*2.4. The Effect of Iron*

The presence of iron ions is another factor that may possibly affect KGA biosynthesis by modulating the activity of iron-dependent enzymes in the Krebs cycle, i.e., aconitase and succinate dehydrogenase [33,34]. In the present study the impact of iron was evaluated in the media supplemented with ammonium iron sulfate hexahydrate in an amount corresponding to 1–4 mg/L of iron ions. The presence of iron had no effect on growth of the examined yeast strain (Figure 4). The biomass level in the cultures supplemented with iron ions was in the range of 19.0–20.4 g/L, which was comparable to the level obtained for the control culture (20.5 g/L). Addition of iron ions resulted in lower production of KGA and enhanced accumulation of PA. In the supplemented cultures yeast produced 53.7–59.0 g/L and 17.0–29.2 g/L of these acids, respectively. Compared to the non-iron-supplemented process, formation of CA was found to be slightly decreased upon the addition of iron ions (2.7–5.7 g/L). The effect of iron on KGA biosynthesis by *Y. lipolytica* was investigated during cultivation on ethanol [35,36]. It was found that iron concentration of 0.5–2.0 mg/L stimulated KGA synthesis, whereas a further increase in iron concentration from 2–3 to 10.0 mg/L caused a gradual decrease in KGA accumulation. The positive impact of iron presence was also reported for KGA production on biodiesel waste by *Y. lipolytica* [24]. Effective biosynthesis of isocitric acid by *Y. lipolytica* from rapeseed oil was noted also upon iron supplementation [37]. In turn, the ability of *Y. lipolytica* to produce erythritol was unaffected by iron ions when yeast was cultivated in bioreactor cultures on glycerol media [38].

**Figure 4.** Impact of iron on 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)2, pH 3.5, 800 rpm, 3 µg/L of thiamine. For abbreviations, see Figure 1. *2.5. The Impact of Sorbitan Monolaurate* Sorbitan monolaurate, known under the trade name Span 20, is a non-ionic, waterinsoluble, lipophilic emulsifier [39]. This surfactant is approved for use as a food additive **Figure 4.** Impact of iron on 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. 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.

#### (E493) and the acceptable daily intake is 10 mg/kg [40]. Literature reports mention a pos-*2.5. The Impact of Sorbitan Monolaurate*

itive effect of Span 20 on the growth and excretion of some metabolites by microorganisms [41–44]. This surfactant, in addition to increasing the dispersion of oil substrates in the culture media, may also increase the permeability of microorganisms' cell membranes. Thus, it facilitates the excretion of metabolites and extracellular proteins from the cell. In this study, Span 20 was applied to the culture media in the concentration ranging from 0.25 to 1 g/L (Figure 5). In comparison to the control, the growth of the yeast was slightly lower in the presence of Span 20 and ranged 18.5–19.4 g/L. Nevertheless, addition of Span 20 had a significant positive impact on KGA biosynthesis. The increase in its addition up to 1 g/L resulted in a gradual increase in KGA production up to 82.4 g/L, corresponding to the yield (Yp/s) of 0.88 g/g and selectivity of 88% (Figures 5 and 6). Comparatively high PA amounts (11.0–18.1 g/L) were obtained in the cultures with Span 20 addition of 0.25– 0.75 g/L. In turn, production of CA was slightly decreased by the presence of Span 20 and was in the range 3.5–5.0 g/L. The positive effect of Span 20 addition was reported previously for production of oxalic acid from fatty acid waste by *Aspergillus niger* [41]. In the process with the addition of 0.75 g/L of the surfactant, the production of oxalic acid increased from 34.7 to 48.4 g/L, compared to the control culture. The addition of 0.25 g/L of Span 20 to the culture of *Y. lipolytica* Wratislavia K1 cultivated on raw glycerol enhanced erythritol production from 149.6 g/L to 165.7 g/L [42]. Moreover, although very low byproduct formation of CA and KGA was noted in this process, synthesis of both acids was enhanced by the presence of the surfactant. Application of Span 20 was also proved to have a positive effect on biomass formation and β-carotene production by *Blakeslea trispora* [43,44]. Sorbitan monolaurate, known under the trade name Span 20, is a non-ionic, waterinsoluble, lipophilic emulsifier [39]. This surfactant is approved for use as a food additive (E493) and the acceptable daily intake is 10 mg/kg [40]. Literature reports mention a positive effect of Span 20 on the growth and excretion of some metabolites by microorganisms [41–44]. This surfactant, in addition to increasing the dispersion of oil substrates in the culture media, may also increase the permeability of microorganisms' cell membranes. Thus, it facilitates the excretion of metabolites and extracellular proteins from the cell. In this study, Span 20 was applied to the culture media in the concentration ranging from 0.25 to 1 g/L (Figure 5). In comparison to the control, the growth of the yeast was slightly lower in the presence of Span 20 and ranged 18.5–19.4 g/L. Nevertheless, addition of Span 20 had a significant positive impact on KGA biosynthesis. The increase in its addition up to 1 g/L resulted in a gradual increase in KGA production up to 82.4 g/L, corresponding to the yield (Yp/s) of 0.88 g/g and selectivity of 88% (Figures 5 and 6). Comparatively high PA amounts (11.0–18.1 g/L) were obtained in the cultures with Span 20 addition of 0.25–0.75 g/L. In turn, production of CA was slightly decreased by the presence of Span 20 and was in the range 3.5–5.0 g/L. The positive effect of Span 20 addition was reported previously for production of oxalic acid from fatty acid waste by *Aspergillus niger* [41]. In the process with the addition of 0.75 g/L of the surfactant, the production of oxalic acid increased from 34.7 to 48.4 g/L, compared to the control culture. The addition of 0.25 g/L of Span 20 to the culture of *Y. lipolytica* Wratislavia K1 cultivated on raw glycerol enhanced erythritol production from 149.6 g/L to 165.7 g/L [42]. Moreover, although very low by-product formation of CA and KGA was noted in this process, synthesis of both acids was enhanced by the presence of the surfactant. Application of Span 20 was also proved to have a positive effect on biomass formation and β-carotene production by *Blakeslea trispora* [43,44].

**Figure 5.** Impact of Span 20 on 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)2, pH 3.5, 800 rpm, 3 µg/L of thiamine. For abbreviations, see Figure 1. **Figure 5.** Impact of Span 20 on 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. 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. **Figure 5.** Impact of Span 20 on 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)2, pH 3.5, 800 rpm, 3 µg/L of thiamine. For abbreviations, see Figure 1.

Culture conditions: 20% Ca(OH)2, pH 3.5, 800 rpm, 3 µg/L of thiamine, 1 g/L of Span 20. Abbreviations: GLY—glycerol; for other abbreviations, see Figure 1. Each arrow indicates addition of one portion of 30 g/L of mixed substrates (see: Materials and Methods, Section 3.2). **3. Materials and Methods** *3.1. Microorganisms* **Figure 6.** Time-course of yeast growth and acid formation during KGA biosynthesis performed by *Y. lipolytica* CBS 146773 in mixed glycerol/oil-based media supplemented with 1 g/L of Span 20. Culture conditions: 20% Ca(OH)2, pH 3.5, 800 rpm, 3 µg/L of thiamine, 1 g/L of Span 20. Abbreviations: GLY—glycerol; for other abbreviations, see Figure 1. Each arrow indicates addition of one portion of 30 g/L of mixed substrates (see: Materials and Methods, Section 3.2). **Figure 6.** Time-course of yeast growth and acid formation during KGA biosynthesis performed by *Y. lipolytica* CBS 146773 in mixed glycerol/oil-based media supplemented with 1 g/L of Span 20. Culture conditions: 20% Ca(OH)<sup>2</sup> , pH 3.5, 800 rpm, 3 µg/L of thiamine, 1 g/L of Span 20. Abbreviations: GLY—glycerol; for other abbreviations, see Figure 1. Each arrow indicates addition of one portion of 30 g/L of mixed substrates (see: Materials and Methods, Section 3.2).

#### The strain *Yarrowia lipolytica* CBS146773, formerly named 1.31.GUT1/6.CIT1/3.E34672 **3. Materials and Methods 3. Materials and Methods**

#### [21], was the subject of this study. The strain was stored on YM agar slants at 4 °C in the *3.1. Microorganisms 3.1. Microorganisms*

culture collection belonging to the Department of Biotechnology and Food Microbiology at Wrocław University of Environmental and Life Sciences (Poland). The strain *Yarrowia lipolytica* CBS146773, formerly named 1.31.GUT1/6.CIT1/3.E34672 [21], was the subject of this study. The strain was stored on YM agar slants at 4 °C in the culture collection belonging to the Department of Biotechnology and Food Microbiology at Wrocław University of Environmental and Life Sciences (Poland). The strain *Yarrowia lipolytica* CBS146773, formerly named 1.31.GUT1/6.CIT1/3.E34672 [21], was the subject of this study. The strain was stored on YM agar slants at 4 ◦C in the culture collection belonging to the Department of Biotechnology and Food Microbiology at Wrocław University of Environmental and Life Sciences (Poland).

#### *3.2. Media Composition and Culture Conditions*

Seed cultures were performed in 300 mL baffled flasks containing 50 mL of an inoculation medium that consisted of (g/L): edible rapeseed oil—20.0; NH4Cl—9.0; KH2PO4—2.0; MgSO4·7 H2O—1.4; CaCO3—10.0; and thiamine—3 µg/L dissolved in distilled water. The pH was 3.5. The medium was inoculated from agar slants and next the culture was grown for 72 h at 29 ◦C and 140 rpm on a rotary shaker (CERTOMAT IS; Sartorius, Germany).

Batch fermentation was conducted in a 5 L bioreactor (BIOSTAT B Plus; Sartorius, Germany) with a working final volume of 2 L. Bioreactor production medium composition was (g/L): pure glycerol (98%; Wratislavia-Bio; Poland)—20.0; NH4Cl—9.0; KH2PO4— 2.0; MgSO4·7 H2O—1.4; and thiamine—3 µg/L, dissolved in tap water. The pH was 3.5. In some cultures, the medium was supplemented with thiamine (1–4 µg/L), biotin (0.5–1.5 mg/L), (NH4)2Fe(SO4)2·6 H2O (7.03–28.12 mg/L) or Span 20 (0.25–1 g/L). The volume of 150 mL of seed culture was used to inoculate production medium in fermenter and the process was conducted for 144 h at 29 ◦C, with agitation rate of 800 rpm, aeration rate of 0.8 vvm and pH 3.5 maintained by the automatic addition of a 20% solution of Ca(OH)<sup>2</sup> (or NaOH/KOH, when indicated). During the cultivation, the process was fed at 24 h intervals (i.e., 24, 48, 72, 96 h, as indicated in Figure 6) with 4 sterile portions of 30 g/L of mixed substrates (glycerol and rapeseed oil in equal amounts of 15 g/L). Any changes in the culture parameters and composition of the medium are indicated for specific experiments in the Results section. All chemicals used in the investigation were of analytical purity (Sigma-Aldrich, Steinheim, Germany). Prior to inoculation, all media were sterilized at 121 ◦C for 30 min.

#### *3.3. Analytical Methods*

The samples collected from the bioreactor fermentation process were analyzed in terms of biomass level (dry matter) and concentration of glycerol and acids—ketoglutaric, pyruvic and citric. The sample preparation and analytical methods were described previously by Rywi ´nska et al. [22]. The results are presented as mean values of the process performed in duplicate. Statistical analysis was performed using one-way analysis of variance (Statistica 13.0 software; StatSoft, Tulsa, OK, USA). The significant differences in the data (X, KGA, PA, CA) were compared by Duncan's multiple range test. (*p* ≤ 0.05).

#### **4. Conclusions**

In this study the effects of selected media components and culture conditions were evaluated in order to enhance the biosynthesis of KGA by *Y. lipolytica* CBS146773. The possible impact of evaluated factors is presented in Figure 7. In the research, the source of carbon and energy was a mixture of glycerol and rapeseed oil. The addition of Span 20 was used to increase the dispersion of oil droplets and increase the permeability of cell membranes, which may facilitate the secretion of the produced metabolites [41,42]. Notably, substrates applied in this investigation are utilized by the yeast cell in different metabolic pathways. Glycerol is first transformed by the action of glycerol kinase to glycerol-3-phosphate, which, after being converted to glyceraldehyde, undergoes further transformations in the glycolytic pathway, resulting in the formation of pyruvate. In the mitochondrion, pyruvate dehydrogenase catalyzes the conversion of pyruvate into acetyl-CoA, which is incorporated into the tricarboxylic acid cycle with the action of citrate synthase. The transformant strain used in this study was characterized by overexpression of genes encoding glycerol kinase and citrate synthase, which was aimed at increasing the efficiency of the above-described metabolic pathway [21]. Moreover, a gene encoding previously uncharacterized mitochondrial organic acid transporter was overexpressed in the yeast strain to investigate whether it might facilitate secretion of organic acids from mitochondrion and increase the extracellular concentration of KGA as a final product.

**Figure 7.** Scheme of metabolic pathways involved in conversion of glycerol and rapeseed oil to KGA by *Yarrowia lipolytica* CBS146773 and the putative effect of selected factors on the efficiency of the process. Abbreviations: **↑:** stimulatory effect; AH: aconitate dehydrogenase; CA: citric acid; *CIT1*: gene encoding citrate synthase; CS: citrate synthase; DHAP: dihydroxyacetone phosphate; *E34672g*: gene *YALI0E34672g* encoding mitochondrial acid transporter; EMP: Embden–Meyerhof–Parnas glycolytic pathway; FA: fumaric acid; GA3P: glyceraldehyde-3-phosphate; GK: glycerol kinase; GLY: glycerol; GLY-3P: glycerol-3-phosphate; *GUT1*: gene encoding glycerol kinase; ICA: isocitric acid; KGA: α-ketoglutaric acid; KGDH: α-ketoglutarate dehydrogenase; MA: malic acid; OAA: oxaloacetic acid; PA: pyruvic acid; PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase; SA: succinic acid; SA-CoA: succinyl-CoA; SDH: succinate dehydrogenase; T: mitochondrial acid transporter; TAG: triacylglyceride; TCA: tricarboxylic acid cycle. **Figure 7.** Scheme of metabolic pathways involved in conversion of glycerol and rapeseed oil to KGA by *Yarrowia lipolytica* CBS146773 and the putative effect of selected factors on the efficiency of the process. Abbreviations: ↑**:** stimulatory effect; AH: aconitate dehydrogenase; CA: citric acid; *CIT1*: gene encoding citrate synthase; CS: citrate synthase; DHAP: dihydroxyacetone phosphate; *E34672g*: gene *YALI0E34672g* encoding mitochondrial acid transporter; EMP: Embden–Meyerhof– Parnas glycolytic pathway; FA: fumaric acid; GA3P: glyceraldehyde-3-phosphate; GK: glycerol kinase; GLY: glycerol; GLY-3P: glycerol-3-phosphate; *GUT1*: gene encoding glycerol kinase; ICA: isocitric acid; KGA: α-ketoglutaric acid; KGDH: α-ketoglutarate dehydrogenase; MA: malic acid; OAA: oxaloacetic acid; PA: pyruvic acid; PC: pyruvate carboxylase; PDH: pyruvate dehydrogenase; SA: succinic acid; SA-CoA: succinyl-CoA; SDH: succinate dehydrogenase; T: mitochondrial acid transporter; TAG: triacylglyceride; TCA: tricarboxylic acid cycle.

The second substrate—rapeseed oil—is hydrolyzed by extracellular lipases to glycerol and fatty acids. In the cell the latter undergo β-oxidation, resulting in the formation of acetyl-CoA—a compound that connects the pathways of glycerol and oil utilization. Formed from acetyl-CoA and oxaloacetate, citrate is further converted to isocitric acid by aconitase, and the subsequent transformation leads to the formation of KGA. Theoreti-The second substrate—rapeseed oil—is hydrolyzed by extracellular lipases to glycerol and fatty acids. In the cell the latter undergo β-oxidation, resulting in the formation of acetyl-CoA—a compound that connects the pathways of glycerol and oil utilization. Formed from acetyl-CoA and oxaloacetate, citrate is further converted to isocitric acid by aconitase, and the subsequent transformation leads to the formation of KGA. Theoretically,

at this stage, metabolism can be stimulated by the presence of iron ions, since aconitase is an iron-dependent enzyme [33,34]. In the presented study, however, we did not observe a positive effect of iron supplementation on KGA biosynthesis. Overproduction of KGA requires inhibition of thiamine-dependent ketoglutarate dehydrogenase, catalyzing KGA conversion to succinyl-CoA. In *Y. lipolytica*, this is possible by limiting exogenous thiamine, as this yeast is unable to synthesize the pyrimidine structure of this vitamin [24]. In accordance with literature reports, in the presented study, the concentration of thiamine was noted as the key factor determining the effective production of KGA by *Y. lipolytica* [22–26]. However, it should be noted that the differences in the amount of thiamine reported as necessary for KGA production might be dependent not only on the strain but also on the kind of substrate applied in the process. In comparison to glycolytic substrates, the use of fatty substrates omits the reaction catalyzed by the second thiamine-dependent enzyme—pyruvate dehydrogenase; hence, the cell's need for this vitamin may be lower in such a process. A side effect of using thiamine limitation in cultures conducted on glycerol media is the inhibition of pyruvate dehydrogenase resulting in accumulation of pyruvate. This effect can be counteracted by increasing the activity of pyruvate carboxylase, which converts pyruvate to oxaloacetate [31]. In this study, two factors that might stimulate the activity of this enzyme were examined: biotin and calcium ions. The presence of calcium ions was found to significantly enhance the biosynthesis of KGA, whereas a positive effect of biotin supplementation was not observed.

The results of the experiments performed in this study to identify the best conditions for effective KGA biosynthesis indicated the following: maintenance of pH at 3.5 by neutralization with the use of Ca(OH)2, an agitation rate of 800 rpm and the addition of 3 µg/L of thiamine and 1 g/L of Span 20. In our earlier investigation, this transformant strain was identified as a good producer of KGA from mixed media which, after preliminary optimization of the process conditions (C:N:P ratio), was able to biosynthesize 53.1 g/L of KGA with productivity of 0.35 g/L h and yield (Yp/s) of 0.53 g/g [21]. In the present work selection of culture conditions enabled the increase in KGA biosynthesis to 82.4 g/L. Moreover, the parameters of KGA biosynthesis were significantly improved—the productivity increased to 0.57 g/L h and the yield (Yp/s) reached 0.59 g/g. A similar amount of the acid was obtained after optimization of the KGA production process performed by *Y. lipolytica* VKM Y-2412 on biodiesel waste containing glycerol and fatty acids [24]. In the optimal conditions, yeast produced 80.4 g/L of KGA with the selectivity of 96.7%. However, the cultivation process was significantly longer (192 h) than in the present research (144 h). In the literature data the highest KGA production was reported for *Y. lipolytica* H355A(PYC1-IDP1) T5 engineered for overexpression of isocitrate dehydrogenase and pyruvate carboxylase, which was able to synthesize 186.0 g/L of KGA from raw glycerol with productivity of 1.75 g/L h [45]. However, the yield of KGA production, which is one of the factors determining the process attractiveness in terms of industrial production, obtained in the process with strain H355A(PYC1-IDP1) T5 reached only 0.36 g/g and was significantly lower than the yield obtained in this research with strain CBS146773.

**Author Contributions:** Conceptualization, L.T.-H. and A.R.; methodology, L.T.-H., A.R. and W.R.; software, L.T.-H.; validation, L.T.-H. and A.R.; formal analysis, L.T.-H.; investigation, W.R., L.T.-H., Z.L. and A.R.; resources, W.R.; data curation, L.T.-H. and W.R.; writing—original draft preparation, L.T.-H.; writing—review and editing, L.T.-H.; visualization, L.T.-H.; supervision, A.R. and W.R.; project administration, W.R.; funding acquisition, A.R., L.T.-H. and W.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by The National Centre for Research and Development under the Project No. POIR.04.01.02-00-0028/18 entitled "Development of an innovative technology for the production of dietary supplements based on alpha-ketoglutaric acid obtained on the biological way with *Yarrowia lipolytica* yeast".

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
