Impact of Sodium Acetate on the growth, biomass, carotenoids, β-carotene, and lipid composition profile of Rhodotorula mucilaginosa

doi:10.21203/rs.3.rs-4865550/v1

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Impact of Sodium Acetate on the growth, biomass, carotenoids, β-carotene, and lipid composition profile of Rhodotorula mucilaginosa

https://doi.org/10.21203/rs.3.rs-4865550/v1

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Lipids, carotenoids, and β-carotene are produced by Rhodotorula mucilaginosa. This has important implications. Further optimization experiments were carried out by analyzing the substrate utilization of yeast medium supplemented with different sources of carbon, nitrogen, phosphate, and sodium acetate. Additional screening was carried out in batch shake flasks to examine the effects of various nitrogen sources, phosphate concentrations, and sodium acetate. Notably, when sodium acetate was supplied as an additional carbon source in the medium, remarkable improvements in the production of key compounds were seen such as the highest yield of cell dry weight (CDW) was 1.65 ± 0.94 gL⁻¹, with a lipid output of 66.53% (w/w), showing a substantial quantity of lipid accumulation in comparison to the biomass. The lipid concentration was 2.48 ± 1.03 gL⁻¹. Furthermore, 5.84 ± 0.05 µg g⁻¹ of β-carotene and 37.66 ± 2.13 µg g⁻¹ of carotenoids were produced. The following were observed in batch reactor fed with sodium acetate 5.02 ± 0.83 gL⁻¹ for cell dry weight (CDW); 3.30 ± 0.27 gL⁻¹ for lipid concentration; 65.73 ± 0.81% (w/w) for lipid content; 40.33 ± 1.84 µg g⁻¹ for carotenoid synthesis; and 17.63 ± 0.32 µg g⁻¹ for β-carotene production. Fed-batch bioreactor using sodium acetate, R. mucilaginosa produced significant levels of biomass, lipids, carotenoids, including β-carotene 48.36 ± 1.14 µg g⁻¹ of carotenoids, 21.38 ± 1.14 µg g⁻¹ of β-carotene, a lipid concentration of 4.06 ± 0.17 gL⁻¹, and a lipid content of 68.58 ± 1.95%.

Analytical Biochemistry

Biochemical Research Methods

Biotechnology and Bioengineering

Lipids

Cell dry weight

Carotenoids

β-Carotene

Rhodotorula mucilaginous

1.1 Yeast a potential source for lipid and carotenoids production

For decades, oleaginous yeasts have been studied as a microbial source of oil (Sitepu et al., 2014). Of the different yeast species, more than 600 are classified and more than 70 are considered oleaginous, and their oleaginicity screenings reveal oil-producing yeast species (Beopoulos et al., 2011, Sitepu et al., 2014). These species are usually from genera of Candida, Cryptococcus, Rhodotorula, Rhizopus, Trichosporon, Lipomyces and Yarrowia and they accumulate 40–70% of lipids of their total biomass depend on the conditions in which they grow and provide a substrate for their growth (Beopoulos et al., 2011). Of all the oil-producing microorganisms, yeast R. glutinis exhibit a high growth rate and lipids that accumulate under low-cost substrates (Xue et al., 2006) compared to algae, fungi and bacteria (Saenge et al., 2011). Lipids extracted from the pure culture of R. glutinis grow on glucose as a carbon source and produce saturated fatty acids (SFA) and unsaturated fatty acids (UFA) most are stearic acid (32%), palmitic acid (25%) and oleic acid (25%) (Easterling et al., 2009), oleic acid 46.9% and palmitic acid 20% (Li et al., 2007).

Carotenoids are mainly produced by filamentous fungi, yeasts, bacteria, algae and lichens. Among the microbial sources of carotenoids, yeasts such as Phafia rhodozyma and Rhodotorula glutinis are beneficial (Bhosale and Gadre, 2001; Buzzini and Martini, 2000). Rhodotorula carotenoids are known for their properties of torulene and torularhodin. It also collects β-Carotene and α-Carotene little quantities (Bhosale and Gadre, 2001; Buzzini and Martini, 2000). Carotenoids are essential natural pigment, lipid-soluble pigment, most of which exhibit C40 terpenoids, yellow, orange and red, which are widely found in microorganisms and plants. Industrially Carotenoid pigments such as β-carotene, lycopene, astaxanthin, Cantaxanthin, lutein and capxanthin are used as natural food dyes or feed additives. Several studies have reported that Carotenoids have anti-cancer, antioxidant and / or provitamin A properties (Bhosale and Gadre, 2001; Buzzini and Martini, 2000). Biorefinery awareness, that is, the conversion of substrates into value-added products, is widely accepted in the research community. Therefore, research for the production of by-products along with many novel and valuable products through fermentation is currently a well-constructed area.

1.2 High yield of lipid and Carotenoids production supply with glucose and sodium acetate

In previous reports the optimized culture media containing different concentrations of glucose or sodium acetate as carbon sources was extensively analyzed for microalgae cultivation (Li et al., 2014; Moon et al., 2013; Wang et al., 2017; Zhang et al., 2011). Silva et al, (2016) reported that the combination of glucose and sodium acetate during mixotrophic cultivation of Neochloris oleoabundans resulted in higher yields of lipids for biomass, protein, and biofuel production. Another study found that the maximum biomass production of 2.01 gL^− 1 was achieved by the Phaeodactylum tricornutum with a ration of 5.0 gL^− 1 glucose, while the sodium acetate concentration was 4.1 gL^− 1 and the biomass 1.15 gL^− 1 (Garcia et al., 2005). Yeast R. mucilaginosa produces high yields under culture conditions that limit nitrogen intake to 15.3 gL^− 1 with 0.17 gg^− 1 xylose (Khot and Ghosh, 2017); the medium biomass with glycerol 30.70 gL^− 1 and lipids (% w/w) 10.2 produced high yields respectively (Gientka et al., 2017). Oleaginous yeasts Cryptococcus humicola collected 15.5 gL^− 1 lipids, with a cellular lipid content of 40% at cell dry weight, in a total of 36 gL^− 1 cellular biomass grown in Ammonia standard hydrolysate (Sitepu et al., 2014). The carbon source, which is primarily studied to control Carotenogenesis and the supplementation of fermented ethanol, acetate and succinate, is carried out by all means by acetyl-CoA oxidation to enter the citric acid cycle and increase the synthesis of Carotenoids (Orosa et al., 2005).

This study focuses on the effect of Sodium acetate on high growth, biomass, lipid, Carotenoids and β-Carotene production.

2.1) Yeast cultivating Media (gL^-1)

Glucose 20

Yeast Extract 5

(NH₄) SO₄ 2

KH₂PO₄ 5

Mg₂SO₄ 1

CaCl₂ 0.2

2.2) YPD Media (for seed culture)

Yeast Extract 1%

Peptone 2%

Dextrose 2%

2.3) Mineral Salt Media (MSM) (gL^-1)

Na₂HPO₄ .12H₂O 9

(NH₄) SO₄ 0.1

Mg₂SO₄ 7H₂O 0.2

CaCl₂. 2H₂O 0.02

KH₂PO₄ 2.3

NaHCO₃ 0.05

Ferric Ammonium Citrate (mg) 1.2

2.4 Growth conditions and screening yeast media

Rhodotorula mucilaginosa, the yeast strain being studied, was acquired from the Biochemical Engineering Lab of the Indian Institute of Technology Guwahati (IITG) in Assam, India. All rights reserved. This strain's unique accession number is KX533469. This yeast has been identified by IITG researchers as a possible option for lipid synthesis (Kurtzman et al., 2011, Gedela at al., 2023).

One milliliter of the R. mucilaginosa sample was added to a medium called YM broth to start the yeast strain growing. Yeast extract (3 gL^-1), peptone (5 gL^-1), and glucose (10 gL^-1) were the ingredients of this broth. Following the inoculation, the cultures were incubated for a full day at 28 °C. After the incubation time, glycerol stocks were made to preserve the resultant cultures for further use. This was accomplished by keeping the cultures at -80 °C and adding 15% glycerol to them. The conserved cultures were brought back to life for additional testing and examination by being moved to a growing medium. Throughout the course of 144 hours, the cultures were stirred at 180 rpm and kept at 30 °C. The yeast cells multiplied and reached their maximum size during this period (Gedela at al., 2023).

The cultivated cells were harvested by centrifugation at 10,000 rpm for 10 minutes to prepare the samples for analysis. After that, the concentrated yeast cells in the resulting pellet were resuspended in distilled water. By utilizing a UV-Vis spectrophotometer, the optical density of the suspension was determined at a wavelength of 600 nm. Researchers were able to evaluate the yeast strain's development and cell density through this method Kurtzman et al., 2011 used several mediums to study yeast colony morphology.

2.5 In batch shake flasks, medium optimization

Through aseptic batch shake flask studies, the growth and lipid production of the yeast strain were investigated. 50 mL of the culture media were contained in each 250 mL shake flask. The culture medium's pH needed to be adjusted to 6 using either 1 M NaOH or 1 M H₂SO₄. According to Kumar et al., 2015, the mineral salt medium (MSM) contained the following ingredients per liter: 20 ml of ferric ammonium citrate solution (0.05 g ferric ammonium citrate dissolved in 20 ml of distilled water), Na₂HPO₄ 2H₂O (9.0 g), KH₂PO₄ (2.3 g), NH₄SO₄ (0.1 g), MgSO₄.7H₂O (0.2 g), CaCl₂ (0.02 g), and 20 ml of ferric ammonium citrate solution. The yeast culture was put to the shake flasks at a volume equal to 5% of the finished broth volume for inoculation. Following that, the flasks were incubated for 144 hours at 30 °C with 180 rpm of agitation. All experiments were carried out in triplicate, which means that each experimental condition was carried out three times, to guarantee the accuracy and consistency of the data. To evaluate the effects of various nutritional sources on biomass growth and lipid formation, the experiments included screening with 1.5% w/v concentrations of sodium acetate, phosphate, and nitrogen (Gedela at al., 2023).

2.6 Bioreactor: batch and fed-batch cultures

A 5 L stirred-tank Sartorius bioreactor (model B-LITE) was used to do a batch fermentation to learn more about the fermentation process. The bioreactor's working volume was established at two liters. Throughout the fermentation process, the culture pH was automatically adjusted to 6.0 by adding 0.5 M H₂SO₄ or 1 M NaOH as needed. The agitation speed was varied between 300 and 400 rpm, and the temperature was kept at 30 °C. The volume of air per unit volume of liquid per minute, or 0.8 vvm, was the aeration rate, and the dissolved oxygen concentration was constantly checked. A peristaltic pump (Miclins, India) was used to regulate the air flow rate needed to maintain the requisite aeration rate; this air flow rate varied from 1 to 3 vvm based on the requirements of the culture. 10% (v/v) of inoculum was used in the fermentation process for inoculation, meaning that 10% of the final culture volume was made up of the inoculum. Throughout the fermentation process, an antifoaming chemical was sporadically applied to stop excessive foaming. Samples were taken for multiple analyses, such as optical density (OD), biomass concentration, lipid content, carotenoids, and β-carotene, at regular 24-hour intervals. A fed-batch fermentation technique was also used in addition to batch fermentation, using a methodology like that of the batch procedure. The culture was fed every time the concentration of sodium acetate decreased by 15 gL^-1 from its starting value. This feeding technique was known as pulse feeding. In accordance with the feeding schedule, feedings were to occur every 36, 48, 72, 96, 120, and 144 hours. Until the batch culture was finished, the substrate concentration was kept constant (Qu et al., 2013).

2.7 Biomass productivity

The following formula was utilized to ascertain the biomass productivity:

Px is equal to (Xi - X0) / (Ti - T0), where Xi is the biomass concentration at time Ti, X0 is the biomass concentration at the initial time (T0), Ti is the time at i (in hours), and T0 is the initial time (T = 0h). Px is the biomass productivity in grams of biomass per liter per hour (g biomass L-1.h-1). Furthermore, the formula Li et al., 2014 provided was used to calculate the specific growth rate (μ) as follows: μ = (ln(x1) - ln(x0)) / (t1 - t0), where μ is the specific growth rate in grams per liter (g/L) per day, and x1 and x0 are the biomass concentrations in grams per liter (g/L) at times t1 and t0, respectively.

2.8 Sodium acetate estimation by HPLC

An HPLC (High-Performance Liquid Chromatography) analysis was conducted to determine the concentration of sodium acetate. The equipment, which was made by Varian in the Netherlands, included a refractive index (RI) detector (model 355) and a solvent delivery system (model 210) for the latter. A 300x 6.5-inch Meta Carb-87H carbohydrate column with an 8 µm particle size was used for the chromatographic separation. The temperature of the column was kept at 60 °C throughout the analysis. At a flow rate of 0.5 mL per minute, an eluent solution containing 9 mM sulfuric acid was employed. The separation of sodium acetate from the other components in the sample was made easier by this eluent. The retention durations of each component were compared with real standards to identify the HPLC peaks. By comparing the known standards to the peaks seen in the sample, the precise retention periods of the compounds were ascertained. Sodium acetate was accurately identified and quantified thanks to this comparison. The research carried out by Deshavath et al., 2017 served as the foundation for the techniques and equipment previously mentioned (Fig.1).

2.9 Total Carotenoids estimation by Spectrophotometrically

The sample underwent a series of processes to extract intracellular carotenoids. To eliminate any remaining materials, the sample was centrifuged first to extract the supernatant, and then the cell pellet rinsed three times with deionized water. The intracellular carotenoids were then removed into the acetone phase by manually disrupting the cells with a bead grinder and resuspending them in acetone. After centrifuging the solution, the carotenoid extract-containing supernatant was separated and saved. The cell pellet was extracted again using fresh acetone until the pellet lost all its color. After gathering the acetone extracts, a spectrophotometer was used to measure the absorbance of the acetone phase at a wavelength of 455 nm. With Sigma's β-carotene serving as the standard reference, the carotenoid content was measured. According to de Cavalho et al., 2012, the following formula was used to determine the sample's total carotenoid content:

Content of carotenoid (µg g-1) is equal to (A × V(ml) × 10^4) / (A^(1%)1cm × P(g)).

Where V is the total extract volume, P is the sample weight, and A^(1%)1cm is the specific extinction coefficient of β-carotene in acetone, which is 2500 (Hiyama and Ke, 1972; Aksu and Eren, 2007; Bhosale, 2001; de Cavalho et al., 2012; Pacia et al., 2016). A represents the absorbance at 455 nm with these values.

2.10 β- Carotenoids standard preparation

To generate calibration curves for β-carotene, 99% pure standard solutions were obtained from Sigma-Aldrich. The following were the concentrations of the β-carotene solutions: 1.8000, 0.9000, 0.4500, 0.2250, and 0.1125 milliliters per milliliter. Using a spectrophotometer, the intensity of β-carotene at a wavelength of 455 nm was plotted to create the calibration curves. The correlation between the β-carotene concentration and the corresponding intensity of the solutions at 455 nm was established. The calibration curves, which gave a quantifiable link between the measured intensity of β-carotene and its concentration, were created using this data. The study by Pacia et al., 2016 contains the specifics of the calibration curves and how they were created based on the concentration of β-carotene at 455 nm.

2.11 Determination of Lipid content

A modified version of Bligh and Dyer's method was used to quantify the amount of lipids in yeast cells. The effectiveness of this modified process in accomplishing total lipid extraction is well established. Using this technique, a 50 mL sample of yeast cells was centrifuged for 5 minutes at 5000 g. To get rid of any extraneous materials, the resultant yeast cell pellet was thrice washed with 50 mL of distilled water. After being cleaned, the cells were treated with 10 mL of 4M HCl and left to incubate for one to two hours at 60 °C. This stage of acid hydrolysis aids in the disintegration of the cellular components and makes lipid extraction easier.

Following the acid hydrolysis, 20 mL of a 1:1 combination of methanol and chloroform was added to the acid-hydrolyzed material at room temperature. For two to three hours, the mixture was agitated to facilitate the extraction of lipids into the organic phase. The organic lower phase and the aqueous upper phase were separated by centrifugation at 2000 g for 5 minutes at room temperature after the extraction. Using a Pasteur pipette, the bottom phase containing the isolated lipids was carefully retrieved. The recovered lower phase was allowed to evaporate under reduced pressure for ten minutes to extract the lipid fraction. The amount of lipid present in the yeast cells was then measured by weighing the resultant dry lipids. The basis for this extraction technique is of Pan et al., 2009.

3.1 Optimization of chemical composition for R. mucilaginosa to upsurge growth, lipid, carotenoid, and β-carotene production

In the preliminary enquiry, yeast medium (YM) enriched with mineral salt medium (MSM) was used to conduct batch shake cultures. These studies on R. mucilaginosa's metabolic capacities have focused on its performance on yeast-based medium. R. mucilaginosa produced lipids, carotenoids, and β-carotene when grown under these conditions (Prabhu et al., 2019; Gedela et al., 2023). Lipids are vital macromolecules with a wide range of applications, including biofuels and nutritional supplements. In yeast medium, R. mucilaginosa has been found to efficiently synthesis and accumulate lipids. This feature makes it a good option for biotechnological applications targeted at the long-term production of biofuels and other lipid-derived products.

Carotenoids are coloured chemicals with antioxidant capabilities that are commonly employed in the food and cosmetic sectors due to their health advantages and brilliant hues. R. mucilaginosa is known for its ability to produce carotenoids (Gedela et al., 2023). These chemicals serve an important function in shielding cells from oxidative stress and can be used in a variety of industrial applications. β-Carotene, a carotenoid precursor of vitamin A, plays a crucial role in human health, especially for eyesight and immunity. R. mucilaginosa produces β-carotene in yeast medium, indicating its potential as a natural supply of this essential nutrient. This brings up the possibility of its usage in dietary supplements and food fortification to fight vitamin A deficiency.

Growing R. mucilaginosa in yeast medium results in the production of lipids, carotenoids, and β-carotene, which has major significances (Gedela et al., 2023). This yeast species could be a versatile and sustainable source of these valuable compounds, meeting the needs of a variety of industries such as biofuel production, nutrition, and cosmetics. Future research and development could optimize culture conditions and increase production processes to fully realize R. mucilaginosa's biotechnological potential. The yeast, R. mucilaginosa will undoubtedly play an important part in producing eco-friendly and cost-effective solutions to fulfil comprehensive requirements as our understanding and capabilities in microbial biotechnology increase.

By examining the substrate utilization of yeast medium supplemented with Carbon sources, Nitrogen sources, Phosphate sources and Sodium acetate, more optimization studies were conducted.

R. mucilaginosa is the subject of study on optimizing culture conditions to boost lipid production, carotenoids, and β-carotene. Experiments were carried out to determine the ideal conditions that would lead to higher yields of these valuable chemicals. The goal of the study was to ascertain the ideal development conditions for R. mucilaginosa and the highest possible accumulation of β-carotene, carotenoids, and lipids (Gedela et al., 2023). In the observed growth profile of R. mucilaginosa, glucose as a carbon source resulted in increased optical density (OD) biomass, carotenoid (57.50 ± 1.54 µgg^-1), and β-carotene (25.50 ± 0.77 µgg^-1) production, but lower lipid production (55.22 ± 2.14 % w/w) than other carbon sources and, while the maximal yield of cell dry weight (CDW) was 3.35 ± 0.07 gL^-1(Table 1, Fig. 2 A, B, C). When sucrose, maltose, lactose, and galactose were employed, OD, biomass, carotenoid, and carotene production decreased, while lipid production (56 % w/w) increased slightly. Specifically, galactose produced somewhat less OD, biomass, carotenoid, and β-carotene than glucose, but more than sucrose, maltose, and lactose (Table 1, Fig.2 A).

When studying the growth profile of R. mucilaginosa using multiple carbon sources, it is critical to understand the microbe's varied metabolic routes for each substrate. The different growth rates and efficiency in utilizing various sugars can be ascribed to their structural complexity and metabolic accessibility. Glucose, as a simple sugar and monosaccharide, is quickly absorbed by microbial cells and enters the glycolytic pathway. This immediate entrance into central metabolism causes rapid energy production and biomass formation. The rapid growth rate reported using glucose as a carbon source could be due to its ease of transport across the cell membrane and the lack of enzymatic degradation before consumption (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018). Sucrose, maltose, and lactose are disaccharides that must be hydrolyzed to form monosaccharides before being digested. Enzymes like sucrase, maltase, and lactase hydrolyze these disaccharides, converting them into glucose and fructose (sucrose), glucose and glucose (maltose), and glucose and galactose (lactose). Because of the need for this enzymatic breakdown, it enters metabolic pathways later than glucose, resulting in slower initial growth rates (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018).

Although galactose is a monosaccharide, it is not as easily processed as glucose. Galactose must be transformed into glucose-1-phosphate via the Leloir route before entering glycolysis. This conversion needs several biochemical steps, slightly postponing the commencement of energy production when compared to straight glucose utilization (Timson, 2007). The desire for glucose can be traced back to evolutionary adaptation, in which microorganisms refined their metabolic machinery for the fastest possible energy production to outcompete other organisms in nutrient-rich settings. This mechanism, known as catabolite repression, guarantees that when glucose is present, enzymes required for the metabolism of alternative carbon sources are suppressed. This regulatory mechanism selects the most efficient energy source to maximize growth and survival (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018).

The examination of the growth profile of R. mucilaginosa with various carbon sources confirms that glucose is the most efficient substrate due to its simple structure and direct utilization pathway (Fig. 2 A). Disaccharides such as sucrose, maltose, and lactose require additional enzymatic processes to digest, resulting in reduced initial growth rates. Galactose, despite being a monosaccharide, requires a conversion mechanism that slows its use. These findings demonstrate R. mucilaginosa's metabolic flexibility while also emphasizing the evolutionary predisposition to prefer glucose due to its quick absorption and immediate metabolic usefulness. The growth profile as determined by optical density (OD) is shown in Image (A), which sheds light on how cell density changes over time. Image (B) shows the percentage of lipids (w/w) collected during the growth phase as well as the dynamic variations in cell dry weight (DCW) in grams per liter (gL^-1). This data demonstrates R. mucilaginosa's capacity to produce lipids under the specified circumstances. Moreover, the production levels of carotenoids (µgg-1) and β-carotene (µgg-1), two important chemicals of relevance, are displayed in image (C). The figure helps to give a thorough grasp of R. mucilaginosa's growth dynamics and production capacity when using media with various nitrogen supplies.

Ammonium sulphate, ammonium acetate, urea, ammonium chloride, and ammonia were among the nitrogen sources that were examined to optimize the chemical composition for the growth of R. mucilaginosa. The findings showed that the growth parameters, such as optical density (OD), biomass, carotenoids (15.6 ± 1.40 µgg^-1), β-carotene (3.23 ± 0.71 µgg^-1), and lipid production (44.44 ± 1.43 % w/w), were marginally increased by ammonium acetate (Table 2, Fig.3 A, B, C). Ammonia can be directly integrated into amino acids and other nitrogen-containing molecules, providing an easily assimilable supply of nitrogen. Its energetically advantageous absorption into cellular metabolism enables effective biosynthesis processes that promote growth and metabolite production. Ammonium acetate offers a supply of carbon (acetate) as well as nitrogen (ammonium) (Prescott et al., 2005). Because of its dual function, absorption into central metabolic pathways can be facilitated more quickly, increasing biomass accumulation and energy output. Acetate can also contribute to the TCA cycle, which will improve the production of lipids and carotenoids, two examples of secondary metabolites (Berg, 2015; Nelson and Cox, 2017).

Prior to being consumed, urea must be hydrolysed by the enzyme urease into ammonia and carbon dioxide. In comparison to ammonia and ammonium acetate, this extra enzymatic step may be less effective, leading to slower growth and decreased metabolite production (Kargi and DeLisa, 2017). Ammonium ions, which are helpful for assimilating nitrogen, are produced by ammonium chloride (Nelson and Cox, 2017). It is less effective than ammonium acetate, which supplies both nitrogen and carbon, because the chloride ions do not offer any extra metabolic advantages (Prescott et al., 2005; Berg, 2015; Nelson and Cox, 2017). Although ammonium sulphate is an excellent source of nitrogen, the medium may become acidic due to the sulphate ions, necessitating the need of extra buffering. When compared to more balanced sources like ammonium acetate, this can somewhat impair growth and metabolite production and complicate the growth environment (Prescott et al., 2005; Berg, 2015; Nelson & Cox, 2017).

According to the study, ammonia and ammonium acetate are better nitrogen sources for maximizing R. mucilaginosa growth and metabolite production. The direct absorption of ammonia into metabolic pathways combined with ammonium acetate's dual function as a nitrogen and carbon source results in increased production of lipids, carotenoids, β-carotene, and OD. Other nitrogen sources, on the other hand, like urea, ammonium chloride, and ammonium sulphate, are less effective because they need extra metabolic processes or have less evenly distributed nutritional contributions (Table 2, Fig.3 A, B, C).

The growth profile is assessed based on various parameters, which are visualized in the figure. R. mucilaginosa growth profile in terms of optical density (OD), which shows the evolution of cell density over time, as shown in image (A). Both the proportion of lipids (w/w) accumulated during the growth phase and the dynamic changes in cell dry weight (DCW) in grams per liter (gL^-1) are displayed in image (B). These measures provide important insights on R. mucilaginosa's capacity for lipid synthesis under the given circumstances. Additionally, picture (C) shows how much β-carotene (µgg^-1) and carotenoids (µgg^-1) were created during the cultivation (Table 2, Fig.4 A, B, C).

The chemical content of the growing medium can influence R. mucilaginosa's growth and metabolism. Key components such as sodium acetate, sodium nitrate, potassium hydrogen phosphate, di-potassium hydrogen phosphate, and di-sodium hydrogen phosphate are essential for optimal development and metabolite production.

According to study, utilizing sodium acetate as a carbon source improves R. mucilaginosa's growth profile. This boost leads to higher optical density (OD), increased biomass, and enhanced synthesis of carotenoids, β-carotene, and lipids. Sodium acetate, as a readily metabolizable carbon source, enters the tricarboxylic acid (TCA) cycle efficiently, increasing cellular energy levels and metabolic capacity (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018). An improved output of OD and biomass when using sodium acetate indicates more effective cell division and growth (Table 2, Fig.4 A). This might be because acetate is directly assimilated into the core metabolic pathways, which lessens the metabolic load from other carbon sources that need more intricate catabolic procedures.

Carotenoids, such as β-carotene, are produced via the energy-demanding mevalonate route. The requisite energy and precursors for the improved synthesis of these pigments can be obtained through the effective use of sodium acetate. Examine has indicated that easily assimilated carbon sources into central metabolism frequently result in higher production of secondary metabolites (Berg, 2015; Nelson and Cox, 2017, Madigan et al., 2018). It is possible to explain the enhanced lipid production seen with sodium acetate by the overflow metabolism, which directs extra carbon toward lipid biosynthesis.

In terms of OD, biomass, carotenoids, β-carotene, and lipid production, sodium acetate is superior than other chemical compositions like potassium hydrogen phosphate, sodium nitrate, di-potassium hydrogen phosphate, and di-sodium hydrogen phosphate (Table 2, Fig.4 A, B, C). While these additional sources are necessary for cellular processes and mainly act as donors of phosphate and nitrogen, they do not offer the same rapid energy benefits as sodium acetate. Despite being a great supply of nitrogen, sodium nitrate must be reduced to ammonium in order to be assimilated. This process uses energy, which could be taken away from the synthesis of metabolites and biomass. Although they play a more supporting function than the primary carbon source, phosphates like potassium, dipotassium, and sodium hydrogen phosphate are essential for nucleotide synthesis and energy transmission (ATP). Despite being essential to growth, they only indirectly contribute to the generation of biomass and secondary metabolites (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018).

To study the impact of various nitrogen sources, phosphate concentrations, and sodium acetate, more screening was done in batch shake flasks. Notably, significant advances in the generation of important chemicals were seen when sodium acetate was added as an extra carbon source together with improved medium. Using sodium acetate in the media to optimize the culture conditions led to noticeable improvements in several metrics. Lipid concentration was 2.48 ± 1.03 gL^-1, while the maximal yield of cell dry weight (CDW) was 1.65 ± 0.94 gL^-1, with a lipid output of 66.53 % (w/w), there was a significant amount of lipid buildup in comparison to the biomass. Furthermore, 37.66 ± 2.13 µgg^-1 of carotenoids and 5.84 ± 0.05 µgg^-1 of β-carotene were produced (Table 2, Fig.4 A, B, C).

It is noteworthy that using sodium acetate as a carbon source led to higher lipid content (w/w), improved growth, improved chemical oxygen demand (COD) removal %, and increased carotenoids and β-carotene synthesis. The image illustrates how sodium acetate and phosphate resources affect R. mucilaginosa growth and output capacities. The outcome showed that increasing the amount of sodium acetate in the yeast medium increased biomass, growth, lipid, carotenoids, and β-carotene synthesis (Table 2, Fig.4 A, B, C). This result showed how important sodium acetate is as a good source of nutrients for R. mucilaginosa cultures to reach maximum production. Silva et al., 2016 reported that the combining glucose and sodium acetate during the mixotrophic cultivation of Neochloris oleoabundans achieved a greater yield for the amounts of biomass, proteins, and lipids for biofuel production. The biomass, lipids, Carotenoids, and β-Carotene contents were considerably enhanced with Sodium acetate. Sodium acetate plays a crucial function in the optimization of R. mucilaginosa growth through chemical composition alteration. Higher OD, biomass, carotenoid, β-carotene, and lipid production are among the superior growth metrics that arise from its effective integration into metabolic pathways. These results highlight how crucial it is to choose the right carbon.

3.2 Effect of Sodium acetate on the composition profile of growth, biomass, lipid, carotenoid, and β-carotene in R. mucilaginosa.

In batch shake flask investigations, preliminary screens were carried out to optimize the content of sodium acetate (0.5%, 1.0%, 1.5%, and 3.5% w/v). The experimental findings showed that the highest yields were obtained at a concentration of 1.5% w/v sodium acetate. To be more precise, the lipid concentration was 1.99 ± 0.01 gL^-1and the maximal cell dry weight (CDW) was 3.01 ± 0.04 gL^-1. With a w/w lipid yield of 66.11 ± 1.75%, there was a significant amount of lipid buildup in comparison to the biomass. Furthermore, 42.16 ± 0.14 µgg^-1 of carotenoids and 6.03 ± 0.07 µgg^-1 of β-carotene were produced (Table 3, Fig.5 A, B, C).

The growth profile as determined by optical density (OD), which shows how cell density changes over time, is shown in Image (A). It offers insight into R. mucilaginosa's growth kinetics. The dynamic variations in cell dry weight (DCW), expressed in grams per liter (gL^-1), are depicted in image (B). This parameter provides information on the accumulation of biomass during the growing phase. Furthermore, provided is the proportion of lipids (w/w) that was accumulated during the growth, which provides insight into the capacity for lipid production.

The amounts of carotenoids (µgg^-1) and β-carotene (µgg^-1) production are shown in Image (C). R. mucilaginosa synthesizes important chemicals known as carotenoids and β-carotene. Keeping an eye on their production gives information about the yeast's productivity and metabolic processes.

R. mucilaginosa's growth dynamics, biomass accumulation, lipid, carotenoids, and β-carotene's synthesis under the impact of sodium acetate and other resources. In their study, Silva et al., 2016 investigated the mixotrophic culture of Neochloris oleoabundans and found that the combination of glucose and sodium acetate increased biomass, protein, and lipid yields all of which are important to produce biofuel. Garcia et al., 2005 conducted a study on Phaeodactylum tricornutum and discovered that a maximum biomass production of 2.01 gL^-1was achieved at a glucose content of 5.0 gL^-1. By contrast, 1.15 gL^-1of biomass was produced at a concentration of 4.1 gL^-1of sodium acetate. Khot and Ghosh, 2017 reported increased biomass yields of 15.3 gL^-1for R. mucilaginosa under nitrogen-limited growth conditions, while consuming 0.17 gg^-1 of xylose. A medium containing glycerol produced a greater biomass yield of 30.70 gL^-1and a lipid content of 10.2% (w/w), as shown by Gientka et al., 2017. Additionally, Sitepu et al., 2014 discovered that 15.5 gL^-1of lipids, or 40% of the cell dry weight, were collected by Cryptococcus humicola. Growing in authentic hydrolysate of ammonia, the yeast attained 36 gL^-1of total cellular biomass.

The purpose of these studies is to learn more about the effects of sodium acetate on yeast lipid synthesis and regulation. Numerous research has investigated the regulation of lipid production in yeast in response to sodium acetate. The effect of sodium acetate on lipid production in several yeast species has been studied by several researchers. These investigations look at the impact of sodium acetate on lipid yield, and associated regulatory processes. Sodium acetate can affect R. mucilaginosa and other microbes in several ways, including growth, biomass, and biochemical composition. Specifically, it is critical to comprehend the metabolic pathways involved while analyzing its impacts on lipids and carotenoids. Microorganisms can use sodium acetate as a source of carbon. The use of it can affect R. mucilaginosa's growth rate and biomass production. The availability of acetate can impact energy generation and cellular metabolism, which in turn can impact total growth and biomass buildup. One of the main steps in the synthesis of lipids is the synthesis of fatty acids, which is facilitated by acetate. Acetate is a resource that microorganisms like R. mucilaginosa can use to produce lipids, such as phospholipids and triglycerides. Acetate's increased availability may encourage cells to store lipids. In microorganisms such as R. mucilaginosa, the process of lipid generation is dependent on multiple metabolic pathways.

Acetyl-CoA is produced from acetate and is a precursor to fatty acid production. The fatty acid synthase (FAS) route uses malonyl-CoA, which is produced when acetyl-CoA is carboxylated. This process is catalyzed by acetyl-CoA carboxylase. Many lipid compounds are synthesized through the FAS pathway, which is a sequence of enzyme processes that lengthen fatty acid chains (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018). R. mucilaginosa cells absorb sodium acetate, which is then transformed into acetyl-CoA by the enzyme acetyl-CoA synthase. This acetyl-CoA is a key molecule that acts as a precursor in a variety of metabolic pathways, including lipid production. Acetyl-CoA carboxylase converts acetyl-CoA into malonyl-CoA. This process is necessary for the elongation of fatty acid chains. The fatty acid synthase complex adds malonyl-CoA and acetyl-CoA units progressively to increasing fatty acid chains, producing long-chain fatty acids. Fatty acids are esterified to glycerol-3-phosphate, creating phosphatidic acid, which is then dephosphorylated to diacylglycerol. Diacylglycerol can be further acylated to produce triacylglycerol (TAG), an important storage lipid. Transcription factors regulate enzyme expression in lipid biosynthesis based on acetyl-CoA levels and other metabolic signals.

Sodium acetate can increase gene expression of critical enzymes in the lipid manufacturing pathway, resulting in increased flux. Acetyl-CoA, a metabolite produced from sodium acetate, can affect enzyme activity in fatty acid synthesis through allosteric regulation. Sodium acetate activates signalling pathways that alter transcription factor activity, resulting in enhanced lipid synthesis (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018). Studies on the action of sodium acetate on R. mucilaginosa have revealed that treatment with sodium acetate considerably increases lipid accumulation. This is most likely owing to the increased availability of acetyl-CoA, a necessary precursor for fatty acid synthesis. Studies have shown that R. mucilaginosa cultures grown in sodium acetate-supplemented medium express greater amounts of acetyl-CoA carboxylase and fatty acid synthase, both of which are required for lipid biosynthesis (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018).

Sodium acetate has a complicated regulatory influence on lipid biosynthesis in R. mucilaginosa, increasing the production of key metabolites necessary for lipid synthesis. Acetyl-CoA availability is raised, and lipid biosynthesis genes are upregulated, both of which lead to increased lipid buildup. This understanding lays the groundwork for optimizing culture conditions to maximize lipid production, which has important implications for biotechnological applications like as biofuel generation and the synthesis of valuable lipid-based chemicals. The role of sodium acetate in the carotenoid biosynthesis pathway and its modulation of enzymes in yeast. R. mucilaginosa provides insight into the metabolic pathways that underpin carotenoid biosynthesis and the factors that influence its synthesis.
Carotenoid biosynthesis, sodium acetate has been shown to stimulate carotenoid production in R. mucilaginosa. Studies have demonstrated that adding sodium acetate to the growth medium promotes the expression of critical enzymes in the carotenoid biosynthesis pathway, resulting in enhanced carotenoids production. Sodium acetate regulates the activity of numerous enzymes in the carotenoid biosynthesis pathway. One of the important enzymes involved is phytoene synthase, which catalyzes the condensation of geranylgeranyl pyrophosphate (GGPP) molecules to produce phytoene, a precursor to carotenoids. Sodium acetate is hypothesized to increase the activity of phytoene synthase, boosting the transit of GGPP toward carotenoid formation (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018). Metabolic Shifts, adding sodium acetate to the growth medium causes metabolic shifts in R. mucilaginosa, resulting in enhanced carbon flux toward carotenoid production. This metabolic reprogramming involves the activation of genes encoding enzymes involved in the carotenoid biosynthesis pathway while downregulating genes associated with other metabolic pathways (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018).

Optimization of Culture variables, according to research, the concentration of sodium acetate in the growth medium, as well as other culture variables such as pH and temperature, might influence carotenoid production in R. mucilaginosa. Optimization of these parameters is critical for maximizing carotenoid synthesis in industrial-scale fermentation systems.
Finally, studying the involvement of sodium acetate in R. mucilaginosa's carotenoid biosynthesis pathway sheds light on how carotenoid production is regulated in this yeast species. Understanding the methods by which sodium acetate affects enzyme activity and metabolic fluxes can help create techniques for increasing carotenoid production in biotechnological applications. Further research into R. mucilaginosa's molecular and physiological responses to sodium acetate supplementation will help to optimize culture conditions for large-scale carotenoid synthesis. Carotenoids are currently used in feed, pharmaceutical and cosmetics industries and have high commercial value with a global market accounted for 766 million dollars expected to increase by 155 million dollars in the current year (Vílchez et al., 2011). Biological properties of carotenoids allow for a wide range of commercial applications. Indeed, recent interest in the carotenoids has been mainly for their nutraceutical properties. Many scientific studies have confirmed the benefits of carotenoids to health and their use for this purpose is growing rapidly. In addition, carotenoids have traditionally been used in food and animal feed for their color properties. Carotenoids are also known to improve consumer perception of quality; an example is the addition of carotenoids to fish feed to impart color to farmed salmon (Vílchez et al., 2011).

The results of this investigation indicate that, in comparison to using yeast medium alone, employing yeast media supplemented with sodium acetate results in increased growth, biomass, lipid, carotenoid, and β-carotene synthesis. Sodium acetate is added as an extra carbon source, which promotes better microbial growth and the synthesis of important chemicals (Gong et al., 2015). The analysis's findings would display the sodium acetate concentration in the culture media at various intervals. It is possible to determine the sodium acetate consumption profile of R. mucilaginosa by charting these concentrations against time. These findings shed important light on R. mucilaginosa's metabolic activities and growth dynamics. The sodium acetate consumption profile shows how well the organism uses this chemical as a carbon source, demonstrating both its metabolic efficiency and potential for use in commercial processes like bioremediation and biofuel production. A thorough comprehension of R. mucilaginosa's metabolic dynamics can be obtained by establishing a correlation between the rate of sodium acetate consumption and the various growth stages of the organism (lag, exponential, stationary, and death phases). It is possible to evaluate the organism's sodium acetate utilization efficiency, which provides information about its potential for use in biotechnology. The HPLC study of R. mucilaginosa's intake of sodium acetate provides important insights about the organism's metabolic activities and ability to use sodium acetate as a carbon source. Understanding the ecological function of R. mucilaginosa in natural habitats and improving yields in bioprocesses, as well as optimizing culture conditions for industrial uses, depend on this investigation. The amount of sodium acetate absorbed by R. mucilaginosa was measured using High Pressure Liquid Chromatography (HPLC) using a solvent delivery system (210), a refractive index (RI) detector (355), and a Meta Carb-87H carbohydrate column (300 x 6.5, particle size 8 µm). The column was filled with the eluent, 9 mM sulfuric acid, at a flow rate of 0.5 mL/min while maintaining a temperature of 60°C. Using authentic standards, HPLC peaks were identified based on the distinct retention duration of each chemical (Fig. 1) (Deshavath et al., 2017). The potent analytical method known as High Performance Liquid Chromatography (HPLC) is used to separate, identify, and quantify the constituents of a mixture. HPLC offers precise and reliable measurements of sodium acetate concentrations throughout time, enabling researchers to track the organism's uptake and use in the context of calculating sodium acetate intake by R. mucilaginosa. HPLC would have been used in the study analysis to measure R. mucilaginosa's sodium acetate consumption while it was growing. The main carbon source in the experimental setup would be sodium acetate, which would be cultivated in a medium with a specified concentration. Periodically, samples from the growing media would be removed and subjected to HPLC analysis (Deshavath et al., 2017) (Fig.1).

3.3 R. mucilaginosa: Production of Lipids, Carotenoids, and β-Carotene by Batch and Fed batch Fermentation Process

This specific bioreactor model was selected for the experiment conducted at the Indian Institute of Technology Guwahati (IITG). The Sartorius B-LITE bioreactor is a commonly used stirred-tank bioreactor with a working volume of 2L. The experimental schematic involved setting the desired culture pH at 6.0, which was achieved by adding either 1 M NaOH or 0.5 M H₂SO₄ as necessary. The temperature of the bioreactor was set at 30 °C to promote the growth of the microbial culture. Agitation was maintained at a speed between 300 and 400 rpm, while aeration was controlled at a rate of 0.8 vvm (volume of air per volume of medium per minute) to ensure sufficient oxygen supply for the microbial cells. The dissolved oxygen concentration was monitored throughout the experiment. To inoculate the culture, a 10% (v/v) volume of the microbial inoculum was added to the bioreactor. This inoculum served as the starting point for the growth of the microbial culture. Throughout the experiment, an antifoaming agent was occasionally added to control foaming, which could disrupt the culture and hinder proper measurements. Samples were collected at regular intervals of 24 hours for analysis. These samples were used to measure various parameters such as optical density (OD), biomass concentration, lipid content, carotenoids, and β-carotene.

In batch reactor, R. mucilaginosa fed sodium acetate the found result were as follows, 5.02 ± 0.83 gL^-1for cell dry weight (DCW); 3.30 ± 0.27 gL^-1for lipid concentration; 65.73 ± 0.81% (w/w) for lipid content; 40.33 ± 1.84 µgg^-1 for carotenoids; and 17.63 ± 0.32 µgg^-1 for β-carotene (Table 5). In this analyze, yeast media supplemented with sodium acetate as an additional carbon source were used to track the growth profile of R. mucilaginosa. The optical density (OD), cell dry weight (CDW) in gL^-1, lipid concentration in gL^-1, carotenoids content in µgg^-1, β-carotene content in µgg^-1, and the use of sodium acetate (gL^-1) as a carbon source are all shown in the graphs in the image.

Furthermore, growth, biomass, lipid, carotenoid, and β-carotene production kinetic characteristics on yeast cultures supplemented with sodium acetate were measured. The productivity of biomass was calculated to be 45.06 mgL^-1Day^-1, lipid productivity to be 33.17 mgL^-1day^-1, and specific growth rate (µmax) to be 1.14 day^-1 (Table 4).

R. mucilaginosa grown in a fed-batch bioreactor with sodium acetate produced considerable amounts of biomass, lipids, and carotenoids, including β-carotene. R. mucilaginosa produces carotenoids at 48.36 ± 1.14 µgg-1, β-carotene 21.38 ± 1.14 µgg-1 and has a 4.06 ± 0.17 gL^-1lipid concentration, 68.58 ± 1.95% lipid content and demonstrating its metabolic plasticity in using sodium acetate as a carbon source for lipid and secondary metabolite synthesis. The fed-batch mode bioreactor culture of R. mucilaginosa supplemented with sodium acetate demonstrates its promise as a viable microbial platform for producing β-carotene, carotenoids, and lipids. R. mucilaginosa is well-suited for large-scale bioprocessing applications due to its high biomass and lipid productivity, as well as its quick growth kinetics. The findings provide important insights into R. mucilaginosa's metabolic capabilities, paving the door for further optimization of culture conditions and strain engineering tactics to improve product yields and process efficiency.

A comparing fed-batch and batch culture bioreactor systems in terms of optical density (OD), biomass, carotenoids, β-carotene, and lipid synthesis sheds light on the efficiency and productivity of various cultivation methods for R. mucilaginosa. Fed-batch culture systems often produce greater OD values and biomass concentrations than batch culture systems. Continuous nutrient input in fed-batch systems promotes microbial growth and greater cell densities during longer cultivation periods. When compared to batch cultures, fed-batch cultures accumulate more biomass due to improved nutrient availability, fewer nutritional constraints, and longer cultivation times. Fed-batch culture bioreactors classically produce more carotenoids, β-carotene, and lipids than batch cultures. The constant feed supply in fed-batch systems stimulates metabolic activity and the buildup of secondary metabolites like carotenoids and lipids.
Higher biomass concentrations in fed-batch cultures create a bigger cellular pool for carotenoid and lipid production, leading to higher total yields. Fed-batch systems have longer cultivation durations, allowing for increased accumulation of carotenoids, β-carotene, and lipids, leading to higher product concentrations than batch cultures.

To increase the amount of lipids, carotenoids, and β-carotene produced by the yeast R. mucilaginosa, this study investigated optimizing the culture conditions. Carbon Source, among the four carbon sources (sucrose, maltose, lactose, and galactose), glucose produced the most biomass but the least amount of fat. Nitrogen Source, of the three nitrogen sources (urea, ammonium chloride, and ammonium sulfate), ammonia and ammonium acetate produced the most growth and metabolites. Sodium Acetate, the production of lipids, carotenoids, and β-carotene was dramatically boosted by sodium acetate supplementation. This most likely results from, elevated acetyl-CoA availability, which is a prerequisite for the synthesis of fatty acids. overexpression of genes related to the pathways to produce lipids and carotenoids. Shift in metabolism for these processes in favor of higher carbon flow.

The study reveals that cultivating R. mucilaginosa with glucose as a carbon source, ammonia or ammonium acetate as a nitrogen source, and supplementing with sodium acetate can increase its synthesis of lipids, carotenoids, and β-carotene. Acetyl-CoA is produced from acetate and is a precursor to fatty acid production. The fatty acid synthase (FAS) route uses malonyl-CoA, which is produced when acetyl-CoA is carboxylated. This process is catalyzed by acetyl-CoA carboxylase. Many lipid compounds are synthesized through the FAS pathway, which is a sequence of enzyme processes that lengthen fatty acid chains (Berg, 2015; Nelson and Cox, 2017; Madigan et al., 2018). R. mucilaginosa cells absorb sodium acetate, which is then transformed into acetyl-CoA by the enzyme acetyl-CoA synthase. This acetyl-CoA is a key molecule that acts as a precursor in a variety of metabolic pathways, including lipid production. Acetyl-CoA carboxylase converts acetyl-CoA into malonyl-CoA. This process is necessary for the elongation of fatty acid chains. The fatty acid synthase complex adds malonyl-CoA and acetyl-CoA units progressively to increasing fatty acid chains, producing long-chain fatty acids.
The study emphasizes the necessity of understanding yeast metabolic pathways while improving growth conditions. R. mucilaginosa has potential for large-scale production of useful chemicals such as biofuels and nutraceuticals.
More study is needed to improve large-scale culturing and better understand the molecular mechanisms underlying sodium acetate's effects.

Declaration of Ethics Statement.
This study does not make use of human or animal participants. This study's analysis and conclusions are purely based on theoretical models, simulations, and/or publicly available data. As a result, no ethical approval or permission were required for this study.

Declaration of Consent for Publication
All authors engaged in this study agree to have their work published in Bioresources and Bioprocessing. The contents of this paper have not previously been published and are not being considered for publication anywhere.

Declaration of Competing Interest
The authors state that they have no known competing financial interests or personal relationships that could have influenced the work presented in this study.

Author’s contribution

Ravi Gadela: methodology, analysis, writing (original draft),
Kannan Pakshirajan: Conceptualization, supervision, review, and editing.

Veeranki Venkata Dasu: Dandles conceptualization, supervision, resource management, review, and editing. All authors reviewed and approved the final manuscript.

Declaration of No Funding.
This study received no grants from public, commercial, or non-profit funding entities.

Declaration of Availability of Data and Materials
The data and materials used and analyzed in the current investigation are available from the corresponding author upon on request.

Acknowledgment

The authors are thankful to the Indian Institute of Technology Guwahati for providing the required facilities to conduct this research work and the Central Instrument Facility (CIF), IIT Guwahati.

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Table 1 Summary of Optimization of various carbon resources for the growth profile of R. mucilaginosa

S.No	Carbon sources	DCW	Total Lipid	Lipid content	Carotenoids	β-carotene
S.No	Carbon sources	(gL^-1)	(gL^-1)	(%, W/W)	(µgg^-1)	(µgg^-1)
1	Glucose	3.35±0.07	1.85±0.03	55.22±2.14	57.50±1.54	25.50±0.77
2	Sucrose	3.17±0.02	1.79±0.04	56.46±1.84	45.00±1.16	22.00±0.86
3	Maltose	3.36±0.04	1.82±0.01	54.16±1.75	44.50±1.01	21.53±0.97
4	Lactose	2.24±0.05	1.18±0.02	52.67±1.21	38.83±1.09	17.57±0.92
5	Galactose	2.50±0.02	1.35±0.02	54.00±1.61	45.40±1.11	20.70±1.12

Table 2 Summary of chemical parameter optimization for the growth profile of R. mucilaginosa

S.No	Parameters	DCW	Total Lipid	Lipid content	Carotenoids	β-carotene
S.No	Parameters	(gL^-1)	(gL^-1)	(%, W/W)	(µgg^-1)	(µgg^-1)
1	Urea	0.10±0.04	0.04±0.05	40.00±1.44	11.15±0.17	2.26±0.14
2	NH₄CL	0.12±0.04	0.05±0.01	41.66±1.71	14.92±0.77	1.71±0.21
3	NH₃	0.19±0.03	0.08±0.02	42.10±2.82	17.53±1.49	3.19±0.07
4	(NH₄)₂SO4	0.10±0.03	0.04±0.03	40.00±1.52	13.63±1.01	2.20±0.04
5	Ammonium Acetate	0.27±0.05	0.12±0.04	44.44±1.43	15.6±1.40	3.23±0.71
6	Sodium Nitrate	0.14±0.01	0.05±0.02	35.71±1.21	13.5±0.93	2.3±0.12
7	Sodium Acetate	2.48±1.03	1.65±0.94	66.53±2.31	37.66±2.13	5.84±0.05
8	Na₂HPO₄	0.16±0.05	0.06±0.01	37.50±1.45	13.21±0.87	3.19±0.15
9	KHPO₄	0.09±0.01	0.03±0.01	33.33±1.01	8.14±0.09	2.67±0.056
10	K₂HPO₄	0.13±0.02	0.05±0.01	38.46±1.09	6.33±0.27	1.65±0.21

Table 3 Summary of the Sodium acetate optimization growth profile in R. mucilaginosa

S.No	Sodium acetate (% w/v)	DCW	Total Lipid	Lipid content	Carotenoids	β-carotene
S.No	Sodium acetate (% w/v)	(gL^-1)	(gL^-1)	(%, W/W)	(µgg^-1)	(µgg^-1)
1	0.5	2.05±0.07	1.09±0.03	53.17±2.14	21.50±0.04	3.00±0.11
2	1	2.35±0.02	1.49±0.04	63.40±0.84	32.63±0.14	5.27±0.01
3	1.5	3.01±0.04	1.99±0.01	66.11±1.75	42.16±0.14	6.03±0.07
4	2.0	2.02±0.05	1.11±0.02	54.95±1.21	11.83±0.02	2.57±0.02

Table 4 Kinetic parameters of the growth of R. mucilaginosa

S.No	Parameters
1	Specific growth rate µ max (day ^-1)^a	1.14
2	Dry cell weight (gL^-1)^a	6.12
3	Biomass productivity (mgL^-1 Day ^-1)^b	45.06
4	Total Lipid (%, w/w DCW)^a	68.67
5	Total lipid productivity (mgL^-1Day ^-1)^c	33.17
6	Total Carotenoids (µgg^-1)	45.16
7	Total β-Carotene (µgg^-1)	8.73

^a All the parameters depicted were the maximum values obtained for R. mucilaginosa growth under different cultivation conditions.

^b The biomass productivity (Px, mgL^-1day ^-1) under different cultivation conditions were calculated based Px = (X_f-X₀)/(t_f - t₀) where, X₀ and X_f were the dry cell weight (gL^-1) obtained at initial (t₀) and final (t_f) time points (in days), respectively.

^c The total lipid productivity (P_TL, mgL^-1day ^-1) under different cultivation conditions were calculated based P_TL = (TL_f -TL₀)/(t_f - t₀) where, TL₀ and TL_f were the dry cell weight (gL^-1) obtained at initial (t0) and final (tf) time points (in days), respectively.

Table 5 The summary of batch and fed-batch reactors growth profile in R. mucilaginosa

Reactor Type

Parameters

DCW

(gL^-1)

Total Lipids

(gL^-1)

Lipid content

(%, W/W)

Carotenoids (µgg^-1)

β-Carotene

(µgg^-1)

Batch

Yeast Media + Sodium Acetate

5.02 ± 0.83

3.30 ± 0.27

65.73 ± 0.81

40.33 ± 1.84

17.63 ± 0.32

Fed-Batch

Yeast Media + Sodium Acetate

5.92 ± 0.63

4.06 ± 0.17

68.58 ± 1.95

48.36 ± 1.14

21.38 ± 0.12

The authors declare no competing interests.

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Impact of Sodium Acetate on the growth, biomass, carotenoids, β-carotene, and lipid composition profile of Rhodotorula mucilaginosa

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1. Introduction

1.1 Yeast a potential source for lipid and carotenoids production

1.2 High yield of lipid and Carotenoids production supply with glucose and sodium acetate

2. Materials and Methods

3. Results and Discussion

4. Conclusions

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