## 3 Results and Discussion
### Impact of the bioreactor Settings on the Yield and Mechanical Properties of Bacterial Nanocellulose
Therefore, for higher volumes of medium, the surface density of bacteria is greater and it drives more extensive biosynthesis yield at early stages in the lag phase [14]. In this context, the s/v ratio should be connected with a parameter defining cell density per surface unit, not the volumetric density. Moreover, the thickness of the BNC membrane increases with time. For long-time culture, a higher amount of medium must be used. On the other hand, the membrane grows only to the stagnation point due to the limitations of oxygen or nutrients [8]. For economically profitable production, the minimum depth of medium in the designed conditions should be only a little higher than maximum predicted thickness of the BNC. In case of a lower amount of medium, less dry weight of cellulose will be obtained. Contrarily, a higher amount of medium will result in the wastage of the culture medium and lower BNC yield per volume unit. The optimum s/v ratio for _K. xylinus_ E25 was reported as 0.71 cm\({}^{-1}\). In our study we used 0.71 cm\({}^{-1}\) and 0.33 cm\({}^{-1}\) which can be calculated as a 1.4 cm and 3.0 cm of medium depth, respectively. The lack of significant effect on both dry weight of cellulose and glucose conversion ratio confirms that the s/v ratio at 0.71 cm\({}^{-1}\) is enough for sufficient production yield. Production of BNC in a SAF bioreactor is connected to two parameters, namely the air-flow ratio and the humidity of air. Both factors affected the dry weight of cellulose and glucose conversion ratio (Figure 4). Detailed study showed that cellulose yield (Figure 4a) and mechanical strength (Figure 4b) were positively regulated by the air-flow ratio. Impact of that parameter was evaluated after 4- and 6-day cultures. A similar bioreactor set was designed by [PERSON] et al. [15], with slight differences. In contrast to our studies, air was introduced by an air distributor from the top, not parallel to the membrane and the air specific flow rate was between 0.21 and 0.63 cm\({}^{3}\)/cm\({}^{2}\)-s, which can be calculated as 126 and 378 L/m\({}^{2}\)-min. The applied air flow ratio was much higher than air flow used in our study. In this condition, authors reported significant effects on water specific use, which is related to water evaporation. Nevertheless, the BNC yield was not affected. Presumably, a lower air-flow ratio is preferable for efficient production. From Figure 4a it can be seen that, after a 4-day culture, the dry weight of cellulose from culture fed with air was 24-28% higher than the control condition. Furthermore, the dry weight of 4-day-BNC from the tested condition was slightly higher than the dry weight of 6-day-BNC from the control condition. On the other hand, the air flow ratio was significant in terms of 6-day culture, but the differences were lower. It can be explained that cellulose growing in the presence of air flow at 4.7-18.8 L/m\({}^{2}\)-min reached the bottom of bioreactor after 4 days and further biosynthesis is limited. For higher yield, the lower s/v ratio should be considered. We can conclude that air flow increases the production rate from 0.55 g/L/day (6-day culture, standard condition) to 0.91 g/L/day (4-day culture, air-flow ratio at 9.4 L/m\({}^{2}\)/min) at the biosynthesis yield 3.31 g/L and 3.63 g/L, respectively. Therefore, in the favorable condition, the productivity was improved by 65%. Acceleration of BNC biosynthesis is probably caused by higher diffusion of nutrients as a consequence of water evaporation. Thus, bacteria trapped in the upper part of the membrane have better access to carbon and nitrogen sources. Data obtained for the humidity of air are in accordance with our conclusions. The moisture in air limited the water evaporation and a negative effect of humid air on cellulose dry weight was observed (Figure 3). Also, continuous supply of fresh air results in ventilation of cultures from carbon dioxide, and oxygen concentration is unchanged during cultivation. Modification of the bioreactor set also caused changes in mechanical parameters of BNC.