## 2 Results and Discussion
### Potential Medium Chain Fatty Acid (MCFA)-Derived Precursors of Ethyl Esters
#### 2.1.1 Free Fatty Acids as Direct Precursors  
C9 and C12 fatty acids were added to model grape juice media (MGJM) fermentations across a range intended to capture the concentrations reported in the literature for comparable molecules in grapes [27, 28]. No significant difference in the production of ethyl nonanoate was seen between the control and the fermentations supplemented with 1 \(\upmu\)M nonanoic acid (Figure 1A). However, there were significant increases when 10 \(\upmu\)M or 100 \(\upmu\)M nonanoic acid was added to the MGJM, with ester concentrations reaching 1120 nmol\(\cdot\)L\({}^{-1}\). The addition of 1 mM nonanoic acid appeared to be toxic to the yeast, as negligible mass was lost throughout the fermentation period. It has been shown in previous studies that free fatty acids can be toxic to yeast at high concentrations [29, 30].  
No significant increase in the production of ethyl dodecanoate was observed after additions of up to 100 \(\upmu\)M dodecanoic acid to the fermentations (Figure 1A). However, there was a large response when 1 mM dodecanoic acid was added, leading to a mean ethyl dodecanoate concentration of 8155 nM (_i.e._, 1.86 mg\(\cdot\)L\({}^{-1}\), a sizeable quantity) compared to a mean of 118 nM in the control wine. An increased lag phase was seen in the 1 mM samples, otherwise the fermentation kinetics appeared not to have been impacted in the dodecanoic acid series of additions (data not shown). The esterification of the fatty acid may be a detoxifying action that occurs above a particular toxicity threshold. The findings of [PERSON] and [PERSON] [31] support this, as esterification of C8 and C10 fatty acids reduced their toxicity to the yeast. The results suggest that a concentration threshold for this toxicity exists, and that grape-derived MCFAs could contribute to these concentrations. It would appear that the threshold is much lower for nonanoic acid than for dodecanoic acid and, since most previous research has focused on the effects of even chain MCFAs [8, 32], the results of this study suggest a potential odd/even carbon number effect that is worthy of further investigation. Alternatively, the stimulation of ethyl nonanoate production by a lower concentration of nonanoic acid than that observed for dodecanoic acid and its corresponding ethyl ester may reflect the size of the endogenous pool of substrate. As odd-numbered MCFAs are less common than the even-numbered ones, a lower concentration would be required to increase the fatty acid substrate concentration to a point where the production of the ethyl ester is significantly greater than that in the control wine. This may also be the case if the fatty acids have differential abilities to diffuse or be actively transported into the yeast where esterification occurs.  
Figure 1: (**A**) Ethyl nonanoate and ethyl dodecanoate concentrations in model wines produced after the addition of increasing amounts of nonanoic acid or decanoic acid to the MGJM; (**B**) Ethyl nonanoate and ethyl dodecanoate concentrations in model wines produced after the addition of increasing amounts of methyl nonanoate or methyl decanoate to the MGJM; (**C**) Ethyl nonanoate and ethyl dodecanoate concentrations in model wines produced after the addition of increasing amounts of nonanoyl carnitine or dodecanoyl carnitine to the MGJM. Histograms represent the mean values (n = 3) and error bars show standard errors. Different letters (e.g., a, b, c, d or a’, b’, c’) denote significant differences between treatments at \(p<0.05\) using ANOVA followed by the Games-Howell _post hoc_ test. Note that the y-axis is in log scale.