## 3 Results and Discussion
### Optimization of Culture Conditions by Single Factor Design
The possible reasons for this were that the increase in shaking speed or the decrease in loading volume increased the shear force and damaged the cells, thus affecting the metabolism of the yeast, and the consequent excessive oxygen concentration caused more methional produced in the Ehrlich pathway to be oxidized to 3-methylthio-1-propanoic acid with less available for producing 3-Met [3]. Thus, it is very important to maintain an appropriate oxygen concentration to ensure the cell biomass and reaction direction in the Ehrlich pathway by regulating the shaking speed and loading volume to ensure a high yield of 3-Met. Inoculum size is also an important parameter often considered when investigating the biosynthetic metabolites during microbial fermentation. It affects not only the yield of the target products but also the fermentation cycle [67]. An appropriate inoculum size can shorten the fermentation cycle while maintaining the yield. A low inoculum volume, such as 0.1% (the cell density was adjusted to a concentration of 1 \(\times\) 10\({}^{6}\) cells/mL by using a blood cell counting plate), can lead to low 3-Met production owing to the fact that longer delay period may lead to lower biomass, whereas a high inoculum volume, such as 6.4%, would also decrease the yield because excessive cell growth may lead to insufficient oxygen supply (Supplementary Figure S2b) [67]. There was no significant difference in 3-Met production when the inoculum size was between 0.2% and 3.2% and the yield was higher because of the superior growth rate and biomass and sufficient nutrients (Supplementary Figure S2b) [43]. Many previous studies have shown that the optimal inoculum size was also in this range, for example, _P. kudriavzevii_ YF1702 for producing 2-phenylethylal [43] and _Bacillus licheniformis_ NCIMB 8059 for producing 2,3-butanediol [68]. The cell membrane is an important component of microorganisms as it maintains cell structure and activity. It is also an important channel for material and energy transfer between cells and the surrounding environment. Changes in the permeability of the cell membrane would cause the absorption of nutrients and the secretion and extravasation of intracellular substances, thus affecting cell metabolism. The cell membrane consists of a phospholipid bilayer, so some amphiphilic surfactants, such as Tween and Triton, would affect its permeability and microbial metabolites [69; 70]. Previous studies have shown that different surfactants have different effects on different metabolites of the same microorganism or on the same metabolites of different microorganisms [43]. Therefore, it was necessary to study the effect of surfactant type and concentration on 3-Met production by strain YHM-G, as no information is available at present. The results showed that both Triton X-100 and the Tween series were able to promote 3-Met production from strain YHM-G in a similar way, while glycerol and Triton X-114 inhibited 3-Met production (Figure 2g). This was because different types of surfactants had different effects on the cell membrane of strain YHM-G, which also confirmed previous reports on the effects of surfactants on microbial metabolism [43]. Tween-80 led to the maximum yield of 3-Met at 20.3% higher than the control (Figure 2g). Varying the Tween-80 concentration had little effect on 3-Met production (Supplementary Figure S2c), possibly because Tween-80 was compatible with the cells of strain YHM-G; even at high concentrations it still increased cell membrane permeability and promoted the dissolved oxygen level in the cells but did not permeabilize the cell membrane [70]. From considering economic factors, a lower Tween 80 concentration was chosen.