## 3 Results and Discussion  
### The Influence of Cultivation Media on Substrate Hydrolysis and Enzymes Stability  
The possibility of BC production using alternative carbon sources other than glucose has been intensively researched for years [10; 28; 29; 30]. These studies indicate that it is possible, but glucose is still the most widely used. However, it is worth remembering that bacteria use this monosaccharide in the biopolymer synthesis pathways, the development and growth of cellular biomass, or both [31]. Moreover, glucose is not an ideal carbon source for the synthesis of BC, and its usage is associated with the possibility of reducing the efficiency of the BC production process. The high concentration of this hexose in the culture medium causes its significant conversion, by oxidation, to gluconic acid (GA) by _K. xylinus_ cells, which finally reduces the effectiveness of the BC synthesis process [32]. Moreover, an undesirable consequence of the overproduction of GA is acidification of the culture medium, which ultimately affects the bacterial metabolism and BC synthesis process. Nevertheless, using various monosaccharides as a carbon source in the cultivation medium allows for maintaining the efficiency of the BC production at a level that is impossible to achieve with different monosaccharide-rich oligo- and polysaccharides used as carbon sources [6; 33]. Most characterised _K. xylinus_ strains do not have the enzymatic potential for converting these kinds of carbon sources to simple sugars that then would be easily assimilated and transformed into precursors necessary for the synthesis of BC.  
Due to the possible influence of medium components and parameters, such as pH or salinity, on enzyme activity and stability, the efficiency of substrate hydrolysis in time was analysed. The glucose liberation profile to media differed depending on the initial amount of used enzyme units. In the culture medium with sucrose and \(\beta\)-D-Fru, 6.0, 5.0, and 3.0 g/L of glucose were obtained after 4 h (Figure 2a). In the medium with lactose and \(\beta\)-galactosidasees expected, 5.0 g/L of glucose was detected within 3 h for all variants of the enzyme units (Figure 2b). In the medium with starch and glucoamylase, glucose concentration was assumed within less than 4 h, with the highest speed in a medium containing 10 U/mL of the enzyme (Figure 2c). The profile of glucose liberation in media with sucrose and \(\beta\)-D-tructofuranosidase suggested the possibility of regulating the dynamics of increasing the accessibility of carbon sources. However, the ability of enzymes to act for an extended time depends on their stability in the reaction environment. The level of remaining activity, measured after finishing cultivation time, indicated that \(\beta\)-D-tructofuranosidase and glucoamylase retained similar initial activity levels, in contrast to \(\beta\)-galactosidasees, which showed significantly lower operational stability (Table 1). The decreasing stability can be explained by the progressive decrease of medium pH that can negatively influence the enzyme's molecular structure. The acidic pH is especially not favourable for maintaining proper enzyme conformation and activity for a long time [34; 35]. The profile of glucose liberation, particularly in media with sucrose and \(\beta\)-D-Fru, suggests the possibility of regulating the gradual increase of the accessibility of carbon sources by the amounts of used enzymes. It could potentially reduce the rate of media acidification and retain significant enzyme activity for a longer time. However, most initial substrateswere converted to monosaccharides in the first hour, when the number of bacterial cells was low and media pH was stable.  
Figure 2: The effect of different amount units of \(\beta\)-D- Fru, \(\beta\)-Gal, and GLA on substrate conversion to simple sugars in HS medium with sucrose (**a**), lactose (**b**), and starch (**c**) as carbon sources.