## 3 Results and Discussion
### Impact of the bioreactor Settings on the Yield and Mechanical Properties of Bacterial Nanocellulose
Despite showing only slight differences in dry  
Figure 4: Impact of air-flow ratio on (**a**) biosynthesis yield and (**b**) tensile parameters of bacterial nanocellulose after 6-day culture. weight of cellulose after 6-day culture, the stress was increased from 4.77 MPa (control condition) to 6.39 MPa (air-flow ratio at 9.4 L/m\({}^{2}\)/min). Additionally, the membranes from cultures with air-flow were more stretchable. The strain for control BNC was only 25%, while for BNC from tested condition was 34-40%. The Young modulus was not significantly changed. The Influence of Inoculum Parameters on the Yield and Mechanical Properties of Bacterial Nanocellulose  
Inoculum parameters, such as inoculation ratio (%) and age of inoculum, should be considered before each biotechnological process. In our study, we tested the volume of inoculum in the range of 2-10% with a 2% interval (Figure 5) and the age of inoculum from 1 to 5 days (Figure 6). In case of inoculum volume, there are no differences for 4-10% of inoculum. Only at 2% we observed a decrease in the BNC yield. From PBD study it was concluded that this parameter has a positive impact on the dry weight of cellulose and glucose conversion ratio. The studied inoculation ratio was 1% (low level) and 10% (high level), thus the data are in agreement. Due to the fact that _K. xylinus_ produces BNC only in the aerobic zone at the air-liquid interface, the defined number of cells is required for efficient production of cellulose. [PERSON] et al. reported that approximately 10% of the total bacterial cells produce BNC during culture [8]. Therefore, the initial inoculum volume 4% is enough for even distribution of active _K. xylinus_ E25 cells in aerobic zone. Other authors also studied the impact of inoculum volume on BNC yield [27; 28]. [PERSON] et al. applied different inoculation ratios (1:5 and 1:10) and did not observe any significant effect on production yield [27], similar to [PERSON] et al. who used 3% and 6% of inoculum in their study [28]. On the other hand, [PERSON] et al. reported a significant difference between 5% and 15% of inoculum which was also assessed in the PBD study [12]. The authors used _K. xylinus_ ATCC 700178 cultivated in SH medium without the ethanol addition. Figure 5: Impact of inoculum volume on (**a**) biosynthesis yield and (**b**) tensile parameters of bacterial nanocellulose. Figure 6: Impact of age of inoculum on (**a**) biosynthesis yield and (**b**) tensile parameters of bacterial nanocellulose. In these conditions there were likely not enough metabolically active cells at 5% of inoculum to spread cells even in the aerobic zone. Mechanical strength (Figure 5b) was not influenced by inoculum volume and tensile parameters correlate with the yield of BNC. In case of the age of inoculum, its impact on BNC biosynthesis was significant. The productivity of bacteria cells is related to the length of the adaptation phase and the time required to initiate BNC production [29]. The adaptation phase of bacteria is affected by cell density, age, physiological state [30], and bacterial strain. The growth pattern for _K. xylinus_ E25 was previously reported [31]. In case of cultures supplemented with ethanol, a rapid exponential growth can be observed in the first days of incubation. Afterwards, the growth increases slower until the maximum cell density is achieved. In the third day of incubation, _K. xylinus_ E25 starts to produce metabolites, such as cellulose and gluconic acid. It is a specific period during which cells become metabolically active. On the fourth day of culture begins the stationary phase. Based on our results, the highest BNC yield can be obtained from 3-day inoculum (Figure 6a). Therefore, a late phase of exponential growth, where bacteria cells are active, is the most favorable timing to transfer inoculum into fermentation medium. Regardless of the impact of the age of inoculum on BNC yield, this parameter influenced the mechanical strength of biomaterial only in the case of 1-day inoculum (Figure 6b).