## 3 Results and Discussion
### Acetification Process Parameters
In a similar manner, comparing the data from Table 1 and Figure 4, it can be observed that in the case of cultures stored for 2 months at 4 \({}^{\circ}\)C, the acidification times varied from 10-20 days to 7-11 days for the wild and mixed cultures, respectively, free and immobilised. In this case, the effect of cell immobilization was obvious for both types of cultures (wild and mixed), which seemed to readapt fast to the acidification conditions after storage and complete the process in shorter times. In the case of immobilised cells, another advantage is that the recovery of the biocatalyst is easier since the whole biocatalyst remains in the bioreactor, while the vinegar can be easily decanted as soon as the fermentation completes and has a better clarity compared to the vinegar made by free cells. The literature indicates that the acidification can take place within 30 h depending on the desired acetic acid content [16]. Other studies have reported that the acidification times to produce vinegar from various fruit using immobilized AAB ranged from 5-28 days [7; 17; 18; 19]. Figure 4: Kinetics of the acidification of wine at 30 \({}^{\circ}\)C, reusing the wild AAB culture, free (FCW-2M) and immobilized (ICW-2M), and the mixed AAB culture (_A. aceti_ and _K. europaeus_), free (FCM-2M) and immobilized (ICM-2M), after 2-months of cold storage at 4 \({}^{\circ}\)C (symbols are the mean values plus standard deviations of the experimental data from 3 batches; curves are sigmoidal Boltzmann’s model fits). The acidification times observed in this study were 3 and 20 days using free cells, and 2 and 10 days using immobilized AAB cells (before and after storage, respectively), to obtain acidity levels in the range 41-50 g/L. Therefore, the acidification times with immobilized cells on DCM were shorter. However, comparisons with data from other studies may be quite ineffective, due to the different method parameters applied (bioreactor design, aeration intensity and air dispersion, composition of the substrate, and the AAB cell density in the bioreactors). The cultures were stored in cold to evaluate their acidification efficiency after storage at low temperature, in the case that the vinegar production process has to be ceased. The optimum growth temperature for AAB is 25-30 \({}^{\circ}\)C. Growth has been observed at lower temperatures but at very low rates [2; 20]. Cold storage of the cultures for 2 months reduced, as expected, the activity of both free and immobilized cells during the first fermentation batch, but the rate of the subsequent batches remained at satisfactory levels. Therefore, the process presents an improvement as the fermentation bathes proceed, ensuring the functional stability of the acetator. This improvement may be attributed to the increased viability of the immobilized cells and their better adaptation to the system, as indicated in previous studies reporting increased fermentation productivity induced by cell immobilization (on DCM or other carriers) [6; 11; 21]. Thus, it can be concluded that the use of sweet wine (partially fermented) as raw material in the production of vinegar is possible using both wild and selected AAB cultures. The cultures were able to convert both the alcohol and the sugar contained in the FSS wines (into acetic acid, cell mass, and other metabolites), indicating that although the presence of sugars seems to promote the activity of AAB, the proposed method is not suitable for the production of new types of \"sweet\" vinegar. Therefore, for sweet vinegar production from FSS (e.g., resembling balsamic vinegar), FSS extract or syrup should be added post-acetification.