# Microbiological Follow-Up of Bioreactor-Assisted Must Alcoholic Fermentation by Flow Cytometry
## 4 Discussion
CFDA-based protocols offer more functional information compared to protocols using DNA-dyes, because CFDA MFI has been related to the esterase activity and, thus, to the general metabolic activity of the cell [16]. We observed that the CFDA MFI of the live population (defined as CFDA + PI-) decreased throughout the fermentation process. It is known that fluorescein derivatives such as CFDA are pH sensitive, and the amount of fluorescence emitted is directly proportional to the pH value. Interestingly, [PERSON] et al. [17] observed that a decrease in yeast intracellular pH (ipH) occurred in the late stationary phase of fermentation. In our case, this phase should correspond to day 5, in which we started to observe decreasing viability. It is noteworthy that day 5 was also the day on which we observed the most dramatic decrease in CFDA MFI. Therefore, we could hypothesise that the observed decrease of CFDA fluorescence was driven by ipH changes. However, because we did not directly measure the enzymatic activity, we could not rule out a direct effect of ipH on esterase activity, the reduction of which could also have led to an overall decrease of CFDA fluorescence [18]. Moreover, it is interesting to note that even after inoculation, the yeast cells displayed low CFDA MFI, although the majority of the cells were PI-, indicating that the cells did not have their membranes compromised (see \"day 0\" in Figure 2). This observation could be explained by the fact that the yeasts were just rehydrated and needed to rewire their metabolism in the new medium. More likely, the yeast cells were not yet capable of bringing the ipH close to neutrality and this possibly affected the CFDA fluorescence emission. From the classical operator-dependent analysis, the population of live cells appeared to be affected by the loss of CFDA fluorescence as a whole, and the distribution of fluorescence values followed a Gaussian distribution (data not shown). However, we could not rule out the presence of functional, discrete clusters possessing different activities during the fermentation process. Thus, to investigate possible \"hidden clusters\", at least from an FCM perspective, we applied unsupervised analysis using the t-SNE algorithm to capture the sample complexity, and to observe the structure of (putative) sub-populations. Our results showed three clusters: two big clusters and one small cloud in the centre. Gating the CFDA-PI plots made recognisable the live cells, the dead cells and the small cluster of damaged cells. We observed, however, that in the t-SNE transformation plot the live cell cluster could be furtherly divided into two sub-clusters separated by a bottleneck end. Thus,we gated these clusters on the CFDA-PI plot and recognised two partially overlapping cell populations also displaying different scatter profiles. It is tempting to speculate that these populations could have different metabolic activity levels and, thus, contribute to fermenting the remaining sugars at different rates. However, it must be emphasised that this was a purely post-analytical attempt to further understand the variability of the yeast population in fermentation and that the results of the t-SNE analysis in terms of new and interesting clusters should be validated with other methods, including cell sorting and functional assays. Although in this paper we mainly present results from an observational study, we also suggest that FCM can be used as a quick and robust tool to follow up the experiments at the bioreactor scale. The staining protocols used are easy to apply, and the sample preparation has been kept as simple as possible. Results can already be seen after 15-30 min, and, thus, the operator can follow-up on the experiments in a timely manner. In the future, FCM can be used to follow yeast (or bacteria) growth in dedicated assays, for example focusing on pH (i.e., for acidifier yeasts) or pressure changes (e.g., their effect on indigenous yeast).