continuing increase in shaking speed or decrease in loading volume, the 3-Met production decreased (Figure 2f).