## 3 Discussion  
In this study, we demonstrated that sound impacts the fermentation performance of brewer's yeast _S. cerevisiae_ CLIB382. Specifically, the growth rate was increased, particularly through the application of high-frequency sound, and the levels of many volatiles were altered in the headspace of the fermentation. Interestingly, the production of several volatile compounds associated with flavour and aroma was affected differently, depending on the frequency of sound applied to fermentations. These results are relevant to industrial fermentation applications that require both high throughput batch cultures and specific flavour profiles, such as the brewing industry. Our results are consistent with several studies that have shown that exposure to sound, compared to a silent control, results in increases in the rate of growth and/or biomass production of plants, bacteria, and yeasts [2; 33; 34; 3]. In microbes, the magnitude of change in observed growth varies significantly, with the growth rate increase varying from 3.2% in _S. cerevisiae_ to more than 300% in _Brevibacterium_ sp. [13; 33]. However, it is unclear how the growth is affected or why the response varies so dramatically between species. In contrast, a previous study employing _S. cerevisiae_[12] showed decreased biomass production following exposure to sound, whereas our study showed no difference in the final biomass concentration between fermentations exposed to either high- or low-frequency sound or silence. This previous study found an increased growth rate of the \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Original Source**} & \multicolumn{3}{c}{**Predicted Source**} \\ \cline{2-4}  & **High-Frequency** & **Low-Frequency** & **Silence** \\ \hline High-frequency & 6 & 0 & 0 \\ Low-frequency & 0 & 6 & 0 \\ Silence & 0 & 0 & 8 \\ \hline \hline \end{tabular} High-frequency = 10 kHz; Low-frequency = 100 Hz; Silence = no sound stimulus.  
\end{table} 
Table 1: Classification table obtained from leave-one-out cross-validation of 20 samples containing 24 aroma-associated volatile metabolites. Figure 3: Aroma compounds identified from SPME GC-MS successfully separated the different sound treatments. Log-transformed relative abundance data of the 24 putatively identified aroma compounds from each ferment are projected onto two-dimensional space (following normalisation against an internal standard), resulting in three distinct clusters. Red = high-frequency (10 kHz), green = low-frequency (100 Hz), blue = silence. yeast in response to sound exposure, similar to ours, albeit with a lower magnitude (-3-10% compared to 28.9% for the high-frequency sound treatment in our study). The previous study was performed with minimal medium, versus the rich medium we employed, and the yeast strain (beer versus wine), aeration (anaerobic versus aerobic), agitation, and vessel (stirring flasks versus stationary tubes) were different, all of which could affect overall biomass production. The composition of volatiles produced during fermentation was shown to be markedly altered by the application of sound in our study. The production of these compounds is directly impacted by both yeast metabolism and the rate of fermentation. For example, increased yeast growth has been shown to increase the production of fused oils and sulphite compounds [34]. Therefore, it is not unexpected that an increased growth rate due to sound could result in changes to the volatile fraction. Furthermore, physiological stress can also alter the production of many of these compounds [35]. Sound, which produces changing zones of high and low pressure, may directly stress the cellular membrane, causing cells to flex and thereby potentially increasing cell membrane tension. Physiological stress on the cell membranes may, in turn, alter the production of secondary metabolites, such as volatiles. However, the mechanism(s) through which sound produces the effects we report has yet to be determined. In _E.