*Article* **Enhancement of** α**-Ketoglutaric Acid Production by** *Yarrowia lipolytica* **Grown on Mixed Renewable Carbon Sources through Adjustment of Culture Conditions**

**Ludwika Tomaszewska-Hetman \* , Anita Rywi ´nska , Zbigniew Lazar and Waldemar Rymowicz**

Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Chełmo ´nskiego Str. 37, 51-630 Wroclaw, Poland

**\*** Correspondence: ludwika.tomaszewska-hetman@upwr.edu.pl

**Abstract:** α-Ketoglutaric acid (KGA) is a valuable compound with a wide range of applications, e.g., in the cosmetics, pharmaceutical, chemical and food industries. The present study aimed to enhance the efficiency of KGA production by *Yarrowia lipolytica* CBS146773 from renewable carbon sources. In the investigation, various factors that may potentially affect KGA biosynthesis were examined in bioreactor cultures performed on a simple medium containing glycerol (20 g/L) and fed with four portions of a substrate mixture (15 + 15 g/L of glycerol and rapeseed oil). It was found that the process may be stimulated by regulation of the medium pH and aeration, application of selected neutralizing agents, supplementation with thiamine and addition of sorbitan monolaurate, whereas presence of biotin and iron ions had no positive effect on KGA biosynthesis. Adjustment of the parameters improved the process efficiency and allowed 82.4 g/L of KGA to be obtained, corresponding to productivity of 0.57 g/L h and yield of 0.59 g/g. In addition, the production of KGA was characterized by a low level (≤6.3 g/L) of by-products, i.e., citric and pyruvic acids. The results confirmed the high potential of renewable carbon sources (glycerol + rapeseed oil) for effective KGA biosynthesis by *Yarrowia lipolytica*.

**Keywords:** α-ketoglutaric acid biosynthesis; *Yarrowia lipolytica*; glycerol; rapeseed oil; culture conditions optimization

## **1. Introduction**

α-Ketoglutaric acid (KGA) is a valuable compound with many demonstrated applications in the cosmetic, pharmaceutical and food industries or as an intermediate in chemical syntheses [1]. The chemical industry uses KGA as a substrate for synthesis of biopolymers [2] and heterocyclic compounds [3]. In food production it is applied as a functional additive in beverages and a nutraceutical in functional food [4–6]. In medicine it has been used in the treatment of various diseases and for the synthesis of pharmaceuticals [7–11]. The health benefits of KGA consumption have been demonstrated not only in humans but also in animals; therefore, its preparations are of interest to feed producers [12–15].

Although KGA is present in the central metabolism of every living cell, it is an intermediate molecule that is synthesized only in an amount that meets the needs of the cells. Therefore, KGA cannot be obtained from easily accessible sources such as food [16]. Industrial production of KGA is achieved mainly by a multi-step chemical method that uses diethyl oxalate and diethyl succinate as the substrates [3,17]. The process yields 75% efficiency, but it causes environmental hazards and has low economic attractiveness of production because of application of raw materials derived from depleting petrochemical resources, use of harmful reagents and generation of toxic wastes and high by-production of impurities. Moreover, the product obtained by this method may be excluded from use in certain applications (e.g., food production) [1,18,19].

**Citation:** Tomaszewska-Hetman, L.; Rywi ´nska, A.; Lazar, Z.; Rymowicz, W. Enhancement of α-Ketoglutaric Acid Production by *Yarrowia lipolytica* Grown on Mixed Renewable Carbon Sources through Adjustment of Culture Conditions. *Catalysts* **2023**, *13*, 14. https://doi.org/10.3390/ catal13010014

Academic Editors: Zhilong Wang and Tao Pan

Received: 28 November 2022 Revised: 19 December 2022 Accepted: 20 December 2022 Published: 22 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The possibility of converting various carbon sources to KGA using microbial fermentation has attracted scientists for several decades [1,4,19]. A wide variety of microorganisms have been found to be capable of synthesizing KGA. Nevertheless, the limiting factors for the use of most of them on a commercial scale are low titer, low yield, formation of by-products, intolerance to low pH and inhibitor presence and high nutritional requirements. Microorganisms that appear to be particularly effective in the biosynthesis of KGA are yeast, among which *Yarrowia lipolytica* has received special attention. In comparison to other yeast species, *Y. lipolytica* enables more efficient production of KGA from a very wide range of substrates and the use of simple culture media that meet low nutritional and vitamin supplementation requirements [1,19,20].

Previously we presented the genetically engineered strain of *Y. lipolytica* as a prospective producer of KGA from mixed renewable carbon sources (glycerol + rapeseed oil), along with simple technology of product recovery from post-culture broth [21]. Moreover, the biomass of this strain was proved to be of nutritional and health-beneficial characteristics especially desired in food and feed applications. The aim of the present work was to increase the efficiency of KGA production from mixed glycerol/rapeseed-oil-based media by the transformant strain *Y. lipolytica* CBS146773 by optimizing the conditions of biosynthesis.

#### **2. Results and Discussion**

In our earlier investigation, the transformant strain *Yarrowia lipolytica* CBS146773, formerly named 1.31.GUT1/6.CIT1/3.E34672 [21], was obtained by genetic engineering. This strain was characterized by overexpression of genes encoding glycerol kinase (*GUT1*), citrate synthase (*CIT1*) and mitochondrial acid transporter (*YALI0E34672g*) and was found to be a good producer of KGA in the process with synergistic co-feeding of glycerol and rapeseed oil [21]. The use of glycerol and rapeseed oil as substrates for *Y. lipolytica* was dictated by ecological and economic reasons. Both substrates are renewable carbon sources suitable for KGA biosynthesis; however, their applications differ in the efficiency of KGA production. The yield of KGA biosynthesis obtained using rapeseed oil might exceed 100%, whereas glycerol application results in by-formation of pyruvic acid (PA), which is linked to the decreased production of KGA and negatively affects the selectivity of the process. However, in practice, the possibility of even partial replacement of the water-insoluble substrate with hydrophilic glycerol can greatly facilitate unit operations during industrialscale processes [21,22]. Since the use of a mixed-substrates feeding strategy has already been demonstrated to be suitable for the process with *Y. lipolytica* CBS146773, in this work we focused on the selection of the conditions of the KGA biosynthesis that are aimed at fully exploiting the genetic potential of the selected strain.

#### *2.1. Maintenance of pH*

The effect of pH-control strategy on the growth and biosynthesis of KGA by the yeast strain *Y. lipolytica* CBS146773 was studied in two aspects: examination of pH value and comparison of different neutralizing agents used for maintaining pH at the appropriate level. The literature includes information about the positive effect of acidic pH on KGA biosynthesis by *Y. lipolytica* yeast [23–27]. In the reports, the pH level optimal for KGA production varied from 2.79 to 4.5 depending on the strain and substrate used in a specific process. In this study the pH impact was tested in the range of 3.0–4.5, using KOH as neutralizing agent. As presented in Figure 1A, the yeast strain presented good growth in the examined pH range. However, the increase in pH value resulted in an increased level of biomass from 18.6 g/L at pH 3.0 to 22.4 g/L when pH 4.5 was applied. It was found that the formation of KGA was dependent on pH. The highest KGA production of 56.8 g/L corresponding to the yield (Yp/s) of 0.41 g/g was obtained in the culture conducted at pH 3.5. Simultaneously, in the same conditions, PA content in the post-culture broth was the lowest and reached 13.9 g/L, whereas in other cultures the concentration of the acid was significantly higher (23.0–38.0 g/L). It was also observed that increasing the pH level from 3.0 to 4.5 resulted in increased citric acid (CA) by-product formation from 1.0 to 13.2 g/L. Taking into account overall production of the acid pool, the highest selectivity of the KGA biosynthesis process (0.76 = 76%) was noted when the pH was maintained at 3.5. Some authors reported that not only the pH value applied but also its control strategy, in which the pH level set at the growth phase was lowered and was controlled at the low level to the end of the cultivation process, enhancing KGA biosynthesis [24,27]. In the process conducted on glycerol with such a two-step pH control strategy, *Y. lipolytica* WSH-Z06 produced 32.5% more KGA (53.4 g/L) than in the process in which a one-step strategy was applied [27]. 3.0 to 4.5 resulted in increased citric acid (CA) by-product formation from 1.0 to 13.2 g/L. Taking into account overall production of the acid pool, the highest selectivity of the KGA biosynthesis process (0.76 = 76%) was noted when the pH was maintained at 3.5. Some authors reported that not only the pH value applied but also its control strategy, in which the pH level set at the growth phase was lowered and was controlled at the low level to the end of the cultivation process, enhancing KGA biosynthesis [24,27]. In the process conducted on glycerol with such a two-step pH control strategy, *Y. lipolytica* WSH-Z06 produced 32.5% more KGA (53.4 g/L) than in the process in which a one-step strategy was

applied [27].

3.5. Simultaneously, in the same conditions, PA content in the post-culture broth was the lowest and reached 13.9 g/L, whereas in other cultures the concentration of the acid was significantly higher (23.0–38.0 g/L). It was also observed that increasing the pH level from

*Catalysts* **2023**, *13*, x FOR PEER REVIEW 3 of 14

**Figure 1.** Impact of pH control by pH value (**A**) and type of neutralizing agent used (**B**) on yeast growth and acids formation during KGA biosynthesis performed by *Y. lipolytica* CBS 146773 in mixed glycerol/oil-based media. Culture conditions: 20% KOH (**A**), pH 3.5 (**B**), 800 rpm, 3 µg/L of thiamine. Abbreviations: X—biomass; KGA—α-ketoglutaric acid; PA—pyruvic acid; CA—citric **Figure 1.** Impact of pH control by pH value (**A**) and type of neutralizing agent used (**B**) on yeast growth and acids formation during KGA biosynthesis performed by *Y. lipolytica* CBS 146773 in mixed glycerol/oil-based media. Culture conditions: 20% KOH (**A**), pH 3.5 (**B**), 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.

In this study the second approach to the pH control strategy was to verify whether a different neutralizing agent may affect the process of KGA formation by the examined

transformant yeast strain. For this purpose, biosynthesis conducted at pH 3.5 was maintained by automatic addition of a 20% solution of NaOH, KOH or Ca(OH)<sup>2</sup> (Figure 1B). In comparison to KOH, application of the two other neutralizing factors resulted in slightly higher biomass growth. The results of this experiment revealed that the type of factor used for neutralization has a significant influence on the efficiency of KGA biosynthesis. When NaOH was used, the yeast produced moderate concentrations of both KGA (39.0 g/L) and PA (24.6 g/L), which, despite the relatively low amount of CA, led to low selectivity of the process (57%). A significant increase in KGA biosynthesis was noted in the culture in which Ca(OH)<sup>2</sup> was applied, as yeast produced 69.1 g/L of KGA with the yield (Yp/s) of 0.49 g/g and selectivity of 83%. These results were in agreement with an earlier investigation that revealed a positive impact of Ca2+ ions on pyruvate carboxylase [28]. In the cultures of *Torulopsis glabrata* CCTCC M202019 growing on glucose, a lower concentration of KGA (1.3 g/L) was obtained when NaOH was used as a neutralizing agent, whereas application of CaCO<sup>3</sup> increased KGA synthesis (11.5–12.0 g/L) [28,29]. This relationship was also confirmed in the culture of *Y. lipolytica* WSK-Z06 performed in glycerol media, where the change of the neutralizing agent from NaOH to CaCO<sup>3</sup> resulted in an increase in the KGA:PA ratio from 22.0:36.9 g/L to 40.3:31.8 g/L [27]. As a consequence of the above described results, all subsequent experiments were carried out at pH 3.5 maintained by addition of Ca(OH)2.
