Effect of Selected Cations and B Vitamins on the Biosynthesis of Carotenoids by Rhodotorula mucilaginosa Yeast in the Media with Agro-Industrial Wastes

Abstract

In recent years, there has been an increase in the search for novel raw materials for the production of natural carotenoids. Among yeasts, Rhodotorula species have the ability to synthesize carotenoids, mainly β-carotene, torulene, and torularhodin, depending on the culture conditions. This study aimed to determine the effect of selected cations (barium, zinc, aluminum, manganese) and B vitamins (biotin, riboflavin, niacin, pantothenic acid) on the biosynthesis of carotenoids by Rhodotorula mucilaginosa MK1 and estimate the percentages of β-carotene, torulene, and torularhodin synthesized by the yeast. The cultivation was carried out in a medium containing glycerol (waste resulting from biodiesel production) as a carbon source and potato wastewater (waste resulting from potato starch production) as a nitrogen source. Carotenoid biosynthesis was stimulated by the addition of aluminum (300 mg/L) or aluminum (300 mg/L) and niacin (100 µg/L) to the medium. The number of carotenoids produced by R. mucilaginosa MK1 in the medium containing only aluminum and in the medium with aluminum and niacin was 146.7 and 180.5 µg/g_d.m., respectively. This content was 101% and 147% higher compared to the content of carotenoids produced by yeast grown in the control medium (73.0 µg/g_d.m.). The addition of aluminum and barium seemed to have a positive effect on the biosynthesis of torulene, and the percentage of this compound increased from 31.86% to 75.20% and 68.24%, respectively. Niacin supplementation to the medium increased the percentage of torularhodin produced by the yeast from 23.31% to 31.59–33.79%. The conducted study showed that there is a possibility of intensifying carotenoid biosynthesis by red yeast and changing the percentages of individual carotenoids fractions by adding cations or B vitamins to the medium. Further research is needed to explain the mechanism of action of niacin on the stimulation of torularhodin biosynthesis.

1. Introduction

Carotenoids are a group of natural pigments that are commonly found in nature. Because of their well-documented health effects, researchers are continually looking for new methods to obtain these compounds. Chemical synthesis processes provide a substantial proportion of carotenoids for industrial applications. However, as the need for natural dyes continues to rise, the production of these compounds with the use of microorganisms is becoming increasingly important [1,2]. Among the microbes that can produce carotenoids, yeasts of the Rhodotorula genus are particularly interesting. The major carotenoids synthesized by these organisms are β-carotene, torulene, and torularhodin [3]. Beta-carotene is the most potent precursor of vitamin A and possesses strong antioxidant properties, and hence has a number of health benefits, such as reducing the risk of diseases and certain types of cancer, protecting against macular degeneration, and strengthening the immune system. It is used as a dye and an ingredient of dietary supplements in the food industry, while in the cosmetic industry, it is applied as an ingredient in sun creams [4]. Torulene, a carotene containing 13 double bonds, exhibits pro-vitamin A and anticancer properties. It has an intense orange color and is indicated as a potential food additive [5]. However, torulene production is not carried out on a large scale because the exact pathway of its biosynthesis and the optimal cultivation conditions are unknown [5,6]. Torularhodin belongs to the group of xanthophylls, and, depending on the concentration, its color ranges from pink to orange [5]. It can be used in medicine, for example, for the prevention of prostate cancer or in pharmaceuticals, which is associated with the apoptosis of neoplastic cells (reduction of Bcl-2 proteins and increased expression of Bax protein and specific caspases [7]. Furthermore, it possesses strong antimicrobial properties and, therefore, can be used as a natural antibiotic [8].

The selection of appropriate strains that can synthesize higher amounts of these compounds, as well as optimization of culture conditions, is essential for the profitable production of carotenoids using microorganisms. Several factors influence the process of carotenoid biosynthesis, of which the most important are carbon and nitrogen source, pH, oxygenation level, and availability of light [2]. Various agri-food wastes have been used for production to reduce the costs of microbiological media. It was shown that glycerol, a by-product of the biodiesel production process, can be successfully used as a carbon source, and potato wastewater, obtained in large amounts from potato starch production, as a nitrogen source [9]. The presence of appropriate micronutrients in the culture medium is also necessary to achieve high yields of carotenoids [10], as these compounds act as cofactors of cellular enzymes involved in their biosynthesis. For example, Mg²⁺ or Mn²⁺ ions act as cofactors of phytoene synthase, and Fe²⁺ ions ensure the proper activity of β-lycopene cyclase [3]. The presence of some metals may also be an environmental stress factor for yeast cells, inducing increased carotenogenesis [11]. Another factor that influences carotenoid biosynthesis in red yeast cells is the presence of vitamins. Among vitamins, B group plays an important role in yeast metabolism [12,13,14,15]. However, no studies have analyzed the effect of these vitamins on the biosynthesis of carotenoids by Rhodotorula yeasts so far. Therefore, this study aimed to investigate the effect of supplementation of the culture medium prepared from two types of industrial wastes (glycerol and potato wastewater) with selected cations and B vitamins on the biosynthetic efficiency of Rhodotorula mucilaginosa as well as the profile of carotenoids produced by this yeast species.

2. Materials and Methods

2.1. Biological Material

The study used the R. mucilaginosa MK1 strain, which was isolated from bee bread and identified by Kieliszek et al. [16]. It is deposited under GenBank Accession Number LC527461.1 (National Center of Biotechnology Information).

2.2. Wastes Used as Components of Media

The culture media used for yeast cultivation were prepared from two industrial wastes, glycerol and potato wastewater. Technical glycerol (ORLEN Południe, Trzebinia, Poland) was used as a carbon source, while potato wastewater, which was prepared in laboratory conditions as described previously [17], was used as a nitrogen source.

2.3. Media and Culture Conditions

Two types of culture media were used in the study. The control medium was prepared from potato wastewater and technical glycerol, in which the final concentration of glycerol was 3% (w/v). One type of experimental media was prepared with zinc (ZnSO₄·7H₂O), aluminum (Al(NO₃)₃·9H₂O), barium (BaCl₂), and manganese (MnSO₄·H₂O) cations, which were added at the concentration range of 50–300 mg/L. Another type of experimental media was prepared with selected B vitamins (niacin, biotin, riboflavin, pantothenic acid), the concentrations of which ranged from 100 to 1000 µg/L. The vitamin solutions prepared for supplementation were sterilized through a sterile membrane filter (0.22 µm) and added at an appropriate amount to the cooled, sterile medium containing potato wastewater and 3% glycerol.

2.4. Bioscreen C Cultures

In the first stage, yeast cultivation was carried out in a Bioscreen C apparatus (Oy Growth Curves Ab Ltd., Finland). For this purpose, a 24-h yeast inoculum was prepared in yeast extract–peptone–dextrose (YPD) medium (200 rpm, 25 °C) and diluted in sterile physiological saline to achieve a cell density of 1 × 10⁶ CFU/mL. Then, 270 µL of the control medium and experimental media were transferred to the microwells on a plate, and 30 µL of the inoculum was added. Cultivation was carried out under intensive shaking at 25 °C. The apparatus monitored changes in the optical density (OD) of the cultures every hour (broadband filter λ = 420–580 nm). Based on the OD values observed after 120 h of cultivation, the duration of the adaptation phase (Δt_lag) and logarithmic phase (Δt_log), the minimum and maximum OD values in the logarithmic growth phase, the specific growth rate coefficient (μ_max = lnOD_max − lnOD_min)/Δt_log), and the total increase in OD were determined.

2.5. Cultures on the Shaker

The control and experimental media were inoculated with a 24-h yeast culture such that the initial cell density was 1 × 10⁵ CFU/mL. Cultivation was performed for 120 h at 25 °C on a rotary shaker (200 rpm; SM-30 Control, Edmund Bühler, Germany).

2.6. Biomass Yield

The cellular biomass yield was determined by weighing. After 120 h, 10 mL of culture was removed and centrifuged at 5000× g for 10 min (Centrifuge 5804R, Eppendorf, Germany). The wet biomass was washed twice with sterile deionized water and dried at 105 °C until a constant weight was reached. The results are expressed in grams of dry matter (g_d.m.) per liter of the medium.

2.7. Content and Profile of Carotenoids in Yeast Biomass

To disintegrate the cellular biomass and extract carotenoids from the yeast cells, 2 mL of dimethyl sulfoxide and 0.5 g of glass beads (diameter: 500 µm) were added to approximately 100 mg of wet biomass. The samples were shaken in a rotator (Multi Bio RS-24, Biosan) at 70 rpm for 1 h. Then, 2 mL of petroleum ether, 2 mL of acetone, and 2 mL of 20% sodium chloride were added, and the samples were shaken again for 1 h. Next, the samples were centrifuged at 3500× g for 5 min (Centrifuge 5804R, Eppendorf, Germany) to separate the phases. The absorbance of the ether fraction was measured at 490 nm (UV1800, Rayleigh). The total carotenoid content (μg/g_d.m.) was calculated from the formula = A_max × D × V/(E × W), where A_max is the absorbance at 490 nm, D is the sample dilution factor, V is the volume of the ether fraction, E is the extinction coefficient of carotenes (0.16), and W is the yeast dry matter (g_d.m.) [6].

2.8. Carotenoids Profile

The individual carotenoids were identified by high-performance liquid chromatography (HPLC). Petroleum ether was evaporated from the ethereal fractions under a nitrogen atmosphere. One milliliter of the HPLC phase consisting of a mixture of acetonitrile, isopropanol, and ethyl acetate (4:4:2, v/v/v) was added to the tube and filtered through a 0.45-µm nylon filter. The samples were analyzed on a C18 analytical column (Bionacom; 250 mm × 4.6 mm, 5 μm). The flow rate of the mobile phase was set to 0.7 mL/min (isocratic). A UV–VIS detector operating at 490 nm was used for detection. β- and γ-carotene were identified based on the retention time of the standards (Sigma-Aldrich), while torulene and torularhodin were based on the retention time of the products separated using thin-layer chromatography [17]. The percentages of these four compounds in the carotenoid mixture were determined based on the area of their peaks.

2.9. Statistical Analysis of the Results

All cultures and analyses were performed in triplicate. Statistical analysis of the results was performed in R program (version i386 2.15.3). The normal distribution of data was verified by the Shapiro–Wilk test and the homogeneity of variance by the Levene test. The significance of differences was determined by one-way analysis of variance and Tukey’s test at a level of α = 0.05.

3. Results

3.1. First Stage—Growth in Bioscreen C

The changes observed in the OD values during cultivation in the Bioscreen C apparatus indicated that R. mucilaginosa MK1 yeast was able to grow in the control medium and the experimental medium enriched with selected cations (

Table 1. Growth characteristics of Rhodotorula mucilaginosa MK1 yeast observed in Bioscreen C during cultivation in media containing different cations.

Type of Medium	Concentration [mg/L]	tlag [h]	tlog [h]	µmax [h–1]	ΔOD
Barium	0	6.0	18.0	0.0341 ± 0.0033 *	1.542 ± 0.204 *
	50	4.0	22.0	0.0334 ± 0.0020 *	1.442 ± 0.060 *
	100	4.0	22.0	0.0313 ± 0.0011 *	1.447 ± 0.210 *
	150	6.0	20.0	0.0342 ± 0.0017 *	1.319 ± 0.098 *
	200	6.0	22.0	0.0314 ± 0.0005 *	1.442 ± 0.082 *
	250	6.0	20.0	0.0338 ± 0.0006 *	1.381 ± 0.044 *
	300	6.0	22.0	0.0298 ± 0.0020 *	1.339 ± 0.147 *
Zinc	0	6.0	18.0	0.0341 ± 0.0033 ab	1.542 ± 0.204 *
	50	6.0	18.0	0.0366 ± 0.0010 a	1.605 ± 0.155 *
	100	6.0	20.0	0.0325 ± 0.0018 ab	1.645 ± 0.096 *
	150	6.0	20.0.	0.0358 ± 0.0019 ab	1.554 ± 0.170 *
	200	6.0	20.0	0.0318 ± 0.0012 ab	1.653 ± 0.320 *
	250	6.0	20.0	0.0324 ± 0.0028 ab	1.600 ± 0.057 *
	300	6.0	20.0.	0.0306 ± 0.0022 b	1.535 ± 0.295 *
Aluminum	0	6.0	18.0	0.0341 ± 0.0033 a	1.542 ± 0.204 *
	50	6.0	20.0	0.0321 ± 0.041 abc	1.344 ± 0.077 *
	100	6.0	20.0	0.0322 ± 0.0030 ab	1.337 ± 0.169 *
	150	6.0	24.0	0.0286 ± 0.0032 abc	1.370 ± 0.053 *
	200	6.0	24.0	0.0265 ± 0.0024 bc	1.306 ± 0.058 *
	250	8.0	26.0	0.0251 ± 0.0021 c	1.339 ± 0.096 *
	300	8.0	28.0	0.0234 ± 0.0022 c	1.305 ± 0.052 *
Manganese	0	6.0	18.0	0.0341 ± 0.0033 *	1.542 ± 0.204 *
	50	6.0	18.0	0.0367 ± 0.0037 *	1.412 ± 0.047 *
	100	6.0	18.0	0.0385 ± 0.0024 *	1.411 ± 0.018 *
	150	6.0	18.0	0.0371 ± 0.0058 *	1.383 ± 0.212 *
	200	6.0	18.0	0.0378 ± 0.0007 *	1.440 ± 0.113 *
	250	6.0	18.0	0.0397 ± 0.0024 *	1.398 ± 0.117 *
	300	6.0	18.0	0.0366 ± 0.0043 *	1.345 ± 0.091 *

Indexes ^{a, b} … denote homogeneous groups determined by Tukey’s test. * means no significant difference. A one-way analysis of variance was performed for the results obtained for each cation separately and compared with the control sample.

).

Statistical analysis showed that the specific growth rate (µ_max) in the media containing barium (50–300 mg/L), zinc (50–300 mg/L), aluminum (50–150 mg/L), and manganese (50–300 mg/L) was comparable to that obtained in the control medium (0.0341 h^–1) (Table 1). A decreased growth rate was noted in the remaining media variants. In particular, a significant reduction in µ_max, as well as an extended logarithmic phase, was observed in cultures supplemented with aluminum at the concentrations of 200–300 mg/L, which proves that this element has a toxic effect on cells and slows down the reproductive process.

The growth characteristics of the studied yeast strain differed in the experimental media supplemented with B vitamins (

Table 2. Characteristics of Rhodotorula mucilaginosa MK1 yeast observed in Bioscreen C during cultivation in media supplemented with B vitamins.

Type of Medium	Concentration [µg/L]	tlag [h]	tlog [h]	µmax [h–1]	ΔOD
Control	0	6.0	18.0	0.0341 ± 0.0033 *	1.542 ± 0.204 a
Biotin	100	8.0	14.0	0.0323 ± 0.0056 *	1.116 ± 0.143 b
	300	8.0	14.0	0.0339 ± 0.0064 *	1.113 ± 0.071 b
	500	8.0	16.0	0.0335 ± 0.0022 *	1.202 ± 0.038 b
	700	8.0	16.0	0.0301 ± 0.0014 *	1.179 ± 0.072 b
	850	8.0	16.0	0.0279 ± 0.0014 *	1.098 ± 0.097 b
	1000	10.0	16.0	0.0293 ± 0.0020 *	1.210 ± 0.193 b
Riboflavin	0	6.0	18.0	0.0341 ± 0.0033 a	1.542 ± 0.204 a
	100	6.0	18.0	0.0306 ± 0.0021 ab	1.173 ± 0.178 b
	300	6.0	18.0	0.0289 ± 0.0017 ab	1.109 ± 0.097 b
	500	6.0	20.0	0.0291 ± 0.0020 ab	1.129 ± 0.137 b
	700	6.0	20.0	0.0283 ± 0.0011 b	1.108 ± 0.079 b
	850	6.0	20.0	0.0268 ± 0.0010 b	1.140 ± 0.040 b
	1000	8.0	20.0	0.0269 ± 0.0022 b	1.153 ± 0.053 b
Niacin	0	6.0	18.0	0.0341 ± 0.0033 a	1.542 ± 0.204 a
	100	8.0	20.0	0.0210 ± 0.0075 b	1.030 ± 0.152 bc
	300	8.0	20.0	0.0221 ± 0.0027 b	1.083 ± 0.138 bc
	500	8.0	20.0	0.0226 ± 0.0022 b	1.163 ± 0.126 b
	700	10.0	20.0	0.0214 ± 0.0007 b	0.868 ± 0.110 bc
	850	10.0	24.0	0.0169 ± 0.0017 b	0.762 ± 0.123 bc
	1000	12.0	26.0	0.0150 ± 0.0020 b	0.694 ± 0.069 c
	0	6.0	18.0	0.0341 ± 0.0033 a	1.542 ± 0.204 a
Pantothenic acid	100	6.0	16.0	0.0324 ± 0.0039 a	1.041 ± 0.154 b
	300	6.0	16.0	0.0318 ± 0.0044 ab	1.064 ± 0.180 b
	500	6.0	16.0	0.0334 ± 0.0031 a	1.142 ± 0.191 ab
	700	8.0	20.0	0.0270 ± 0.0033 bc	0.986 ± 0.037 b
	850	10.0	20.0	0.0224 ± 0.0043 bc	0.941 ± 0.204 b
	1000	10.0	20.0	0.0199 ± 0.0023 c	0.850 ± 0.097 b

Indexes ^{a, b} … denote homogeneous groups determined by Tukey’s test. * means no significant difference. A one-way analysis of variance was performed for the results obtained for each vitamin separately and compared with the control sample.

). Specific growth rate values above 0.03 h^–1 were estimated in cultures containing biotin at the concentrations of 100–700 µg/L, riboflavin at the lowest concentration of 100 µg/L, and pantothenic acid at the concentrations of 100–500 µg/L. Importantly, growth was found to be delayed in the media supplemented with 100 µg/L niacin. In all other media supplemented with vitamins, the total increase in OD was significantly lower (0.694–1.202) compared to the control media (1.542).

3.2. Second Stage—Growth and Biosynthesis of Carotenoids in Shaker Culture in Media Supplemented with Cations or B Vitamins

After cultivation in the Bioscreen C apparatus, the yeast cultures were transferred to shaker flasks. At the beginning of cultivation, the biomass yield, which is defined as the amount of dry yeast cell biomass in 1 L of the medium, was determined (

Table 3. Biomass yield and content and profile of carotenoids after 120 h of cultivation in media enriched with selected cations.

Type of Medium	Concentration [mg/L]	Biomass Yield [gd.m./L]	Total Carotenoid Content in Biomass [µg/gd.m.]	Volumetric Yield of Carotenoids [mg/L]	Percentages of Carotenoids
					Torularhodin	Torulene	β-Carotene	Others
Barium	0	21.48 ± 1.24 a	73.06 ± 10.08 *	1.56 ± 0.13 a	22.79 ± 2.03 a	31.85 ± 0.71 b	43.08 ± 1.52 a	2.29 ± 0.20 *
	50	15.65 ± 0.07 b	71.96 ± 8.85 *	1.13 ± 0.14 ab	21.29 ± 2.60 a	34.34 ± 2.81 b	41.38 ± 0.43 a	3.00 ± 0.23 *
	100	15.65 ± 1.56 b	83.71 ± 1.14 *	1.31 ± 0.15 ab	19.95 ± 2.42 a	36.39 ± 1.51 b	41.30 ± 3.64 a	2.37 ± 0.28 *
	150	14.80 ± 0.21 b	73.70 ± 12.83 *	1.09 ± 0.21 ab	19.37 ± 1.55 a	35.60 ± 1.92 b	43.28 ± 3.58 a	1.76 ± 0.11 *
	200	15.10 ± 0.85 b	77.28 ± 14.15 *	1.16 ± 0.15 ab	16.79 ± 0.60 a	37.56 ± 4.51 b	43.03 ± 4.56 a	2.63 ± 0.65 *
	250	11.70 ± 1.48 bc	83.07 ± 16.96 *	0.96 ± 0.08 b	8.89 ± 0.35 b	63.77 ± 4.04 a	24.94 ± 3.37 b	2.41 ± 0.33 *
	300	9.10 ± 1.27 c	81.98 ± 3.83 *	0.75 ± 0.14 b	7.32 ±1.32 b	68.24 ± 6.00 a	21.74 ± 3.39 b	2.71 ± 1.29 *
Zinc	0	21.48 ± 1.24 *	73.06 ± 10.08 *	1.56 ± 0.13 *	22.79 ± 2.03 *	31.85 ± 0.71 *	43.08 ± 1.52 *	2.29 ± 0.20 *
	50	22.60 ± 2.62 *	64.65 ± 6.12 *	1.45 ± 0.03 *	22.07 ± 2.03 *	36.77 ± 2.16 *	38.92 ± 3.63 *	2.25 ± 0.56 *
	100	24.80 ± 2.90 *	61.46 ± 10.21 *	1.51 ± 0.08 *	20.02 ± 2.04 *	35.57 ± 5.57 *	42.01 ± 2.50 *	2.41 ± 1.03 *
	150	21.18 ± 1.59 *	75.90 ± 13.01 *	1.60 ± 0.15 *	18.93 ± 0.80 *	34.75 ± 2.06 *	44.17 ± 2.43 *	2.16 ± 0.42 *
	200	24.07 ± 2.09 *	68.29 ± 15.28 *	1.63 ± 0.23 *	22.32 ± 2.04 *	32.99 ± 3.15 *	40.74 ± 0.87 *	3.96 ± 0.24 *
	250	24.53 ± 1.73 *	64.82 ± 13.41 *	1.58 ± 0.22 *	20.62 ± 2.45 *	32.80 ± 3.08 *	43.63 ± 0.93 *	2.96 ± 0.30 *
	300	20.17 ± 0.53 *	68.80 ± 9.80 *	1.39 ± 0.23 *	20.78 ± 0.83 *	32.98 ± 3.65 *	44.10 ± 3.25 *	2.15 ± 0.42 *
Aluminum	0	21.48 ± 1.24 a	73.06 ± 10.08 b	1.56 ± 0.13 *	22.79 ± 2.03 a	31.85 ± 0.71 d	43.08 ± 1.52 a	2.29 ± 0.20 *
	50	22.42 ± 1.59 ab	72.58 ± 23.83 b	1.61 ± 0.42 *	10.74 ± 0.71 bc	58.57 ± 2.40 b	28.36 ± 1.41 bc	2.34 ± 0.28 *
	100	21.83 ± 2.58 ab	66.73 ± 10.28 b	1.47 ± 0.40 *	13.54 ± 1.06 b	50.78 ± 0.41 c	33.58 ± 1.56 b	2.10 ± 0.91 *
	150	20.48 ± 1.03 ab	61.89 ± 6.08 b	1.27 ± 0.19 *	9.26 ± 1.37 bcd	62.75 ± 3.17 b	26.24 ± 1.65 c	1.76 ± 0.15 *
	200	17.83 ± 1.10 ab	64.19 ± 12.47 b	1.14 ± 0.15 *	8.27 ± 1.24 cd	64.15 ± 1.55 b	25.08 ± 1.03 cd	2.51 ± 1.33 *
	250	16.85 ± 0.99 ab	92.74 ± 23.34 ab	1.57 ± 0.49 *	5.94 ± 0.45 d	71.36 ± 1.58 a	20.27 ± 1.62 de	2.45 ± 0.40 *
	300	15.71 ± 2.46 b	146.73 ± 14.12 a	2.32 ± 0.58 *	4.50 ± 0.90 d	75.20 ± 0.64 a	17.80 ± 0.64 e	2.51 ± 0.90 *
Manganese	0	21.48 ± 1.24 b	73.06 ± 10.08 *	1.56 ± 0.13 *	22.79 ± 2.03 a	31.85 ± 0.71 *	43.08 ± 1.52 *	2.29 ± 0.20 ab
	50	25.90 ± 1.27 a	76.96 ± 13.85 *	1.98 ± 0.26 *	12.85 ± 3.39 b	35.09 ± 6.70 *	48.91 ± 3.74 *	3.17 ± 0.43 a
	100	26.57 ± 0.46 a	73.93 ± 18.68 *	2.02 ± 0.59 *	16.31 ± 2.06 ab	28.64 ± 5.31 *	53.25 ± 2.82 *	1.81 ± 0.42 b
	150	26.08 ± 1.10 a	78.63 ± 5.34 *	2.05 ± 0.23 *	13.82 ± 1.96 b	29.61 ± 0.72 *	53.86 ± 1.56 *	2.72 ± 0.33 ab
	200	25.18 ± 2.79 a	69.41 ± 4.06 *	1.75 ± 0.30 *	15.81 ± 2.18 ab	31.11 ± 5.18 *	50.74 ± 3.54 *	2.35 ± 0.54 ab
	250	25.15 ± 2.12 a	69.38 ± 9.89 *	1.73 ± 0.10 *	14.35 ± 1.26 b	32.35 ± 4.41 *	51.13 ± 2.67 *	2.18 ± 0.48 ab
	300	25.47 ± 0.67 a	80.43 ± 3.08 *	2.05 ± 0.13 *	14.10 ± 3.46 b	34.30 ± 3.46 *	49.30 ± 2.89 *	2.32 ± 0.91 ab

Indexes ^{a, b} … denote homogeneous groups determined by Tukey’s test. * means no significant difference. A one-way analysis of variance was performed for the results obtained for each cation separately and compared with the control sample.

). In the control medium, the biomass yield after cultivation was estimated at 21.48 g_d.m./L. Statistical analysis indicated that the addition of zinc at a concentration of 50–300 mg/L did not influence the biomass yield. On the other hand, the addition of other cations caused a difference in the values of this index. The biomass yield determined after cultivation in the media containing manganese at doses from 50 to 300 mg/L was found to be significantly higher than that in the control medium and ranged from 25.15 to 26.57 g_d.m./L. In the media variants containing barium, a decrease in the growth of cellular biomass was found with an increase in the dose of the cation. The determined cellular biomass yields ranged from 15.65 to 9.10 g_d.m./L. The addition of aluminum at a concentration of 300 mg Al³⁺/L caused a significant reduction in the biomass yield (15.71 g_d.m./L). Among the tested vitamins, supplementation with biotin and riboflavin in the entire concentration range did not cause any significant difference in the determined values of the biomass yield, which ranged from 19.52 to 23.23 g_d.m./L. The addition of niacin at the doses of 700, 850, and 1000 µg/L caused a significant reduction in biomass yield, which ranged from 15.35 to 12.12 g_d.m./L. The same phenomenon was observed with pantothenic acid at concentrations of 850 and 1000 µg/L, and the values of biomass yield were 16.55 and 14.68 g_d.m./L, respectively.

The content of total carotenoids in the yeast cell biomass obtained after cultivation in the medium with potato wastewater and 3% glycerol was, on average, 73.06 µg/g_d.m. (Table 3). A significant increase in the carotenoid content (146.73 µg/g_d.m.) was noted only in the medium containing 300 mg Al³⁺/L. Supplementation of medium with barium, zinc, and manganese, as well as B vitamins, did not cause an increase in the overall carotenoid content in the cellular biomass. Based on the values of cellular biomass yield and the total content of carotenoids, the volumetric efficiency of biosynthesis (defined as the number of carotenoids obtained from 1 L of the culture medium) was determined. After cultivation in the control medium, the volumetric efficiency amounted to an average of 1.56 mg/L, while after cultivation in the medium containing 300 mg/L aluminum, it increased to 2.32 mg/L.

Supplementation of the culture medium with barium, aluminum, and manganese cations or niacin caused changes in the content of synthesized carotenoids (Table 3 and

Table 4. Biomass yield and content and profile of carotenoids after 120 h of cultivation in media enriched with selected B vitamins.

Type of Medium	Concentration [mg/L]	Biomass Yield [gd.m./L]	Total Carotenoid Content in Biomass [µg/gd.m.]	Volumetric Yield of Carotenoids [mg/L]	Percentages of Carotenoids
					Torularhodin	Torulene	β-Carotene	Others
Biotin	0	21.48 ± 1.24 *	73.06 ± 10.08 *	1.56 ± 0.13 *	22.79 ± 2.03 *	31.85 ± 0.71 *	43.08 ± 1.52 *	2.29 ± 0.20 *
	100	20.32 ± 3.29 *	75.02 ± 13.22 *	1.50 ± 0.02 *	17.91 ± 0.63 *	41.49 ± 0.11 *	38.72 ± 0.81 *	1.89 ± 0.07 *
	300	23.23 ± 3.50 *	79.48 ± 7.62 *	1.86 ± 0.46 *	20.84 ± 0.28 *	37.36 ± 4.22 *	39.32 ± 2.87 *	2.49 ± 1.63 *
	500	22.90 ± 2.12 *	77.40 ± 9.49 *	1.76 ± 0.05 *	19.02 ± 0.64 *	35.64 ± 1.49 *	41.91 ± 0.33 *	3.45 ± 1.80 *
	700	20.38 ± 2.51 *	84.18 ± 7.34 *	1.71 ± 0.06 *	21.70 ± 0.92 *	34.51 ± 0.30 *	41.75 ± 2.06 *	2.05 ± 0.83 *
	850	21.13 ± 0.39 *	82.60 ± 5.70 *	1.74 ± 0.09 *	21.80 ± 1.27 *	32.57 ± 1.27 *	43.00 ± 0.35 *	2.65 ± 0.29 *
	1000	21.80 ± 1.34 *	70.32 ± 6.61 *	1.54 ± 0.24 *	21.39 ± 2.58 *	37.20 ± 5.28 *	39.76 ± 2.45 *	1.66 ± 0.25 *
Riboflavin	0	21.48 ± 1.24 *	73.06 ± 10.08 *	1.56 ± 0.13 *	22.79 ± 2.03 *	31.85 ± 0.71 *	43.08 ± 1.52 *	2.29 ± 0.20 *
	100	22.72 ± 2.65 *	78.66 ± 14.24 *	1.77 ± 0.11 *	20.40 ± 1.34 *	36.04 ± 1.91 *	41.80 ± 0.47 *	1.77 ± 0.09 *
	300	21.67 ± 3.08 *	77.57 ± 16.31 *	1.66 ± 0.11 *	22.90 ± 1.98 *	35.23 ± 2.83 *	39.53 ± 0.01 *	2.34 ± 0.85 *
	500	21.30 ± 1.48 *	84.52 ± 4.44 *	1.80 ± 0.03 *	19.31 ± 0.78 *	36.30 ± 1.37 *	41.80 ± 0.78 *	2.60 ± 0.20 *
	700	21.00 ± 2.83 *	80.33 ± 10.61 *	1.67 ± 0.01 *	22.61 ± 0.95 *	35.49 ± 1.56 *	39.60 ± 1.35 *	2.32 ± 0.74 *
	850	20.52 ± 2.58 *	79.08 ± 8.22 *	1.61 ± 0.04 *	22.29 ± 0.49 *	32.99 ± 1.18 *	43.00 ± 1.82 *	1.73 ± 0.14 *
	1000	19.52 ± 1.45 *	85.55 ± 10.62 *	1.68 ± 0.33 *	22.08 ± 1.42 *	33.78 ± 0.66 *	41.68 ± 2.17 *	2.47 ± 0.08 *
Niacin	0	21.48 ± 1.24 ab	73.06 ± 10.08 *	1.56 ± 0.13 ab	22.79 ± 2.03 b	31.85 ± 0.71 b	43.08 ± 1.52 a	2.29 ± 0.20 *
	100	20.85 ± 0.28 ab	83.28 ± 12.64 *	1.73 ± 0.24 a	33.79 ± 2.04 a	42.51 ± 0.81 a	21.74 ± 1.97 b	1.98 ± 0.88 *
	300	24.50 ± 0.85 a	70.47 ± 3.52 *	1.73 ± 0.03 a	31.83 ± 3.25 a	43.70 ± 2.99 a	21.70 ± 0.62 b	2.78 ± 0.35 *
	500	23.25 ± 3.96 a	75.61 ± 7.18 *	1.74 ± 0.13 a	31.59 ± 0.78 a	43.90 ± 1.85 a	22.19 ± 1.34 b	2.34 ± 0.28 *
	700	15.35 ± 1.27 bc	87.99 ± 10.75 *	1.34 ± 0.05 ab	33.34 ± 2.67 a	44.03 ± 0.78 a	20.75 ± 1.97 b	1.89 ± 0.08 *
	850	13.73 ± 0.32 c	83.51 ± 15.46 *	1.14 ± 0.19 ab	29.79 ± 2.04 a	45.89 ± 1.50 a	22.52 ± 0.87 b	1.81 ± 0.33 *
	1000	12.12 ± 1.10 c	79.84 ± 7.00 *	0.97 ± 0.17 b	33.18 ± 1.48 a	40.63 ± 1.41 a	23.97 ± 2.84 b	2.23 ± 0.06 *
Pantothenic acid	0	21.48 ± 1.24 ab	73.06 ± 10.08 *	1.56 ± 0.13 *	22.08 ± 1.42 *	33.78 ± 0.66 *	41.68 ± 2.17 *	2.47 ± 0.08 *
	100	22.77 ± 3.01 a	77.96 ± 4.47 *	1.77 ± 0.13 *	25.80 ± 1.53 *	27.35 ± 1.62 *	44.35 ± 2.68 *	2.51 ± 0.47 *
	300	21.35 ± 2.40 a	75.96 ± 7.73 *	1.61 ± 0.02 *	26.92 ± 1.24 *	30.05 ± 1.39 *	41.20 ± 2.77 *	1.84 ± 0.15 *
	500	22.58 ± 1.45 a	79.02 ± 8.01 *	1.78 ± 0.07 *	25.43 ± 7.14 *	31.35 ± 4.09 *	40.72 ± 2.88 *	2.51 ± 0.17 *
	700	18.78 ± 0.60 ab	73.25 ± 16.32 *	1.37 ± 0.26 *	21.33 ± 2.43 *	32.39 ± 0.65 *	44.62 ± 0.90 *	1.67 ± 0.88 *
	850	16.55 ± 0.71 b	86.91 ± 12.43 *	1.44 ± 0.27 *	24.10 ± 0.65 *	32.44 ± 0.16 *	41.42 ± 0.78 *	2.05 ± 0.29 *
	1000	14.68 ± 1.38 b	81.50 ± 16.10 *	1.21 ± 0.35 *	25.14 ± 4.74 *	32.84 ± 3.35 *	40.02 ± 1.48 *	2.01 ± 0.08 *

Indexes ^{a, b} … denote homogeneous groups determined by Tukey’s test. * means no significant difference. A one-way analysis of variance was performed for the results obtained for each vitamin separately and compared with the control sample.

). The proportions of torularhodin, torulene, and β-carotene determined after cultivation in the control medium were 22.79%, 31.85%, and 43.08%, respectively. With an increase in the concentration of barium cations in the medium from 50 to 300 mg/L, a decrease was observed in the proportion of torularhodin from 21.29% to 7.32%, respectively. However, an increase in torulene was noted from 34.34% to 68.24%. The proportion of β-carotene in the biomass obtained after cultivation in the medium supplemented with 300 mg/L barium cations was 21.74%, which was twofold lower than that in the control sample. The addition of aluminum cations to the media containing potato wastewater and glycerol caused similar changes in the percentages of carotenoids, such as barium. With an increase in the concentration of Al³⁺ ions in the medium, the proportion of torulene increased from 31.85% to 75.20%. This was due to a decrease in the biosynthesis of torulene (from 22.79% to 4.50%) and β-carotene (from 43.08% to 17.80%). Similarly, the addition of manganese at doses of 250 and 300 mg/L resulted in a decrease in torularhodin to 14.20–14.35%, while the content of β-carotene increased (49.30–51.13%). Among the examined B group vitamins, only the addition of niacin to the medium caused significant changes in the composition of carotenoids. This vitamin stimulated the biosynthesis of torularhodin, the percentage of which increased from 22.79% (control culture) to 29.79–33.79% in the media containing niacin at concentrations from 100 to 1000 µg/L. At the same time, an increase in torulene content was also noted in the niacin-supplemented media (from 31.85% in the control culture to 40.63–45.89%).

3.3. Third Stage—Growth and Biosynthesis of Carotenoids in Shaker Culture in Media Supplemented with Both Cations and B Vitamins

In the third stage, the cultivation was carried out in the media simultaneously supplemented with selected cations and vitamins. Barium cations (300 mg/L) and niacin (100 µg/L) were added to the first experimental medium, and aluminum cations (300 mg/L) and niacin (100 µg/L) to the second. After cultivation, the cellular biomass yield obtained in the medium supplemented with barium and niacin was 9.59 g_d.m./L, while in the medium containing aluminum and niacin, the yield was 16.46 g_d.m./L. In both cases, the mean carotenoid content in the cell biomass was found to be higher than that in the control medium and in the media supplemented with single compounds and amounted to 106.44 µg/g_d.m. (Ba²⁺ + niacin) and 180.50 µg/g_d.m. (Al³⁺ + niacin). Based on the cellular yield, the mean volumetric efficiency of carotenoid biosynthesis was estimated at 1.02 and 2.97 mg/L, respectively. Simultaneous enrichment of media with cations and vitamins also resulted in changes in the percentages of the synthesized carotenoids. In the medium supplemented with barium and niacin, a significant reduction in the percentage of β-carotene was found (from 43.08% in the control sample to 7.30%), which resulted in an increase in the proportions of torularhodin (26.69%) and torulene (63.58%).

4. Discussion

Of the four cations tested, barium, which belongs to the group of alkaline earths, was found to have the most toxic effect on the cells of R. mucilaginosa MK1. Unfortunately, the literature lacks data concerning its influence on the growth and biosynthesis of carotenoids by yeasts. Among the few studies focusing on the effect of barium on yeast growth, the study by Alvino et al. [18] showed that barium added in the form of barium ferrite nanoparticles (BaFeNP) at doses ranging from 15 to 500 µg/mL did not cause any significant effect on the growth of Candida albicans ATCC 10,231 yeast strain.

Zinc, which is a chemical element from the zinc group, is a component of many enzymes, transcription factors, and regulatory proteins. It ensures optimal metabolism of nucleic acids and proteins and plays an important role in the growth, division, and functioning of cells. In yeast cells, zinc is present in both free and bound forms, with the latter being dominant [19]. In the present study, the addition of this element did not influence the growth and biosynthesis of carotenoids by R. mucilaginosa MK1 yeast at the studied concentrations. In a study by El-Banna et al. [20], the addition of ZnSO₄ salt at a dose of 0.1 g/L stimulated carotenoid synthesis by Rhodotorula glutinis yeast which produced 638 µg/g_d.m. of carotenoids (volume yield: 2.81 mg/L), while the content and volume yield determined in the medium lacking this salt after cultivation were, respectively, 292 µg/g_d.m. and 1.13 mg/L. A study by Rusinov-Videva et al. also confirmed that carotenoid biosynthesis was stimulated in a medium supplemented with zinc ions in the form of ZnSO₄ [21]. The yeast Sporobolomyces salmonicolor AL₁ was found to produce 16-fold more β-carotene in the medium containing 112 ppm of Zn²⁺ ions (from 11.2 to 189.2 µg/g_d.m.) compared to the medium lacking these ions. Interestingly, in the presence of Zn²⁺, the biosynthesis of coenzyme Q₁₀ was completely inhibited. It was assumed that zinc ions activated the enzyme catalytic centers responsible for isoprenoid synthesis and thus contributed to the biosynthesis of β-carotene. Some authors analyzed whether supplementation of zinc in the form of nanoparticles, rather than inorganic salt, caused an increase in the efficiency of biosynthesis of these compounds. Ibrahim and Mahmoud [22] reported that the addition of zinc in the form of ZnO nanoparticles to the culture medium resulted in a high content of carotenoids in the yeast cell biomass. After 72 h of cultivation, the volumetric yield of biosynthesis by Rhodotorula toruloides MH023518 yeast strain increased from 124 mg/L (control medium) to 264 mg/L (medium containing 50 ppm ZnO). It is worth noting, however, that cultivation was carried out in the YPD model medium, which contains optimal nutrients. Nanoparticles generate cellular stress leading to the formation of reactive oxygen species (ROS). This, in turn, increases the biosynthesis of carotenoids to neutralize ROS [22].

The above-presented results prove that it is necessary to select the appropriate doses of zinc, as well as the ideal composition of the propagating medium, for each strain. If higher doses of zinc ions were used in this study, they would have stimulated carotenoid biosynthesis by the R. mucilaginosa MK1 yeast strain. However, a high concentration of this element in the medium may significantly reduce the yeast growth rate and inhibit carotenoid biosynthesis. This was demonstrated in the study by Rovinaru et al. [23], who showed that after 120 h of cultivation of the yeast R. glutinis CCY 020-002-033 in the medium supplemented with 500 mg/L zinc, the volumetric efficiency of β-carotene biosynthesis and the cellular biomass yield were 0.92 mg/L and 0.16 g_d.m./L, respectively, while after cultivation in the control medium the values were 2.4 mg/L and 1.94 g_d.m./L, respectively. The authors reported that the synthesis of β-carotene (7.1 mg/L) was highest in the medium containing 250 mg Zn²⁺/L.

At acidic pH, aluminum ions are toxic to both plant and animal cells. Many studies suggest that they increase the peroxidation of phospholipids and proteins in cell membranes and induce oxidative stress in cells [11,24]. It was also indicated that the addition of Al³⁺ to the medium at doses from 200 to 300 mg/L resulted in a significant reduction of the biomass yield (Table 3). Among the cations tested in the present study, only aluminum ions increased the content of carotenoids in the cell biomass of the R. mucilaginosa MK1 strain and changed the proportions of β-carotene, torulene, and torularhodin synthesized by the yeast. The share of torulene increased significantly from 31.85% in the control sample to 75.20% in the biomass obtained after cultivation in the medium supplemented with 300 mg Al³⁺/L. A study by Elfeky et al. [11] showed that the enrichment of the culture medium with aluminum sulfate (0.7 mM) led to an increase in the synthesis of carotenoid pigments and torulene, which confirms the role of aluminum ions in the induction of carotenoid biosynthesis in yeast cells. Wang et al. [25] found that the addition of aluminum ions in the medium resulted in an increase in the activity of malate dehydrogenase and the intracellular accumulation of citric acid by Rhodotorula taiwanensis RS1 yeast strain.

Manganese is an important trace element that belongs to the group of transition metals. As a cell component, it plays a key role among enzymes involved in various metabolic processes and is also part of an antioxidant—superoxide dismutase, which protects cells by inactivating free radicals. Manganese also acts as an electron store, from where electrons are transferred to reaction sites, and as an activator of many enzymes involved in oxidation, carboxylation, carbohydrate metabolism, and the series of phosphorus and citric acid reactions. Similar to magnesium, this element is involved in the binding of ATP with enzyme complexes (phosphotransferase and phosphokinase) and activates the enzymes taking part in the Krebs cycle, such as dehydrogenase and decarboxylase, as well as RNA polymerase [26]. Hence, this study tested the influence of magnesium on carotenoids biosynthesis by yeast. The results showed no increase in the number of carotenoids in cellular biomass in the control medium, but after cultivation in the medium supplemented with manganese at a dose of 300 mg/L, the proportion of torularhodin in the total pool of carotenoids decreased from 22.8% (control) to 14.1%. Interestingly, Buzzini et al. [27] observed the opposite effect in their study on carotenoid biosynthesis by Rhodotorula graminis DBVPG 7021 strain, which showed that the presence of manganese ions inhibited the biosynthesis of torulene and torularhodin. The authors hypothesized that manganese cations had a possible negative influence on the activity of desaturases involved in carotenoid biosynthesis by yeast.

Similar to micronutrients, B vitamins are critical for the proper functioning and metabolism of yeast cells. For example, biotin is the prosthetic group of acetyl-CoA carboxylase, an enzyme that catalyzes the formation of malonyl-CoA, a substrate in de novo lipid biosynthesis [28]. Riboflavin is essential for the activity of enzymes involved in the TCA cycle, as well as for the electron transport chain, β-oxidation, and various biosynthetic processes [15]. Pantothenic acid (vitamin B₃) is a metabolic precursor of coenzyme A and many acyl carrier proteins, which are cofactors of many enzymes [12,13]. Niacin is involved in the biosynthesis of nicotinamide adenine dinucleotide [14]. Due to the significant role played by B vitamins in yeast cell metabolism, this study investigated their influence on carotenoid biosynthesis. To the best of the authors’ knowledge, there are no data on this subject in the scientific literature. It was found that among the tested vitamins, only niacin caused a change in the profile of carotenoids synthesized by R. mucilaginosa MK1. A significant increase in the content of torulene and torularhodin was observed, while the proportion of β-carotene was decreased. It was also noted that supplementation of the medium with a concentration of niacin significantly slowed down the growth of yeast, indicating that the vitamin inhibits certain metabolic processes. Perhaps the high concentration of niacin decreased the activity of lycopene cyclase, which catalyzes the conversion of γ-carotene to β-carotene, resulting in an increase in the content of torulene and torularhodin in the total carotenoid pool. This is the first study to have examined the effect of niacin on the biosynthesis of carotenoids, and therefore, further studies are needed to understand the mechanism underlying the effect of niacin on the enzymes involved in the carotenoid biosynthesis pathway in yeast cells.

5. Conclusions

The study proved that the profile of carotenoids synthesized by the yeast R. mucilaginosa MK1 in media containing potato juice water and glycerol could be changed by supplementing the medium with selected cations and B vitamins. The total carotenoid content increased significantly after yeast cultivation in the medium supplemented with aluminum at a dose of 300 mg/L. In addition, the amount of torulene increased to 72.2% (from 31.8% for the control medium) in the presence of aluminum. Torulene biosynthesis was also stimulated by enriching the medium with barium at an amount of 300 mg/L (percentage: 68.24%). The addition of niacin to the medium increased the production of torularhodin from 23.31% to 31.59–33.79%. The highest content of carotenoids (180.50 µg/g_d.m.) was observed after cultivation in the medium supplemented with both aluminum ions (300 mg/L) and niacin (100 µg/L). This is the first study to show the effect of niacin on the biosynthesis of carotenoids by red yeast cells, and therefore, further research is needed in this area to understand the mechanism underlying the influence of niacin.