## 3 Results and Discussion
### The Effect of Culture Condition on the Yield and Mechanical Properties of Bacterial Nanocellulose
After a 6-day culture in medium, there were only 0.69, 1.39 and 2.39 g/L glucose for initial concentrations of 5, 10, and 15 g/L, respectively (data not shown). Therefore, glucose was exhausted and BNC biosynthesis was limited. Because of the lower yield, the mechanical strength was decreased. For 40 g/L the value of pH was 3.0 at the end of the culture, which can be ascribed to higher oxidation of glucose to gluconic acid. In this condition, only a slight decrease in the yield was noted, but tensile properties were significantly affected similar to the culture with an initial pH at 3.5. Other authors reported similar effects of glucose concentration on BNC yield, but do not correlate it with the mechanical strength of membranes. [PERSON] et al. observed that glucose between 15 and 25 g/L did not affect cellulose production [24]. [PERSON] and [PERSON] reported that BNC yield decreased with an increase in the initial glucose concentration between 40 and 200 g/L and connected that effect with the diminished pH value below 3.0 [37]. Negative impact of high glucose content was also observed in shaking conditions [33], but the inhibiting concentration was higher (50 g/L) than in stationary culture. Figure 8: Impact of glucose concentration on (**a**) biosynthesis yield and (**b**) tensile parameters of bacterial nanocellulose. Figure 9a,b shows both the BNC yield and productivity, with time course of cultivation. As it can be seen at Figure 9a, the yield is increasing during the entire incubation time. On the first day, only 0.47 g/L was achieved, whereas after a 7-day culture the yield was over 4 g/L. On the other hand, the highest productivity was noted on the fifth day of incubation. For longer cultivation times, the productivity decreased. There are several possibilities why the productivity decreases after 5 days of cultivation. [PERSON] et al. described the wall effect as a factor limiting BNC production [38]. According to this research, the maximum thickness of membrane in glass cuboid bioreactor is 20 mm, but stagnation in BNC growth should be observed earlier. In our study, we used an s/v ratio of 0.71 cm\({}^{-1}\), which was 1.4 cm medium high, so the maximum thickness of the pellicle was less than 1.4 cm and in standard conditions the medium was exhausted after 6-7 days. On the other hand, in cultures with introduced air above the membrane, BNC reaches the bottom in the fourth day. Thus, we cannot discard the possibility of limiting the biosynthesis by the wall effect in favorable conditions. Secondly, the final pH was 3.50 on the fifth day of culture and for longer cultures the pH decreased below 3.5, which also had an impact on the production rate. Also important is the physiological state of cells during the incubation time. As we mentioned, the highest production rate is related to the late exponential stage, but the stationary phase starts from the fourth day of cultivation. Furthermore, bacteria switches its metabolism into acetic acid consumption instead of glucose [31], despite the relatively high concentration of remaining glucose. Reduction of glucose metabolism results in lower UDP-glucose biosynthesis which is a priming metabolite for cellulose polymerization. Time of culture also influences the mechanical properties of the membrane (Figure 9c). Tensile stress was changed until the third day of culture and stayed constant. Inverse relationship was observed for strain. Initially, the pellicle was able to stretch up to 35-40%, but after the third day of incubation, the value of stress was significantly lower  
Figure 9: Impact of culture time on (**a**) biosynthesis yield, (**b**) productivity, and (**c**) tensile parameters of bacterial nanocellulose. For 5- to 7-day cultures, the strain was twice lower than of 1-day BNC. Young modulus was increasing until 5 days. The low value of the Young modulus for membranes at short cultivation times was a consequence of low stress and high strain of BNC.