*2.2. Manipulation of Aeration Level*

In order to evaluate the impact of dissolved oxygen level cultures were performed at various agitation rates (400–900 rpm) while maintaining a constant air flow of 0.8 vvm. A significant effect of agitation rate on growth of the yeast was observed (Figure 2). The biomass level reached a maximum of 25.1 g/L at 900 rpm and decreased depending on the decrease in the agitation rate to 15.7 g/L at 400 rpm. In the range of 400–700 rpm biosynthesis of KGA (38.1–54.5 g/L) was accompanied by comparatively high production of PA (25.5–38 g/L). As a result, KGA production yield (Yp/s) and selectivity were at the level of 0.27–0.39 g/g and 53–59%, respectively. The highest concentration of KGA and the best parameters of its biosynthesis were obtained when the agitation rate reached 800 rpm, and a further increase to 900 rpm resulted in a decrease in the KGA biosynthesis efficiency. In these cultures, the selectivity of the process was found to be significantly higher (72–83%) than in the process that was conducted at a lower agitation speed, i.e., 400–700 rpm. The agitation rate is a parameter affecting the amount of oxygen dissolved in the culture broth. In this study, the application of an agitation rate in the range of 400–900 rpm corresponded to 20–60% pO2, measured in the KGA production phase of the performed cultures. The aeration level has been identified as an important factor influencing KGA biosynthesis by *Y. lipolytica* growing on ethanol, rapeseed oil and biodiesel waste [23–25]. Similar to the results obtained in our study with the use of glycerol/oil media, the process of KGA production by *Y. lipolytica* VKM Y-2412 conducted on biodiesel waste (a substrate containing 70.8% glycerol and 23.9% fatty acids) was also promoted by high aeration [24]. The increase in aeration from 5% pO<sup>2</sup> to 50% pO<sup>2</sup> enabled an increase in the production of KGA from 56.8 to 80.4 g/L. In contrast, high aeration was not necessary for KGA biosynthesis from ethanol. In comparison to the culture with high aeration (50% pO2), 1.3-times higher KGA formation (49.0 g/L) was observed when low aeration was applied (5% pO2) [25]. As high aeration was found to stimulate KGA biosynthesis by the examined yeast strain, an agitation rate of 800 rpm was applied in all further experiments.

**Figure 2.** Impact of agitation on yeast growth and acids formation during KGA biosynthesis process performed by *Y. lipolytica* CBS 146773 in mixed glycerol/oil-based media. Culture conditions: 20% Ca(OH)2, pH 3.5, 3 µg/L of thiamine. For abbreviations, see Figure 1. *2.3. Availability of Exogenous Vitamins* Yeast reported as producers of KGA have been characterized as auxotrophic for one, two or several vitamins. These vitamins are co-factors of enzymes in the Krebs cycle, and **Figure 2.** Impact of agitation on yeast growth and acids formation during KGA biosynthesis process performed by *Y. lipolytica* CBS 146773 in mixed glycerol/oil-based media. Culture conditions: 20% Ca(OH)<sup>2</sup> , pH 3.5, 3 µg/L of thiamine. Abbreviations: X—biomass; KGA—α-ketoglutaric acid; PA pyruvic acid; CA—citric acid; Y—yield of KGA with respect to biomass formed (p/x) and utilized substrates (p/s); S—selectivity of KGA relative to sum of acids formed (KGA/(KGA + PA + CA)). Mean values for a specific product concentration marked with different letters (a, b, c, . . . ) differ significantly at *p* ≤ 0.05. Error bars indicate standard deviations.

#### their exogenous level is one of the crucial factors affecting accumulation of KGA in auxo-*2.3. Availability of Exogenous Vitamins*

trophic cells [19]. *Y. lipolytica* is auxotrophic only to thiamine, limitation of which is known to reduce the activity of α-ketoglutarate dehydrogenase and therefore determines KGA oversynthesis [24]. It should be noted that auxotrophy only for one vitamin gives an advantage to the process performed by *Y. lipolytica* because it requires very precise control of only one vitamin. In order to obtain thiamine limitation in the cultures with *Y. lipolytica*  CBS146773, the vitamin concentration was applied in the very low range of 1–4 μg/L (Figure 3A). It was clearly apparent, that thiamine had a significant impact on yeast growth, as the biomass level increased from 5.1 to 26.2 g/L with increased thiamine concentration. Increasing the vitamin addition from 1 to 3 μg/L resulted in rapid changes in both KGA and PA concentrations, but the opposite trend was observed for these acids—an increase from 11.0 to 69.1 g/L in the case of KGA and a decrease from 43.3 to 7.6 g/L in the case of PA. Thus, the selectivity of the process increased from 20% to 83% with the change of thiamine availability from 1 to 3 μg/L. No further improvement in KGA concentration or parameters of its biosynthesis was observed after the addition of 4 μg/L of thiamine. Because of the big differences in yeast growth observed between all the cultures it is worth paying attention to the parameter of yield of KGA calculated with respect to biomass formed (Yp/x). This parameter was the highest (3.37 g/g) when application of 3 μg/L of thiamine resulted in the highest amount of KGA produced. However, its value was very similar in the cultures with thiamine supplementation of 1 and 4 μg/L, where it reached 2.16 and 2.35 g/g, respectively, despite the amount of produced KGA (11.0—61.5 g/L, respectively) differing significantly between these processes. As mentioned above, the appropriate thiamine concentration is a crucial factor for effective KGA biosynthesis by the yeast belonging to the species *Y. lipolytica*. The optimal concentration for KGA biosynthesis requires a balance between the amount necessary for growth and the amount determining the decreased activity of α-ketoglutarate dehydrogenase and is a strain-dependent feature. It is reported in the literature that increasing availability of thiamine (up to 200 μg/L) stimulates the growth of the yeast, whereas for KGA synthesis a "peak" is observed at a certain low vitamin concentration (0.15–4 μg/L) specific to the kind of substrate, substrate feeding method and yeast strain applied for the process [22–26]. Moreover, it should Yeast reported as producers of KGA have been characterized as auxotrophic for one, two or several vitamins. These vitamins are co-factors of enzymes in the Krebs cycle, and their exogenous level is one of the crucial factors affecting accumulation of KGA in auxotrophic cells [19]. *Y. lipolytica* is auxotrophic only to thiamine, limitation of which is known to reduce the activity of α-ketoglutarate dehydrogenase and therefore determines KGA oversynthesis [24]. It should be noted that auxotrophy only for one vitamin gives an advantage to the process performed by *Y. lipolytica* because it requires very precise control of only one vitamin. In order to obtain thiamine limitation in the cultures with *Y. lipolytica* CBS146773, the vitamin concentration was applied in the very low range of 1–4 µg/L (Figure 3A). It was clearly apparent, that thiamine had a significant impact on yeast growth, as the biomass level increased from 5.1 to 26.2 g/L with increased thiamine concentration. Increasing the vitamin addition from 1 to 3 µg/L resulted in rapid changes in both KGA and PA concentrations, but the opposite trend was observed for these acids—an increase from 11.0 to 69.1 g/L in the case of KGA and a decrease from 43.3 to 7.6 g/L in the case of PA. Thus, the selectivity of the process increased from 20% to 83% with the change of thiamine availability from 1 to 3 µg/L. No further improvement in KGA concentration or parameters of its biosynthesis was observed after the addition of 4 µg/L of thiamine. Because of the big differences in yeast growth observed between all the cultures it is worth paying attention to the parameter of yield of KGA calculated with respect to biomass formed (Yp/x). This parameter was the highest (3.37 g/g) when application of 3 µg/L of thiamine resulted in the highest amount of KGA produced. However, its value was very similar in the cultures with thiamine supplementation of 1 and 4 µg/L, where it reached 2.16 and 2.35 g/g, respectively, despite the amount of produced KGA (11.0—61.5 g/L, respectively) differing significantly between these processes. As mentioned above, the appropriate thiamine concentration is a crucial factor for effective KGA biosynthesis by the yeast belonging to the species *Y. lipolytica*. The optimal concentration for KGA biosynthesis requires a balance between the amount necessary for growth and the amount determining the decreased activity of α-ketoglutarate dehydrogenase and is a strain-dependent feature. It is reported in the literature that increasing availability of thiamine (up to 200 µg/L) stimulates the growth

of the yeast, whereas for KGA synthesis a "peak" is observed at a certain low vitamin concentration (0.15–4 µg/L) specific to the kind of substrate, substrate feeding method and yeast strain applied for the process [22–26]. Moreover, it should be noted that by-product formation of PA also might be affected by thiamine concentration when yeast is grown on glycolytic carbon sources (glucose, fructose, glycerol, etc.), which are utilized via pyruvate because of modulation of thiamine-dependent pyruvate dehydrogenase activity [30]. *Catalysts* **2023**, *13*, x FOR PEER REVIEW 6 of 14 be noted that by-product formation of PA also might be affected by thiamine concentration when yeast is grown on glycolytic carbon sources (glucose, fructose, glycerol, etc.), which are utilized via pyruvate because of modulation of thiamine-dependent pyruvate

dehydrogenase activity [30].

**Figure 3.** Impact of thiamine (**A**) and biotin (**B**) on the yeast growth and acids formation during KGA biosynthesis performed by *Y. lipolytica* CBS 146773 in mixed glycerol/oil-based media. Culture **Figure 3.** Impact of thiamine (**A**) and biotin (**B**) on the yeast growth and acids formation during KGA biosynthesis performed by *Y. lipolytica* CBS 146773 in mixed glycerol/oil-based media. Culture conditions: 20% Ca(OH)<sup>2</sup> , pH 3.5, 800 rpm, 3 µg/L of thiamine (**B**). Abbreviations: X—biomass; KGA—α-ketoglutaric acid; PA—pyruvic acid; CA—citric acid; Y—yield of KGA with respect to biomass formed (p/x) and utilized substrates (p/s); S—selectivity of KGA relative to sum of acids formed (KGA/(KGA + PA + CA)). Mean values for a specific product concentration marked with different letters (a, b, c, . . . ) differ significantly at *p* ≤ 0.05. Error bars indicate standard deviations.

conditions: 20% Ca(OH)2, pH 3.5, 800 rpm, 3 µg/L of thiamine (**B**). For abbreviations, see Figure 1. The impact of exogenous biotin addition (0–1.5 mg/L) and all subsequent experiments were performed in media supplemented with 3 μg/L of thiamine. Biotin is another vitamin which may induce accumulation of KGA by affecting the activity of pyruvate carboxylase [30–32]. In this study, biotin addition to the culture stimulated yeast growth— The impact of exogenous biotin addition (0–1.5 mg/L) and all subsequent experiments were performed in media supplemented with 3 µg/L of thiamine. Biotin is another vitamin which may induce accumulation of KGA by affecting the activity of pyruvate carboxylase [30–32]. In this study, biotin addition to the culture stimulated yeast growth—in the processes supplemented with the vitamin, biomass was at the level of 21.5–24.2 g/L,

> in the processes supplemented with the vitamin, biomass was at the level of 21.5–24.2 g/L, whereas in the control culture its concentration reached 20.5 g/L (Figure 3B). However, no

whereas in the control culture its concentration reached 20.5 g/L (Figure 3B). However, no positive effect of biotin addition on KGA production was observed. The amount of KGA in the post-culture broth decreased from 69.1 to 51.1 g/L after culture supplementation with 1.5 mg/L of biotin. Simultaneously, PA concentration increased from 7.6 g/L in the culture not supplemented with biotin to 28.6 g/L in the process where 1.5 mg/L of the vitamin was used. Theoretically, biotin presence increases the activity of pyruvate carboxylase, which catalyzes the conversion of pyruvate to oxaloacetate. Therefore, biosynthesis of KGA from substrates metabolized by the glycolysis pathway (e.g., glycerol) should be enhanced by biotin supplementation. In the shake-flask culture of *Y. lipolytica* WSH-Z06, addition of 0.8 mg/L of biotin had only small positive effect on KGA biosynthesis from glycerol whereas PA production was unaffected [26]. Interesting observations were reported by Otto et al. [30], who studied the changes in the by-product spectrum during KGA biosynthesis from glycerol by *Y. lipolytica* H355A(PYC1) T3—a strain that overexpressed pyruvate carboxylase. In comparison to the mother strain H355, higher activity of the enzyme in the transformant strain resulted in a higher biomass level, a decrease in KGA production from 133.0 to 126.9 g/L, and a simultaneous slight increase in the formation of PA and other by-products. The positive effect on yeast growth was explained by accumulation of precursor molecules (oxaloacetic, malic, succinic and fumaric acids) caused by an imbalance between enhanced activity of pyruvate carboxylase and inhibited activity of pyruvate dehydrogenase (due to thiamine limitation). Assuming that, in the present study, the addition of biotin increased the activity of pyruvate carboxylase, the same tendency was noticeable—stimulation of yeast growth and PA production with a decrease in KGA formation. Similarly, the growth of *Y. lipolytica* VKM Y-2412 was slightly increased whereas KGA production was decreased and no effect on PA formation was noted when the process was conducted on biodiesel waste media upon supplementation with biotin (10–40 µg/L) [24].
