## 3 Results and Discussion
### Impact of Yeast Extract Reduction on Erythiritol Production and Purity in Y. lipolytica  
For the second stage of this project, a nitrogen source concentration of 3.1 g/L was selected. The aim of this experiment was to compare erythroid production parameters at different concentrations of YE, which is commonly used as a source of thiamine in media for erythroid production, as well as for the biosynthesis of citric acid by _Y. lipolytica_. _Y. lipolytica_ yeast strains are natural thiamine auxotrophs, as they cannot synthesize the pyrimidine moiety of the thiamine molecule. Without thiamine, they fail to grow [18]. According to the literature data, the concentration of this vitamin required for proper growth is 200 \(\upmu\)g/L [19]. However, other studies have highlighted varying sensitivities of strains to thiamine deficiency/concentration [20]. In microbiological media, pure thiamine is often replaced with natural ingredients such as corn steep liquor (CSL) or YE. CSL originates from a waste stream in the corn starch wet-milling process. It is rich in organic nitrogen, amino acids, lactic acid, minerals, vitamins, and reducing sugars [21]. However, CSL's batch-to-batch variability, high solids' content, and strong coloration are significant disadvantages for its use as a nutrient source in fermentation processes [22]. YE is a product primarily derived from waste brewer's yeast. It is rich in nucleotides, proteins, amino acids, sugars, and various trace elements. Its advantages include low production costs and abundant raw material supply. However, the presence of YE can result in yellow discoloration of erythritol crystals. Therefore, reducing or even eliminating YE in the medium composition could eliminate the decolorization step, improving crystallization efficiency and erythritol purity. Decolorization is also widely used for other polyols, such as xylitol [23]. Two commonly employed approaches involve decolorization during either the recovery or purification stage, typically using activated carbon. In both cases, the key parameters identified include dose, temperature, contact time, and mixing speed [24; 25]. Nevertheless, a virtually colorless post-culture liquid can be obtained by minimizing the addition of coloring components to the culture--in this case, YE. Studies on erythroid biosynthesis from glycerol by _Y. lipolytica_[20] identified a YE concentration of 1.0 g/L as optimal. Therefore, earlier experiments were conducted at this concentration (Figure 2). However, in the following study, erythroid production parameters were examined with a reduced YE content (Figure 3). Four cultivation processes were conducted in the presence of 3.1 g/L (NH4)2 SO4, with YE concentrations ranging from 0.00 to 0.75 g/L (Table 1--Production medium 2). Any potential thiamine deficiency in cultures with 0.00-0.75 g/L YE was supplemented with the addition of 200 \(\upmu\)g/L of pure vitamin. For comparison, Figure 3 also includes results from the cultivation conducted at YE = 1.0 g/L (from the previous experiment). The time required for complete glucose utilization ranged narrowly from 168 to 182 h, and the final erythroid concentration varied from 141.9 g/L (YE = 0.75 g/L) to 155.0 g/L (YE = 0.25 g/L) (Figure 3A). No statistically significant differences were observed in the erythritol production parameters; however, the highest values of yield (Y = 0.52 g/g) and volumetric productivity (Q = 0.85 g/L), as well as the highest selectivity (87%), were noted at a YE concentration of 0.25 g/L (Figure 3B). Based on these results, it can be concluded that the medium for erythroid production from glucose does not necessarily require YE in its composition, but only 200 \(\upmu\)g/L of thiamine. However, due to the lower biomass level in the cultivation without YE addition (Figure 3A) and the slightly higher erythroid production parameters achieved at 0.25 g/L YE (Figure 3B), this YE concentration was chosen for subsequent experiments.