## 3 Results and Discussion
### The Effect of Culture Condition on the Yield and Mechanical Properties of Bacterial Nanocellulose  
After the PBD study, the effect of pH on the dry weight of cellulose was insignificant, unlike its impact on the glucose conversion ratio (Figure 2). In the next step, we determined the influence of pH on the BNC yield and mechanical strength in the wide range of values (Figure 7). From Figure 7a it can be seen that the highest BNC production was obtained over a pH range between 4.5 and 6.0 without a specific maximum. For higher pH values a slight decrease in the yield was noted, while for more acidic medium (pH 3.5) the yield was reduced to 0.68 g/L. Also, at pHs below 3.5 and over 7.5, _K. xylinus_ E25 did not produce cellulose. The value of pH had a similar effect on tensile parameters (Figure 7b). For membranes produced in media with pH 3.5 the stress and Young modulus were decreased about 2 and 3 times, respectively. Strain significant differences were observed, but there was not any specific trend noted. For media with higher pH, the mechanical strength was unaffected. Generally, cellulose-producing bacteria produce good quality BNC in the pH range of 4.0-7.0 [32]. The optimum is dependent on the bacteria strain. _K. xylinus_ ATCC 700178 produces BNC with similar yield in media with pH between 4.5 and 6.2 [12]. [PERSON] et al. reported that in case of strain _Acetobacter sp._ A9, a high level of cellulose production occurred at pH 4.5 and 7.5 with the maximum at 6.5 [33]. The medium with pH 5.5 was optimal for _K. hansenii_ AS.5 and for another pH values the decrease in cellulose production was observed [24]. According to [PERSON],  
Figure 7: Impact of pH on (a) biosynthesis yield and (b) tensile parameters of bacterial nanocellulose. Roayaei, and Qezelbash, the highest BNC yield in culture of _Acetobacter sp_. 4B-2 was observed at pH 7.0 [34], while for the _K. medellensis_ strain from Colombian vinegar the optimal pH was 3.5 [35]. Most of the strains are more sensitive to lower pH and below 3.0 cells cease to produce cellulose, thus normally pH of culture media is adjusted to 5.0 or 6.0 [36]. In addition, during the culture in a medium containing glucose, gluconic acid is produced and released into medium as a product of glucose oxidation. That results in pH decrease. Therefore, for low initial pH, the yield is diminished and the mechanical strength is not satisfactory. In our study (data not shown), the final pH for media with a pH range between 4.5 and 7.0 was >3.5. In the case of initial pHs of 4.0 and 3.5, the value of pH dropped to 3.4 and 3.2, respectively, which could inactivate BNC biosynthesis. Glucose concentration strongly affects both the yield (Figure 8a) and tensile stress of BNC (Figure 8b). The yield is increased with the increase of glucose concentration from 5 to 20 g/L. For higher concentrations up to 35 g/L there are no significant differences, but the slight decrease in the yield was observed at 40 g/L of glucose. The mechanical properties of membranes correlate with the yield in the range of glucose concentrations between 5 and 30 g/L. The highest stress (about 3.5 MPa) was achieved for BNC synthesized in medium containing 15-30 g/L of the carbon source. For lower and higher glucose concentrations, the stress is diminished to 2.0 and 2.5 MPa in case of 5 and 40 g/L glucose, respectively. BNC membranes are more stretchable for 20-35 g/L of glucose than for other concentrations. In case of the Young modulus (%) the trend has the shape of a bell curve, but due to high standard deviations we cannot clearly correlate these mechanical parameters with the glucose concentration in medium. Although the yield and mechanical properties are influenced by glucose concentration, the relationships between the factor and studied responses are not the same. For low concentrations of glucose, the yield was connected with a lack of carbon source in medium at the end of the culture.