Supercritical carbon-dioxide (SC-CO2) extraction of lipids and carotenoids from Rhodotorula toruloides CBS 14 in comparison with conventional extraction methods

Supercritical carbon-dioxide (SC-CO2) extraction of lipids and carotenoids from Rhodotorula toruloides CBS 14 in comparison with conventional extraction methods | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Supercritical carbon-dioxide (SC-CO2) extraction of lipids and carotenoids from Rhodotorula toruloides CBS 14 in comparison with conventional extraction methods Yashaswini Nagavara Nagaraj, Johanna Blomqvist, Sabine Sampels, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5460903/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2025 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted 8 You are reading this latest preprint version Abstract Background We conducted extractions using supercritical carbon dioxide (SC-CO 2 ) and conventional solvent methods to extract lipids and carotenoids from R. toruloides CBS 14 cells grown on wheat straw hydrolysate. The lipid extracts were analyzed using gas chromatography (GC), and the carotenoids were identified and quantified using ultra-high performance liquid chromatography (UHPLC). Results Four main carotenoids in the extracts from both extraction methods were identified including β-carotene, γ-carotene, torularhodin, and torulene. Interestingly, torularhodin was the major carotenoid extracted using SC-CO 2 extraction, followed by torulene. This was different from the conventional acetone extraction method, where β-carotene was the main carotenoid. After the conventional extraction, torularhodin and torulene underwent degradation due to the saponification step, which was necessary to remove lipids before UHPLC analysis. The total carotenoid concentration obtained from SC-CO 2 extraction was 332.09 ± 27.32 μg/g dry weight compared to 19.9 ± 2.74 μg/g dry weight in acetone extraction. A small amount of carotenoids was observed to be lost into the lipid extract, but this loss was not as substantial as that seen with acetone extraction. Additionally, the total lipid content in samples extracted using SC-CO 2 was significantly lower than that obtained using the conventional Folch method. GC analysis revealed that oleic acid was the major fatty acid in both lipid extracts, followed by palmitic acid and linoleic acid. Notably, the proportion of unsaturated fatty acids was higher in the extracts from the SC-CO 2 method compared to the conventional method. Conclusion These findings indicate that the SC-CO 2 extraction method outperformed conventional methods by preserving the integrity of unsaturated lipids and retaining an abundance of carotenoids, resulting in high-quality extracts. R. toruloides CBS 14 supercritical carbon dioxide extraction lipids carotenoids Folch method acetone extraction saponification Figures Figure 1 Figure 2 Introduction In recent years, the consumption of fats and oils as food ingredients has significantly increased, constituting more than 80% of their overall usage (1). Lipids play a vital role in cooking, baking, and food preparation. Additionally, they find application as animal feeds, providing improved nutritional value and energy density to livestock diets (2). As the demand continues to rise, alternative sources for plant oils are being explored. One focus has been targeted on microbial oil (MO) as sustainable fat source. MO is synthesized and accumulated by specific microorganisms, known as oleaginous microorganisms, through cultivation processes that utilize renewable resources as substrates (3). Lignocellulosic biomass presents a promising substrate due to its renewability and abundant nature, featuring substantial quantities of readily usable sugars (4, 5). Many oleaginous yeasts possess the capability to convert sugars, polysaccharides, glycerol, and other compounds into oil (3). The major constituents of the produced lipids are primarily neutral triacylglycerols (TAGs), which contain energy-rich fatty acids resembling the fatty acid composition of vegetable oil and, therefore have the potential to be utilized in the production of food, feed, biofuels, and various biochemicals (3, 6). Some oleaginous yeasts can also produce carotenoids as secondary metabolites (7, 8, 9). Carotenoids are used as natural colorants and antioxidants in the food industry to enhance the visual appeal of products (10). They are also employed as dietary supplements, either in their natural form or as purified extracts, due to their antioxidant and health-promoting properties (11). To fully utilize the biotechnological potential of oleaginous yeasts for the production of lipids and carotenoids, it is crucial to develop effective extraction processes. This is because the lipids and carotenoids produced by these yeast cells are sensitive to peroxidation which can be induced by for example heat and light (12). Therefore, cautious extraction methods need to be adopted to ensure efficient recovery of these valuable compounds preserving their quality and bioactivity to ensure the full benefit of oleaginous yeasts for the production of high-value lipids and carotenoids (13, 14). Traditionally, the lipids and carotenoids have been extracted from oleaginous yeasts using organic solvents. However, this conventional method has limitations due to the significant use of harmful solvents, multiple extraction steps, and reduced effectiveness caused by the barriers presented by cell envelopes (15, 16, 17). To overcome these challenges, various physical techniques such as heat treatment, ultrasound, high-pressure homogenization, bead-milling, and vigorous shaking have been employed before or during extraction (18). These procedures aim to disrupt the cell envelopes, allowing solvents to penetrate the cells and solubilize the lipids and carotenoids, thereby improving the extraction yield (16, 19). These physical approaches enhance the accessibility of solvents to intracellular components, leading to more efficient extraction processes with reduced solvent consumption and increased recovery of lipids and carotenoids (18). Supercritical carbon dioxide (SC-CO 2 ) extraction has emerged as a gentle technique for extracting natural substances aimed at applications in the food and pharmaceutical sector (20). Supercritical fluids, are characterized by operating above the critical temperature and pressure of the compound, resulting in specific properties (21). In the supercritical state, carbon dioxide exhibits unique properties including high compressibility, liquid-like density, enhanced diffusivity, low viscosity, and low surface tension, similar to an organic solvent. The properties of supercritical fluids, particularly SC-CO 2 , play a significant role in their ability to permeate and extract compounds from matrices compared to conventional organic solvents (16). Researchers have indicated that, compared to conventional organic solvents, SC-CO 2 exhibits higher diffusivity and lower density, viscosity, and surface tension. Additionally, these properties can be widely adjusted by altering the operational conditions (22, 23). These characteristics enable SC-CO 2 to penetrate both micro and macro porous components, allowing for selective extraction, fractionation, and purification of target compounds (22). SC-CO 2 extraction offers several advantages over traditional methods. Firstly, it is non-toxic and eliminates the need for potentially harmful organic solvents, making it a safer option for both operators and consumers (24, 25). This quality renders it suitable for food and feed-related applications. It also preserves the integrity of the extracted compounds, as it operates under milder conditions, minimizing thermal degradation (26, 27, 28). Researchers have focused on optimizing the extraction process by adjusting key parameters such as temperature, pressure, extraction time, and CO 2 flow rate. By fine-tuning these variables, optimal extraction conditions can be achieved, ensuring maximum yield and quality of the extracted natural products (29). The objective of this study was to investigate the potential of supercritical carbon dioxide (SC-CO 2 ) extraction to obtain lipids and carotenoids from an oleaginous red yeast, cultivated on lignocellulose hydrolysate. Materials and methods Rhodotorula toruloides cultivation: Inoculum preparation: R. toruloides CBS 14 was obtained from the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands. Cells were stored in frozen stocks at −80 °C. The inoculum was prepared as described before by Nagaraj et al. (8). Briefly, cells from YPD-agar plates (glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L, agar 15 g/L) were inoculated into 300 mL of YPD (glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L) in a 3 L Erlenmeyer flask and incubated at 25 °C for 48–72 h at 150 rpm. The cells were harvested by centrifugation (4000 g, 10 min), washed twice with sterile saline solution (NaCl, 9 g/L), resuspended in saline, and inoculated into the fermenters. All chemicals were purchased from Sigma-Aldrich (Europe) unless otherwise stated. Yeast cultivation: The cells from the initial inoculum were added to 1.5 L of growth medium in Minifors 2, Bench-Top bioreactors (INFORS HT, Switzerland, working volume 2 L). The yeast cells were cultivated in the bioreactors as described previously (8). In brief, the prepared yeast inoculum was introduced to nitrogen-limited growth medium within the bioreactors, containing filter-sterilized wheat straw hydrolysate and YNB (yeast nitrogen base without amino acids and ammonium sulphate) 1.7 g/L, (NH 4 ) 2 SO 4 2 g/L, KH 2 PO 4 7 g/L, NaH 2 PO 4 2 g/L, MgSO 4 .7H 2 O 1.5 g/L, and yeast extract 1 g/L. Immediately after inoculation, 100 mL of culture broth was collected. After 96 hours, when all the sugar was consumed, the fermentation was stopped, and the cells were harvested, washed, French-pressed (Constant Systems LTD, Daventry, UK) at 40 kPa and at -5 °C, freeze-dried, and stored at -20 °C until further processing. Extraction methods: 1. Conventional solvent extraction a. Lipid extraction by Folch method Lipids from the yeast cells were extracted using the Folch method (30) with some modifications as explained previously (8). All analyses were performed in triplicates. In brief, freeze-dried cells underwent lipid extraction through sequential treatments involving 1 M HCl, 0.8% KCl, Folch solution (chloroform: methanol, 2:1 v/v ratio), and pure chloroform. The cell material was heated in the HCl at 75 °C for 1 hour to enable a more effective cell disruption (8). The separation was executed using a separatory funnel, with the lower lipid-rich phase collected in a pre-weighed glass tube. Subsequently, the glass tube containing the lipid phase was subjected to nitrogen gas to remove chloroform through evaporation. The resulting tube containing the lipids was then weighed. Finally, the dried lipid samples were resuspended in 1 mL hexane and stored at -20 °C until further processing for methylation. b. Carotenoid extraction using acetone The carotenoid extractions were carried out in dark conditions by using the method described by Reif et al .(31) with some modifications as explained in detail in our previous work (8). The extractions were performed in triplicates. In summary, French-pressed yeast biomass was subjected for the acetone extraction process and at the end of the extraction, the acetone collected in the glass tube was evaporated under nitrogen, leaving behind the carotenoid extract, which was further subjected to saponification using ethanol, butylated hydroxytoluene (BHT, 0.2 mg/mL in methanol), methanol, and methanolic potassium hydroxide (2M KOH in methanol). This saponification was done to remove the lipids from the extracts, as they could interfere with the UHPLC analysis. The final saponified carotenoid extracts were diluted with methanol: acetone (1:1, v/v) before further analysis. 2. Supercritical carbon-dioxide (SC-CO 2 ) extraction All the extractions were carried out in dark conditions using a supercritical extractor (Jasco Supercritical Extractor, SFE 4000 series, Kovalent AB, Italy), with freeze dried biomass of R. toruloides CBS 14 loaded into the extraction vessel (10 mL capacity with possibility to extract 1- 5 g of biomass) mixed with a layer of silica beads (SiLibeads Typ ZSA 2.2-2.5 mm) serving as solvent flow distributors. The extractor was equipped with a high-pressure modifier pump and a backpressure regulator for a stable and trouble-free constant flow of CO 2 . The fractions were collected in the automatic fraction collectors. The entire extraction process was controlled and operated using the software ChromNAV, version 2.3C for precise monitoring and regulation. All the extractions were made in triplicates. a. Lipid extraction The lipids from the freeze-dried biomass were extracted using a similar method as described by Milanesio et al. (32) with some modifications. The freeze-dried yeast cells were mixed with silica beads (yeast cell: beads, 3:2, w/w) and loaded into the extraction vessel. Extraction pressure was 300 bar, at a temperature of 45 °C and with a CO 2 flow rate of 2 mL/min. The total extraction time was 180 min. Subsequently, the residue was subjected for carotenoid extraction. The lipids were collected in pre-weighed brown bottles and then dissolved in 1 mL hexane and stored at −20 °C until further analysis. b. Carotenoid extraction Once the lipid extraction using pure supercritical carbon dioxide (SC-CO 2 ) was completed, the carotenoids were extracted from the biomass extraction using a method similar to that described by Lim et al. (33) with some modifications. Ethanol (99.5% v/v) was used as a co-solvent in the process. The carotenoid extraction process was conducted at a pressure of 300 bar, a temperature of 50 °C, a CO 2 flow rate of 2 mL/min, and a co-solvent flow rate of 0.2 mL/min. The extraction duration was set for a total of 180 minutes. To maintain the integrity of the extracted compounds, the resulting extracts dissolved in ethanol were stored in brown bottles at a temperature of -20°C until further analysis. Analyses: a. Fatty acid analysis The extracted lipid samples were methylated, using the procedure with boron trifluoride reagent described previously by Nagaraj et al .(8). Further, the methylated fatty acids were analyzed in a GC system (CP-3800, CTC Analytics AG, Switzerland) equipped with a split injector and a flame ionization detector and fitted with a 50 m long ´ 22 mm i.d., 0.25 μm film thickness, BPX 70 fused-silica capillary column. The GC was programmed to start at 158 °C, with temperature increasing at a rate of 2 °C min −1 until 220 °C and a final constant time of 13 min at 220 °C. The peaks were identified by comparing their retention times with those of the standard mixture GLC-68D (Nu-Chek Prep, Elysian, MN, USA) and other authentic standards. The response factors were also evaluated by comparison with the GLC-68D standard. All samples were applied in triplicates. b. Carotenoid profile analysis in lipids using HPLC-DAD Pigments were analysed by HPLC. Analysis was carried out with an HP 1100 chromatograph (Agilent) equipped with a DAD. Pigment extracts in hexane (10 µL) were injected onto a LiChrospher® 100 RP-18 (5 µm) column (Merck). The samples were separated by an elution system at a flow rate of 1 mL/min. The elution system was composed of solvents A, acetonitrile/water/ formic acid 86:10:4 (v/v/v), and B, ethyl acetate/formic acid 96:4 (v/v), with a gradient of 100% A at 0 min, 100% B at 20 min, and 100% A at 30 min (34). The carotenoid content was expressed as mg of β-carotene equivalent (β-EQ)/g of dried yeast weight. c. Carotenoid profile in carotenoid extract analysis using UHPLC-PDA Carotenoid composition analysis was performed using a Shimadzu UHPLC-Nexera instrument from Kyoto, Japan. The instrument setup included an autosampler (SIL-20AC), quaternary pumps (LC-20AD), a column oven (CTO-20AC), and a PDA detector (Shimadzu, model SPD-M20A) connected in series. LabSolutions software was employed for instrument control, data acquisition, and data processing. Carotenoid separation was accomplished using an analytical RP C18 Kinetex 100 column (100 mm length, 4.6 mm internal diameter, 2.6 μm particle size; Phenomenex) with a binary gradient system. The mobile phase A consisted of a mixture of acetonitrile and methanol in a ratio of 7:3 (v/v), while the mobile phase B comprised ultrapure water with 0.1% formic acid. The gradient elution program was as follows: 0-3 minutes, 60% B; 3-7 minutes, 100% B; 7-30 minutes, 100% B; and 30-35 minutes, 60% B. The flow rate was set at 0.3 mL/min, the column temperature was maintained at 40°C, and a sample volume of 20 μL was injected. UV-visible spectra were acquired in the range of 250 to 600 nm using the PDA detector. Specific wavelengths were selected for the detection of individual carotenoids after screening of commercial standards (company) for the max absorption: β-carotene and torularhodin were detected at 450 nm, γ-carotene at 462 nm, and torulene at 478 nm (8). The analysis was made in triplicates. Statistical analysis: Experiments were performed in triplicate, and the presented results are mean ± standard deviation. Student’s t-Test was performed to evaluate the significance of differences between the mean values using RStudio (Version 2023.06.1+524) software package. The differences were considered significant at p ≤ 0.05. Results and discussion 1. Lipid extraction Lipid extraction from the yeast samples was performed using the Folch method and SC-CO 2 extraction (Figure 1). The results depicted in the figure indicate that the lipid content in the samples extracted with SC-CO 2 was significantly lower (p=0.01). Similar results have been found by Milanesio et al . (32) when extracting lipids from the yeast Yarrowia lipolytica with different methods. They compared Soxhlet and accelerated solvent extraction (ASE) using a mixture of chloroform and methanol in the same ratio as Folch and SC-CO 2 extraction with and without ethanol. They found significantly lower lipid yield with SC-CO 2 and also showed that pretreatment of the yeast mass with ethanol increased extraction capacity of SC-CO 2 . Another aspect of lipid yield by different extraction methods is the polarity of the used solvents. Duarte et al. (35) highlighted that SC-CO 2 extraction will not yield the certain fractions of lipids, namely the waxes, phospholipids, sterols and pigments and hence result in a lower lipid recovery. In contrast, in Folch method, a mixture of polar and non-polar solvents, improves extraction of polar lipids (36) and thereby enhances the lipid yield as earlier seen in algae (15, 37). This argument is strengthened by the results of Milanesio et al. (32), showing a better extraction with a polar co-solvent added to the non-polar SC-CO 2 . In the present study using ethanol as a co-solvent for lipid extraction was not an option as we wanted to separate the carotenoids from the lipids which would have co-eluted when using ethanol from the start of extraction. 2. Fatty acid analysis The lipid profiles of the two different extracts are presented in Table 1. The analysis showed that oleic acid (C18:1(n-9)) was the predominant fatty acid in the lipid extracts obtained from both extraction methods. Following oleic acid, palmitic acid (C16:0) and linoleic acid (C18:2(n-6)) were found to be the major fatty acids in the yeast lipids. This general composition is in line with our and others' earlier findings (8, 38, 39). Moreover, the presence and amounts of linoleic acid and linolenic acid (C18:3(n-3)) were in line with our earlier results being a significant proportion of polyunsaturated fatty acids (PUFAs) in the yeast lipids. The composition and ratio of saturated to unsaturated fatty acids (SFAs/UFAs) are crucial indicators for assessing the nutritional and functional properties of oils (29). In this study, the SFA/UFA ratios were determined for the extracts obtained through the Folch and SC-CO 2 methods, resulting in ratios of 0.48 and 0.34, respectively. SC-CO 2 extraction method resulted in lipid extracts with a significantly higher content of UFAs, accounting for approximately 70% of the total fatty acids, compared to the Folch method resulting in 62.3%. Table 1 . Quantification of different fatty acids in R. toruloides CBS 14 samples extracted using Folch and SC-CO 2 methods (n=3). Data are presented as mean % ± standard deviation. Asterisks indicate statistically significant differences (p ≤ 0.05). Fatty acid profile (%) of the total fatty acids Folch method SC-CO 2 method C14:0 1.46 ± 0.08 1.18 ± 0.09 C16:0 23.8 ± 0.63 19.2 * ± 0.76 C18:0 4.56 ± 0.29 3.31 * ± 0.22 C24:0 0.38 ± 0.03 0.22 * ± 0.02 C18:1 (n-9) 46.6 ± 1.55 50.9 * ± 1.39 C18:2 (n-6) 14.0 ± 1.49 17.2 * ± 1.33 C18:3 (n-3) 1.69 ± 0.24 1.85 ± 0.34 Total SFA 30.2 ± 0.55 23.9 * ± 0.48 Total UFA 62.3 ± 0.70 70.0 * ± 0.67 SFA/UFA 0.48 0.34 Abbreviations: SFA – saturated fatty acids; UFA – unsaturated fatty acids This difference is most probably connected to the above-described difference in lipid recovery. As mentioned, the polarity of the solvent will affect the composition of extracted lipids. To explore these effects further, there is a need to separate and quantify the lipid classes from the extracts and determine the fatty acid composition of the fractions. Another aspect is the temperature. In the employed Folch method, the cells are treated with HCl at 75°C for 1 hour. This elevated temperature can lead to thermal degradation of the PUFAs. The thermal degradation of PUFAs results in a decrease in their abundance, ultimately affecting the overall composition of the lipid extract (29, 40). In addition, also the carotenoids, which can act as antioxidants and protect the PUFAs from oxidative degradation, are sensitive to heat (41, 42). If they are oxidized during the extraction process their antioxidative capacity, protecting the PUFAs is lost, most probably leading to an increased oxidation of lipids. In contrast, the SC-CO 2 extraction method operates at lower temperatures, which helps to minimize the thermal damage inflicted on UFAs and PUFAs present in the oil. Also, in SC-CO 2 extraction, the process is conducted in the absence of oxygen, which further contributes to the protection of bioactive compounds from oxidation (29, 43). Han et al. (29) achieved comparable outcomes, wherein they conducted a comparative analysis of the lipid composition of Berberis dasystachya Maxim. seed oil extracted by both SC-CO 2 method and the conventional organic solvent method (using petroleum ether extraction). Their findings indicated that the SC-CO 2 approach yielded greater proportions of UFAs and PUFAs when contrasted with the organic solvent method. Findings from Kayathi et al. (44) also confirmed that the lipid extracted from mango kernel using SC-CO 2 was particularly rich in UFAs. 3. Lipid extract analysis for carotenoid profile The extracted lipids using SC-CO 2 were found to contain some carotenoids as well. These carotenoids were profiled using HPLC-DAD, revealing detectable amounts of β- and γ-carotene in the lipid extract (Figure 2). Additionally, trace amounts of 15-cis-β-carotene, torulene and torularhodin were also present. A total of 19.85 ± 4.64 mg β-EQ/g d.w. carotenoid content was identified in the lipid extract. The presence of carotenoids in the lipid extract can be attributed to the non-polar, lipophilic nature of SC-CO 2 , which makes it an effective solvent for extracting low-polarity compounds like β- and γ-carotene and also contrasts with the lower solubility of torulene and torularhodin in SC-CO 2 (45, 46, 47). The use of co-solvents can enhance the solubility of target compounds and improve extraction selectivity (23). As a result, when using pure SC-CO 2 , some amount of β- and γ-carotene are co-extracted along with the lipids. 4. Carotenoid analysis of the carotenoid extract Carotenoids produced in R. toruloides CBS 14 were extracted in the SC-CO 2 using the mixture of carbon-dioxide and ethanol. The inclusion of ethanol to SC-CO 2 has been found to enhance the solvent effectiveness of the supercritical fluid. This leads to the swelling of the sample matrix, causing an increase in internal volume and surface area. It therefore contributes to the decomposition of the cellular wall, potentially improving the bioavailability of carotenoids during the extraction process (41). Additionally, by introducing ethanol, the polarity of CO 2 can be modified, leading to enhanced solubility of carotenoids within the supercritical environment (16, 48, 49). The obtained carotenoid extracts were further analyzed using UHPLC-PDA. The quantities of the four major carotenoids identified are presented in table 2: β-carotene, γ-carotene, torulene, and torularhodin. With the SC-CO 2 extraction torularhodin was found to be the most abundant carotenoid, followed by torulene. Table 2. Quantification of individual carotenoids in R. toruloides CBS 14 extracted using SC-CO 2 and acetone-extraction methods. Data are presented as mean ± standard deviation of carotenoid content in the samples from all three fermenters. Carotenoids Quantity of carotenoids ( m g /g d.w.) SC-CO 2 method Acetone extraction method * β -carotene 25.4 ± 4.41 14.8 ± 0.28 γ -carotene 0.99 ± 0.11 4.20 ± 0.59 Torulene 105 ± 13.15 < LOQ Torularhodin 200 ± 12.94 0.90 ± 0.28 Total carotenoids 332 ± 2 7.32 19.9 ± 2.74 Abbreviations: d.w. – Dry weight; LOQ – limit of quantification; Asterisks indicate data taken from previous work (8) In a previous experiment, using acetone-extraction method, β-carotene was showing the highest proportion in R. toruloides CBS 14 (8). However, we have argued that torulene and torularhodin were not detected most-likely caused by degradation during the saponification step employed (Table 2). The saponification step was crucial for eliminating unwanted lipids from the carotenoid extracts before UHPLC analyses. This resulted in a two-fold reduction of the total carotenoid content of the saponified carotenoid extract when compared to the unsaponified samples (8), strengthening our hypothesis that torulene and torularhodin were degraded, as those carotenoids have been found in R. toruloides earlier (38). In contrast, the SC-CO 2 method involved the extraction of lipids from the sample prior to carotenoid extraction. Consequently, the carotenoid extracts obtained were free from lipids, eliminating the need for saponification. These lipid-free extracts were directly injected into the UHPLC system. Torularhodin and torulene were observed in even higher concentrations than β-carotene, confirming our previous hypothesis that torularhodin and torulene were earlier degraded during saponification. This could also be seen in an increased amount of extracted total carotenoids with SC-CO 2 extraction. In a study conducted by Martinez et al. (16) similar results were obtained, where upon SC-CO 2 extraction of carotenoids from Rhodotorula glutinis with ethanol as the co-solvent, torularhodin was identified as the primary carotenoid. This finding is consistent with other studies that have also confirmed torularhodin as the most abundant carotenoid in various Rhodotorula species, accounting for approximately 60-70% of the total carotenoids extracted (50, 51). Also, Hosseini et al. (52) confirmed enhanced carotenoid recovery from Dunaliella salin a extracts using SC-CO 2 extraction, compared to the conventional extraction method (Soxhlet method). However, there might still be a variation in the content of these carotenoids, depending on strain and substrate as Qi et al. (38) found more than 10 times higher values of both total carotenoids in different strains of R. toruloides , grown on tea waste. Zheng et al. (39) on the other hand found β-carotene as the major carotenoid in different R. toruloides strains grown on cane molasses. Although some carotenoid loss into the lipid extract occurred with SC-CO 2 extraction, it was less substantial compared to the loss observed with acetone extraction. This suggests that SC-CO 2 extraction offers a more effective recovery of carotenoids than acetone extraction. Conclusion Supercritical carbon-dioxide (SC-CO 2 ) extraction emerged as preferable method to extract high amounts of carotenoids and increased proportion of UFA from R. toruloides . Gas chromatography analysis showed that lipids extracted using SC-CO 2 exhibited higher levels of UFA compared to the lipids extracted using the Folch method. SC-CO 2 extraction revealed that torularhodin and torulene were the major carotenoids in R. toruloides CBS14. This finding can be of significance since these carotenoids are of high potential value since they have a higher antioxidant potential than β- and γ-carotene. Up to now, natural sources for their isolation are limited, thus, the established extraction may enable future biotechnological production of these carotenoids The SC-CO 2 extraction method demonstrated the ability to preserve the integrity of unsaturated lipids and abundance of carotenoids, resulting in high-quality extracts. However, it is important to note that despite the lower lipid recovery, the SC-CO 2 extraction method offers the advantage of extraction without any organic solvents, eliminating the need for potentially harmful and environmentally unfriendly solvents. These findings hold great potential for future industrial applications and can serve as a starting point for further research in this area. Abbreviations GC Gas chromatography HPLC High pressure liquid chromatography DAD Diode array detector PDA Photodiode array PUFAs Polyunsaturated fatty acids SFAs Saturated fatty acids UFAs Unsaturated fatty acids SC-CO 2 Supercritical carbon-dioxide SFE Supercritical fluid extraction TAGs Triacylglycerols UHPLC Ultra-high pressure liquid chromatography Declarations Funding The study was financially supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas) (Grant Number 2018-01877) and Nordforsk-SAFE/Swedish Research council for Environment, Agricultural Sciences and Spatial Planning (Formas), (Grant Number 2020-02637). Author information: Authors and Affiliations 1. Department of Molecular Science, Swedish University of Agricultural Sciences, Uppsala BioCentre, P.O. Box 7051, SE-750 07 Uppsala, Sweden. Yashaswini Nagavara Nagaraj (YNN), Johanna Blomqvist (JB), Sabine Sampels (SaS), Jana Pickova (JP), Mats Sandgren (MS) and Volkmar Passoth (VP). 2. Institute of Biotechnology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37, Bratislava, Slovakia. Milan Čertík (MC) and Peter Gajdoš (PG). Authors’ contribution Experimental work, methodology: YNN, JB, SaS and PG; Result evaluation: YNN, JB, MC, SaS, JP, and VP; Conceptualization: SaS, JP, and VP.; Writing - original draft: YNN; Writing review and editing: SaS, JP, JB, MS, VP and MC; Supervision: SaS, JP, JB, MS, VP.; Funding acquisition: MS and VP; Project administration: MS and VP. All authors have read and agreed to the published version of the manuscript. Corresponding author Correspondence to Volkmar Passoth. 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Biochemical Engineering Journal. 2002;11(2-3):181-7. Klempova T, Basil E, Kubatova A, Certik M. Biosynthesis of gamma‐linolenic acid and beta‐carotene by Zygomycetes fungi. Biotechnology Journal. 2013;8(7):794-800. Duarte SH, dos Santos P, Michelon M, de Pinho Oliveira SM, Martínez J, Maugeri F. Recovery of yeast lipids using different cell disruption techniques and supercritical CO 2 extraction. Biochemical engineering journal. 2017;125:230-7. Dong T, Knoshaug EP, Pienkos PT, Laurens LM. Lipid recovery from wet oleaginous microbial biomass for biofuel production: a critical review. Applied Energy. 2016;177:879-95. Kim D-Y, Vijayan D, Praveenkumar R, Han J-I, Lee K, Park J-Y, et al . Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus . Bioresource technology. 2016;199:300-10. Qi F, Shen P, Hu R, Xue T, Jiang X, Qin L, et al . Carotenoids and lipid production from Rhodosporidium toruloides cultured in tea waste hydrolysate. Biotechnology for biofuels. 2020;13(1):1-12. Zheng X, Hu R, Chen D, Chen J, He W, Huang L, et al . Lipid and carotenoid production by the Rhodosporidium toruloides mutant in cane molasses. Bioresource Technology. 2021;326:124816. Hadaruga D, Ünlüsayin M, Gruia A, Rusu G, Hadaruga N. Thermal and oxidative stability of Atlantic salmon oil ( Salmo salar L.) and complexation with beta-cyclodextrin. Beilstein Journal of Organic Chemistry. 2016;12. Saini RK, Keum Y-S. Carotenoid extraction methods: A review of recent developments. Food chemistry. 2018;240:90-103. Šovljanski O, Saveljić A, Tomić A, Šeregelj V, Lončar B, Cvetković D, et al . Carotenoid-Producing Yeasts: Selection of the Best-Performing Strain and the Total Carotenoid Extraction Procedure. Processes. 2022;10(9):1699. Temelli F. Perspectives on supercritical fluid processing of fats and oils. The Journal of Supercritical Fluids. 2009;47(3):583-90. Kayathi A, Chakrabarti PP, Bonfim-Rocha L, Cardozo-Filho L, Jegatheesan V. Selective extraction of polar lipids of mango kernel using supercritical carbon dioxide (SC–CO 2 ) extraction: Process optimization of extract yield/phosphorous content and economic evaluation. Chemosphere. 2020;260:127639. Ambrico A, Larocca V, Trupo M, Martino M, Magarelli RA, Spagnoletta A, et al . A New Method for Selective Extraction of Torularhodin from Red Yeast Using CO 2 -SFE Technique. Applied Biochemistry and Biotechnology. 2024. Vafaei N, Rempel CB, Scanlon MG, Jones PJ, Eskin MN. Application of supercritical fluid extraction (SFE) of tocopherols and carotenoids (hydrophobic antioxidants) compared to non-SFE methods. AppliedChem. 2022;2(2):68-92. Friedrich J, List G, Heakin A. Petroleum-free extraction of oil from soybeans with supercritical CO 2 . Journal of the American Oil Chemists’ Society. 1982;59(7):288-92. Sánchez-Camargo AP, Meireles MÂA, Lopes BLF, Cabral FA. Proximate composition and extraction of carotenoids and lipids from Brazilian redspotted shrimp waste ( Farfantepenaeus paulensis ). Journal of Food Engineering. 2011;102(1):87-93. Takahashi M, Watanabe H, Kikkawa J, Ota M, Watanabe M, Sato Y, et al . Carotenoids extraction from Japanese persimmon (Hachiya-kaki) peels by supercritical CO 2 with ethanol. Analytical Sciences. 2006;22(11):1441-7. Mihalcea A, Onu A, Tucureanu C, Ungureanu C, Raileanu S, Salageanu A, et al . Extraction of torularhodin from Rhodotorula rubra yeast using sunflower oil. studies. 2015;1:16. Park P, Kim E, Chu K. Chemical disruption of yeast cells for the isolation of carotenoid pigments. Separation and Purification Technology. 2007;53(2):148-52. Hosseini SRP, Tavakoli O, Sarrafzadeh MH. Experimental optimization of SC-CO 2 extraction of carotenoids from Dunaliella salina . The Journal of Supercritical Fluids. 2017;121:89-95. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5460903","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":390245940,"identity":"bb17d986-614d-4d54-824b-3f022c8477b8","order_by":0,"name":"Yashaswini Nagavara Nagaraj","email":"","orcid":"","institution":"Swedish University of Agricultural Sciences, Uppsala BioCentre","correspondingAuthor":false,"prefix":"","firstName":"Yashaswini","middleName":"Nagavara","lastName":"Nagaraj","suffix":""},{"id":390245941,"identity":"09a439be-142d-4574-9576-b16b8197e2f1","order_by":1,"name":"Johanna Blomqvist","email":"","orcid":"","institution":"Swedish University of Agricultural Sciences, Uppsala BioCentre","correspondingAuthor":false,"prefix":"","firstName":"Johanna","middleName":"","lastName":"Blomqvist","suffix":""},{"id":390245942,"identity":"005e6ddd-86d6-4f4b-8773-ee20a5eaa465","order_by":2,"name":"Sabine Sampels","email":"","orcid":"","institution":"Swedish University of Agricultural Sciences, Uppsala BioCentre","correspondingAuthor":false,"prefix":"","firstName":"Sabine","middleName":"","lastName":"Sampels","suffix":""},{"id":390245943,"identity":"9b9b4e3d-65f8-4ce0-8de3-601038c7d7a4","order_by":3,"name":"Jana Pickova","email":"","orcid":"","institution":"Swedish University of Agricultural Sciences, Uppsala BioCentre","correspondingAuthor":false,"prefix":"","firstName":"Jana","middleName":"","lastName":"Pickova","suffix":""},{"id":390245944,"identity":"d6621bbe-96f5-4d9c-aeb3-ea5009f2d4c4","order_by":4,"name":"Mats Sandgren","email":"","orcid":"","institution":"Swedish University of Agricultural Sciences, Uppsala BioCentre","correspondingAuthor":false,"prefix":"","firstName":"Mats","middleName":"","lastName":"Sandgren","suffix":""},{"id":390245945,"identity":"e30d26ea-e968-49c5-9a2a-1e4c4c592d8b","order_by":5,"name":"Peter Gajdoš","email":"","orcid":"","institution":"Slovak University of Technology in Bratislava","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Gajdoš","suffix":""},{"id":390245946,"identity":"c4f5e47e-4457-4066-accb-eaec084048d7","order_by":6,"name":"Milan Čertík","email":"","orcid":"","institution":"Slovak University of Technology in Bratislava","correspondingAuthor":false,"prefix":"","firstName":"Milan","middleName":"","lastName":"Čertík","suffix":""},{"id":390245947,"identity":"eed635a2-1b14-41c9-af5a-7b6c974c2946","order_by":7,"name":"Volkmar Passoth","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYFAC5mZk3gE5BnaCWhiRtSQcMGZgJlVLYgMhLQbHG5uNbtTcYzBnP/74w8cfd9LnN/MYMP6owKPlzMHm5JxjxQyWPTlmkjMSnuVuOMxjwMxzBrcWyRmJzYdz2BIYDA7ksDHzJBzO3cAM1MLYRkjLP6CW888ffwZqSZcHOeznP9xa+CUSm5Nz24BabiQYSAO1JDAAHcbA24BHC8/BZuPcvgQeyxlvgH5JO2y44TBbwWGeY7i1sLE3H5bO+ZYgZ86f/vjDB5vD8vLtzRsf/qjBrQUGgI5BAgcIawACA8JKRsEoGAWjYKQCANJLUMa1q0UKAAAAAElFTkSuQmCC","orcid":"","institution":"Swedish University of Agricultural Sciences, Uppsala BioCentre","correspondingAuthor":true,"prefix":"","firstName":"Volkmar","middleName":"","lastName":"Passoth","suffix":""}],"badges":[],"createdAt":"2024-11-15 13:38:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5460903/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5460903/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13068-025-02632-7","type":"published","date":"2025-03-21T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71481234,"identity":"f273ab65-651a-414f-bfad-2dd078190447","added_by":"auto","created_at":"2024-12-16 06:01:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18137,"visible":true,"origin":"","legend":"\u003cp\u003eAverage lipid content in dry cells of \u003cem\u003eR. toruloides\u003c/em\u003e CBS 14 after extraction by Folch and SC-CO\u003csub\u003e2\u003c/sub\u003e methods (n=3). The difference in the extracted amount of lipid was significant (p=0.01).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5460903/v1/93eaf1e40f50892d29124fc5.png"},{"id":71481232,"identity":"cee1e2b8-a845-410a-8b62-747c290e4253","added_by":"auto","created_at":"2024-12-16 06:01:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29988,"visible":true,"origin":"","legend":"\u003cp\u003eIndividual carotenoid concentration in the lipid extract of \u003cem\u003eR. toruloides\u003c/em\u003e CBS 14 as analysed by HPLC-DAD.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5460903/v1/db9bcc2044504a67c0d84aee.png"},{"id":79120615,"identity":"db2d8a8e-9f4e-4db2-bdb9-34fc06a6e239","added_by":"auto","created_at":"2025-03-24 16:10:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":901566,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5460903/v1/65cf79c6-acd1-41d6-98c8-269ab440a9b4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Supercritical carbon-dioxide (SC-CO2) extraction of lipids and carotenoids from Rhodotorula toruloides CBS 14 in comparison with conventional extraction methods","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, the consumption of fats and oils as food ingredients has significantly increased, constituting more than 80% of their overall usage (1). Lipids play a vital role in cooking, baking, and food preparation. Additionally, they find application as animal feeds, providing improved nutritional value and energy density to livestock diets (2). As the demand continues to rise, alternative sources for plant oils are being explored. One focus has been targeted on microbial oil (MO) as sustainable fat source. MO is synthesized and accumulated by specific microorganisms, known as oleaginous microorganisms, through cultivation processes that utilize renewable resources as substrates (3). Lignocellulosic biomass presents a promising substrate due to its renewability and abundant nature, featuring substantial quantities of readily usable sugars (4, 5). Many oleaginous yeasts possess the capability to convert sugars, polysaccharides, glycerol, and other compounds into oil (3). The major constituents of the produced lipids are primarily neutral triacylglycerols (TAGs), which contain energy-rich fatty acids resembling the fatty acid composition of vegetable oil and, therefore have the potential to be utilized in the production of food, feed, biofuels, and various biochemicals (3, 6). Some oleaginous yeasts can also produce carotenoids as secondary metabolites (7, 8, 9). Carotenoids are used as natural colorants and antioxidants in the food industry to enhance the visual appeal of products (10). They are also employed as dietary supplements, either in their natural form or as purified extracts, due to their antioxidant and health-promoting properties (11).\u003c/p\u003e \u003cp\u003eTo fully utilize the biotechnological potential of oleaginous yeasts for the production of lipids and carotenoids, it is crucial to develop effective extraction processes. This is because the lipids and carotenoids produced by these yeast cells are sensitive to peroxidation which can be induced by for example heat and light (12). Therefore, cautious extraction methods need to be adopted to ensure efficient recovery of these valuable compounds preserving their quality and bioactivity to ensure the full benefit of oleaginous yeasts for the production of high-value lipids and carotenoids (13, 14).\u003c/p\u003e \u003cp\u003eTraditionally, the lipids and carotenoids have been extracted from oleaginous yeasts using organic solvents. However, this conventional method has limitations due to the significant use of harmful solvents, multiple extraction steps, and reduced effectiveness caused by the barriers presented by cell envelopes (15, 16, 17). To overcome these challenges, various physical techniques such as heat treatment, ultrasound, high-pressure homogenization, bead-milling, and vigorous shaking have been employed before or during extraction (18). These procedures aim to disrupt the cell envelopes, allowing solvents to penetrate the cells and solubilize the lipids and carotenoids, thereby improving the extraction yield (16, 19). These physical approaches enhance the accessibility of solvents to intracellular components, leading to more efficient extraction processes with reduced solvent consumption and increased recovery of lipids and carotenoids (18).\u003c/p\u003e \u003cp\u003eSupercritical carbon dioxide (SC-CO\u003csub\u003e2\u003c/sub\u003e) extraction has emerged as a gentle technique for extracting natural substances aimed at applications in the food and pharmaceutical sector (20). Supercritical fluids, are characterized by operating above the critical temperature and pressure of the compound, resulting in specific properties (21). In the supercritical state, carbon dioxide exhibits unique properties including high compressibility, liquid-like density, enhanced diffusivity, low viscosity, and low surface tension, similar to an organic solvent.\u003c/p\u003e \u003cp\u003eThe properties of supercritical fluids, particularly SC-CO\u003csub\u003e2\u003c/sub\u003e, play a significant role in their ability to permeate and extract compounds from matrices compared to conventional organic solvents (16). Researchers have indicated that, compared to conventional organic solvents, SC-CO\u003csub\u003e2\u003c/sub\u003e exhibits higher diffusivity and lower density, viscosity, and surface tension. Additionally, these properties can be widely adjusted by altering the operational conditions (22, 23). These characteristics enable SC-CO\u003csub\u003e2\u003c/sub\u003e to penetrate both micro and macro porous components, allowing for selective extraction, fractionation, and purification of target compounds (22). SC-CO\u003csub\u003e2\u003c/sub\u003e extraction offers several advantages over traditional methods. Firstly, it is non-toxic and eliminates the need for potentially harmful organic solvents, making it a safer option for both operators and consumers (24, 25). This quality renders it suitable for food and feed-related applications. It also preserves the integrity of the extracted compounds, as it operates under milder conditions, minimizing thermal degradation (26, 27, 28). Researchers have focused on optimizing the extraction process by adjusting key parameters such as temperature, pressure, extraction time, and CO\u003csub\u003e2\u003c/sub\u003e flow rate. By fine-tuning these variables, optimal extraction conditions can be achieved, ensuring maximum yield and quality of the extracted natural products (29).\u003c/p\u003e \u003cp\u003eThe objective of this study was to investigate the potential of supercritical carbon dioxide (SC-CO\u003csub\u003e2\u003c/sub\u003e) extraction to obtain lipids and carotenoids from an oleaginous red yeast, cultivated on lignocellulose hydrolysate.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRhodotorula toruloides\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;cultivation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInoculum preparation:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eR. toruloides\u003c/em\u003e CBS 14 was obtained from the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands. Cells were stored in frozen stocks at −80\u0026nbsp;°C. The inoculum was prepared as described before by Nagaraj\u0026nbsp;\u003cem\u003eet al.\u003c/em\u003e(8). Briefly, cells from YPD-agar plates (glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L, agar 15 g/L) were inoculated into 300 mL of YPD (glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L) in a 3 L Erlenmeyer flask and incubated at 25\u0026nbsp;°C for 48–72 h at 150 rpm. The cells were harvested by centrifugation (4000 g, 10 min), washed twice with sterile saline solution (NaCl, 9 g/L), resuspended in saline, and inoculated into the fermenters.\u0026nbsp;All chemicals were purchased from Sigma-Aldrich (Europe) unless otherwise stated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast cultivation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells from the initial inoculum were added to 1.5 L of growth medium in Minifors 2, Bench-Top bioreactors (INFORS HT, Switzerland, working volume 2 L). The yeast cells were cultivated in the bioreactors as described previously (8). In brief, the prepared yeast inoculum was introduced to nitrogen-limited growth medium within the bioreactors, containing filter-sterilized wheat straw hydrolysate and YNB (yeast nitrogen base without amino acids and ammonium sulphate) 1.7 g/L, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 2 g/L, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 7 g/L, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 2 g/L, MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO 1.5 g/L, and yeast extract 1 g/L. Immediately after inoculation, 100 mL of culture broth was collected. After 96 hours, when all the sugar was consumed, the fermentation was stopped, and the cells were harvested, washed, French-pressed (Constant Systems LTD, Daventry, UK) at 40 kPa and at -5 °C, freeze-dried, and stored at -20 °C until further processing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction methods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e1.\u0026nbsp; \u0026nbsp;\u003c/em\u003e\u003cem\u003eConventional solvent extraction\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea.\u0026nbsp; \u0026nbsp;Lipid extraction by Folch method\u003c/p\u003e\n\u003cp\u003eLipids from the yeast cells were extracted using the Folch method (30) with some modifications as explained previously (8). All analyses were performed in triplicates. In brief, freeze-dried cells underwent lipid extraction through sequential treatments involving 1 M HCl, 0.8% KCl, Folch solution (chloroform: methanol, 2:1 v/v ratio), and pure chloroform. The cell material was heated in the HCl at 75 °C for 1 hour to enable a more effective cell disruption (8). The separation was executed using a separatory funnel, with the lower lipid-rich phase collected in a pre-weighed glass tube. Subsequently, the glass tube containing the lipid phase was subjected to nitrogen gas to remove chloroform through evaporation. The resulting tube containing the lipids was then weighed. Finally, the dried lipid samples were resuspended in 1 mL hexane and stored at -20 °C until further processing for methylation.\u003c/p\u003e\n\u003cp\u003eb.\u0026nbsp; \u0026nbsp;Carotenoid extraction using acetone\u003c/p\u003e\n\u003cp\u003eThe carotenoid extractions were carried out in dark conditions by using the method described by Reif \u003cem\u003eet al\u003c/em\u003e.(31) with some modifications as explained in detail in our previous work (8). The extractions were performed in triplicates. In summary, French-pressed yeast biomass was subjected for the acetone extraction process and at the end of the extraction, the acetone collected in the glass tube was evaporated under nitrogen, leaving behind the carotenoid extract, which was further subjected to saponification using ethanol, butylated hydroxytoluene (BHT, 0.2 mg/mL in methanol), methanol, and methanolic potassium hydroxide (2M KOH in methanol). This saponification was done to remove the lipids from the extracts, as they could interfere with the UHPLC analysis. The final saponified carotenoid extracts were diluted with methanol: acetone (1:1, v/v) before further analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.\u0026nbsp; \u0026nbsp;Supercritical carbon-dioxide (SC-CO\u003csub\u003e2\u003c/sub\u003e) extraction\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll the extractions were carried out in dark conditions using a supercritical extractor (Jasco Supercritical Extractor, SFE 4000 series, Kovalent AB, Italy), with freeze dried biomass of\u0026nbsp;\u003cem\u003eR. toruloides\u003c/em\u003e CBS 14 loaded into the extraction vessel (10 mL capacity with possibility to extract 1- 5 g of biomass) mixed with a layer of silica beads (SiLibeads Typ ZSA 2.2-2.5 mm) serving as solvent flow distributors. The extractor was equipped with a high-pressure modifier pump and a backpressure regulator for a stable and trouble-free constant flow of CO\u003csub\u003e2\u003c/sub\u003e. The fractions were collected in the automatic fraction collectors. The entire extraction process was controlled and operated using the software ChromNAV, version 2.3C for precise monitoring and regulation. All the extractions were made in triplicates.\u003c/p\u003e\n\u003cp\u003ea.\u0026nbsp; \u0026nbsp;Lipid extraction\u003c/p\u003e\n\u003cp\u003eThe lipids from the freeze-dried biomass were extracted using a similar method as described by Milanesio \u003cem\u003eet al.\u003c/em\u003e(32) with some modifications. The freeze-dried yeast cells were mixed with silica beads (yeast cell: beads, 3:2, w/w) and loaded into the extraction vessel. Extraction pressure was 300 bar, at a temperature of 45\u0026nbsp;°C and with a CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eflow rate of 2 mL/min. The total extraction time was 180 min. Subsequently, the residue was subjected for carotenoid extraction. The lipids were collected in pre-weighed brown bottles and then dissolved in 1 mL hexane and stored at −20\u0026nbsp;°C until further analysis.\u003c/p\u003e\n\u003cp\u003eb.\u0026nbsp; \u0026nbsp;Carotenoid extraction\u003c/p\u003e\n\u003cp\u003eOnce the lipid extraction using pure supercritical carbon dioxide (SC-CO\u003csub\u003e2\u003c/sub\u003e) was completed, the carotenoids were extracted from the biomass extraction using a method similar to that described by Lim \u003cem\u003eet al.\u003c/em\u003e(33) with some modifications. Ethanol (99.5% v/v) was used as a co-solvent in the process. The carotenoid extraction process was conducted at a pressure of 300 bar, a temperature of 50 °C, a CO\u003csub\u003e2\u003c/sub\u003e flow rate of 2 mL/min, and a co-solvent flow rate of 0.2 mL/min. The extraction duration was set for a total of 180 minutes. To maintain the integrity of the extracted compounds, the resulting extracts dissolved in ethanol were stored in brown bottles at a temperature of -20°C until further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalyses:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea.\u0026nbsp; \u0026nbsp;Fatty acid analysis\u003c/p\u003e\n\u003cp\u003eThe extracted lipid samples were methylated, using the procedure with boron trifluoride reagent described previously by Nagaraj\u0026nbsp;\u003cem\u003eet al\u003c/em\u003e.(8). Further, the methylated fatty acids were analyzed in a GC system (CP-3800, CTC Analytics AG, Switzerland) equipped with a split injector and a flame ionization detector and fitted with a 50 m long\u0026nbsp;´\u0026nbsp;22 mm i.d., 0.25 μm film thickness, BPX 70 fused-silica capillary column. The GC was programmed to start at 158\u0026nbsp;°C, with temperature increasing at a rate of 2\u0026nbsp;°C min\u003csup\u003e−1\u003c/sup\u003e until 220\u0026nbsp;°C and a final constant time of 13 min at 220\u0026nbsp;°C. The peaks were identified by comparing their retention times with those of the standard mixture GLC-68D (Nu-Chek Prep, Elysian, MN, USA) and other authentic standards. The response factors were also evaluated by comparison with the GLC-68D standard. All samples were applied in triplicates.\u003c/p\u003e\n\u003cp\u003eb.\u0026nbsp; \u0026nbsp;Carotenoid profile analysis in lipids using HPLC-DAD\u003c/p\u003e\n\u003cp\u003ePigments were analysed by HPLC. Analysis was carried out with an HP 1100 chromatograph (Agilent) equipped with a DAD. Pigment extracts in hexane (10 µL) were injected onto a LiChrospher® 100 RP-18 (5 µm) column (Merck). The samples were separated by an elution system at a flow rate of 1 mL/min. The elution system was composed of solvents A, acetonitrile/water/ formic acid 86:10:4 (v/v/v), and B, ethyl acetate/formic acid 96:4 (v/v), with a gradient of 100% A at 0 min, 100% B at 20 min, and 100% A at 30 min (34). The carotenoid content was expressed as\u0026nbsp;mg of β-carotene equivalent (β-EQ)/g of dried yeast weight.\u003c/p\u003e\n\u003cp\u003ec.\u0026nbsp; \u0026nbsp;Carotenoid profile in carotenoid extract analysis using UHPLC-PDA\u003c/p\u003e\n\u003cp\u003eCarotenoid composition analysis was performed using a Shimadzu UHPLC-Nexera instrument from Kyoto, Japan. The instrument setup included an autosampler (SIL-20AC), quaternary pumps (LC-20AD), a column oven (CTO-20AC), and a PDA detector (Shimadzu, model SPD-M20A) connected in series. LabSolutions software was employed for instrument control, data acquisition, and data processing. Carotenoid separation was accomplished using an analytical RP C18 Kinetex 100 column (100 mm length, 4.6 mm internal diameter, 2.6 μm particle size; Phenomenex) with a binary gradient system. The mobile phase A consisted of a mixture of acetonitrile and methanol in a ratio of 7:3 (v/v), while the mobile phase B comprised ultrapure water with 0.1% formic acid. The gradient elution program was as follows: 0-3 minutes, 60% B; 3-7 minutes, 100% B; 7-30 minutes, 100% B; and 30-35 minutes, 60% B. The flow rate was set at 0.3 mL/min, the column temperature was maintained at 40°C, and a sample volume of 20 μL was injected. UV-visible spectra were acquired in the range of 250 to 600 nm using the PDA detector. Specific wavelengths were selected for the detection of individual carotenoids after screening of commercial standards (company) for the max absorption: β-carotene and torularhodin were detected at 450 nm, γ-carotene at 462 nm, and torulene at 478 nm (8). The analysis was made in triplicates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were performed in triplicate, and the presented results are mean ± standard deviation. Student’s t-Test was performed to evaluate the significance of differences between the mean values using RStudio (Version 2023.06.1+524) software package. The differences were considered significant at p ≤ 0.05.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cem\u003e1.\u0026nbsp; \u0026nbsp;Lipid extraction\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLipid extraction from the yeast samples was performed using the Folch method and SC-CO\u003csub\u003e2\u003c/sub\u003e extraction (Figure 1). The results depicted in the figure indicate that the lipid content in the samples extracted with SC-CO\u003csub\u003e2\u003c/sub\u003e was significantly lower (p=0.01). Similar results have been found by Milanesio \u003cem\u003eet al\u003c/em\u003e. (32) when extracting lipids from the yeast \u003cem\u003eYarrowia lipolytica\u003c/em\u003e with different methods. They compared Soxhlet and accelerated solvent extraction (ASE) using a mixture of chloroform and methanol in the same ratio as Folch and SC-CO\u003csub\u003e2\u003c/sub\u003e extraction with and without ethanol. They found significantly lower lipid yield with SC-CO\u003csub\u003e2\u003c/sub\u003e and also showed that pretreatment of the yeast mass with ethanol increased extraction capacity of SC-CO\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother aspect of lipid yield by different extraction methods is the polarity of the used solvents. Duarte \u003cem\u003eet al.\u003c/em\u003e(35)\u0026nbsp;highlighted that SC-CO\u003csub\u003e2\u003c/sub\u003e extraction will not yield the certain fractions of lipids, namely the waxes, phospholipids, sterols and pigments and hence result in a lower lipid recovery. In contrast, in Folch method, a mixture of polar and non-polar solvents, improves extraction of polar lipids\u0026nbsp;(36)\u0026nbsp;and thereby enhances the lipid yield\u0026nbsp;as earlier seen in algae\u0026nbsp;(15, 37). \u0026nbsp;This argument is strengthened by the results of Milanesio \u003cem\u003eet al.\u003c/em\u003e (32), showing a better extraction with a polar co-solvent added to the non-polar SC-CO\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study using ethanol as a co-solvent for lipid extraction was not an option as we wanted to separate the carotenoids from the lipids which would have co-eluted when using ethanol from the start of extraction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2. \u0026nbsp; Fatty acid analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe lipid profiles of the two different extracts are presented in Table 1. The analysis showed that oleic acid (C18:1(n-9)) was the predominant fatty acid in the lipid extracts obtained from both extraction methods. Following oleic acid, palmitic acid (C16:0) and linoleic acid (C18:2(n-6)) were found to be the major fatty acids in the yeast lipids. This general composition is in line with our and others\u0026apos; earlier findings (8, 38, 39). Moreover, the presence and amounts of linoleic acid and linolenic acid (C18:3(n-3)) were in line with our earlier results being a significant proportion of polyunsaturated fatty acids (PUFAs) in the yeast lipids. The composition and ratio of saturated to unsaturated fatty acids (SFAs/UFAs) are crucial indicators for assessing the nutritional and functional properties of oils (29). In this study, the SFA/UFA ratios were determined for the extracts obtained through the Folch and SC-CO\u003csub\u003e2\u003c/sub\u003e methods, resulting in ratios of 0.48 and 0.34, respectively. SC-CO\u003csub\u003e2\u003c/sub\u003e extraction method resulted in lipid extracts with a significantly higher content of UFAs, accounting for approximately 70% of the total fatty acids, compared to the Folch method resulting in 62.3%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Quantification of different fatty acids in \u003cem\u003eR. toruloides\u003c/em\u003e CBS 14 samples extracted using Folch and SC-CO\u003csub\u003e2\u003c/sub\u003e methods (n=3). Data are presented as mean % \u0026plusmn; standard deviation. Asterisks indicate statistically significant differences (p \u0026le; 0.05).\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"654\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 450px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFatty acid profile (%) of the total fatty acids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFolch method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSC-CO\u003csub\u003e2\u003c/sub\u003e method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC14:0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e1.46 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e1.18 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC16:0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e23.8 \u0026plusmn; 0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e19.2\u003csup\u003e*\u003c/sup\u003e \u0026plusmn; 0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC18:0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e4.56 \u0026plusmn; 0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e3.31\u003csup\u003e*\u003c/sup\u003e \u0026plusmn; 0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC24:0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e0.38 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e0.22\u003csup\u003e*\u003c/sup\u003e \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC18:1 (n-9)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e46.6 \u0026plusmn; 1.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e50.9\u003csup\u003e*\u003c/sup\u003e \u0026plusmn; 1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC18:2 (n-6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e14.0 \u0026plusmn; 1.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e17.2\u003csup\u003e*\u003c/sup\u003e \u0026plusmn; 1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eC18:3 (n-3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e1.69 \u0026plusmn; 0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e1.85 \u0026plusmn; 0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal SFA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.2 \u0026plusmn; 0.55\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e23.9\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e*\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u0026plusmn; 0.48\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal UFA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e62.3 \u0026plusmn; 0.70\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e70.0\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e*\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u0026plusmn; 0.67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 204px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSFA/UFA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 210px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.48\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 240px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eAbbreviations: SFA \u0026ndash; saturated fatty acids; UFA \u0026ndash; unsaturated fatty acids\u003c/p\u003e\n\u003cp\u003eThis difference is most probably connected to the above-described difference in lipid recovery. As mentioned, the polarity of the solvent will affect the composition of extracted lipids. To explore these effects further, there is a need to separate and quantify the lipid classes from the extracts and determine the fatty acid composition of the fractions.\u003c/p\u003e\n\u003cp\u003eAnother aspect is the temperature. In the employed Folch method, the cells are treated with HCl at 75\u0026deg;C for 1 hour. This elevated temperature can lead to thermal degradation of the PUFAs. The thermal degradation of PUFAs results in a decrease in their abundance, ultimately affecting the overall composition of the lipid extract (29, 40). In addition, also the carotenoids, which can act as antioxidants and protect the PUFAs from oxidative degradation, are sensitive to heat (41, 42). If they are oxidized during the extraction process their antioxidative capacity, protecting the PUFAs is lost, most probably leading to an increased oxidation of lipids. In contrast, the SC-CO\u003csub\u003e2\u003c/sub\u003e extraction method operates at lower temperatures, which helps to minimize the thermal damage inflicted on UFAs and PUFAs present in the oil. Also, in SC-CO\u003csub\u003e2\u003c/sub\u003e extraction, the process is conducted in the absence of oxygen, which further contributes to the protection of bioactive compounds from oxidation (29, 43). Han \u003cem\u003eet al.\u003c/em\u003e(29) achieved comparable outcomes, wherein they conducted a comparative analysis of the lipid composition of \u003cem\u003eBerberis dasystachya\u003c/em\u003e Maxim. seed oil extracted by both SC-CO\u003csub\u003e2\u003c/sub\u003e method and the conventional organic solvent method (using petroleum ether extraction). Their findings indicated that the SC-CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eapproach yielded greater proportions of UFAs and PUFAs when contrasted with the organic solvent method. Findings from Kayathi \u003cem\u003eet al.\u003c/em\u003e(44) also confirmed that the lipid extracted from mango kernel using SC-CO\u003csub\u003e2\u003c/sub\u003e was particularly rich in UFAs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3. \u0026nbsp; Lipid extract analysis for carotenoid profile\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe extracted lipids using SC-CO\u003csub\u003e2\u003c/sub\u003e were found to contain some carotenoids as well. These carotenoids were profiled using HPLC-DAD, revealing detectable amounts of \u0026beta;- and \u0026gamma;-carotene in the lipid extract (Figure 2). Additionally, trace amounts of 15-cis-\u0026beta;-carotene, torulene and torularhodin were also present. A total of 19.85 \u0026plusmn; 4.64 mg \u0026beta;-EQ/g d.w. carotenoid content was identified in the lipid extract. The presence of carotenoids in the lipid extract can be attributed to the non-polar, lipophilic nature of SC-CO\u003csub\u003e2\u003c/sub\u003e, which makes it an effective solvent for extracting low-polarity compounds like \u0026beta;- and \u0026gamma;-carotene and also contrasts with the lower solubility of torulene and torularhodin in SC-CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(45, 46, 47). The use of co-solvents can enhance the solubility of target compounds and improve extraction selectivity (23). As a result, when using pure SC-CO\u003csub\u003e2\u003c/sub\u003e, some amount of \u0026beta;- and \u0026gamma;-carotene are co-extracted along with the lipids.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4. \u0026nbsp; Carotenoid analysis of the carotenoid extract\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCarotenoids produced in \u003cem\u003eR. toruloides\u003c/em\u003e CBS 14 were extracted in the SC-CO\u003csub\u003e2\u003c/sub\u003e using the mixture of carbon-dioxide and ethanol. The inclusion of ethanol to SC-CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ehas been found to enhance the solvent effectiveness of the supercritical fluid. This leads to the swelling of the sample matrix, causing an increase in internal volume and surface area. It therefore contributes to the decomposition of the cellular wall, potentially improving the bioavailability of carotenoids during the extraction process (41). Additionally, by introducing ethanol, the polarity of CO\u003csub\u003e2\u003c/sub\u003e can be modified, leading to enhanced solubility of carotenoids within the supercritical environment (16, 48, 49). The obtained carotenoid extracts were further analyzed using UHPLC-PDA. The quantities of the four major carotenoids identified are presented in table 2: \u0026beta;-carotene, \u0026gamma;-carotene, torulene, and torularhodin. With the SC-CO\u003csub\u003e2\u003c/sub\u003e extraction torularhodin was found to be the most abundant carotenoid, followed by torulene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Quantification of individual carotenoids in \u003cem\u003eR. toruloides\u003c/em\u003e CBS 14 extracted using SC-CO\u003csub\u003e2\u003c/sub\u003e and acetone-extraction methods. Data are presented as mean \u0026plusmn; standard deviation of carotenoid content in the samples from all three fermenters.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCarotenoids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 412px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eQuantity of carotenoids\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003e\u003cstrong\u003em\u003c/strong\u003e\u003cstrong\u003eg\u003c/strong\u003e\u003cstrong\u003e/g d.w.)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSC-CO\u003csub\u003e2\u003c/sub\u003e method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcetone extraction method\u003csup\u003e*\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003cstrong\u003e-carotene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e25.4 \u0026plusmn; 4.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e14.8 \u0026plusmn; 0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026gamma;\u003c/strong\u003e\u003cstrong\u003e-carotene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e0.99 \u0026plusmn; 0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e4.20 \u0026plusmn; 0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTorulene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e105 \u0026plusmn; 13.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e\u0026lt; LOQ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTorularhodin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e200 \u0026plusmn; 12.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e0.90 \u0026plusmn; 0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal carotenoids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e332 \u0026plusmn; 2\u003c/strong\u003e\u003cstrong\u003e7.32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e19.9 \u0026plusmn; 2.74\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eAbbreviations: d.w. \u0026ndash; Dry weight; LOQ \u0026ndash; limit of quantification; Asterisks indicate data taken from previous work\u003cem\u003e\u0026nbsp;\u003c/em\u003e(8)\u003c/p\u003e\n\u003cp\u003eIn a previous experiment, using acetone-extraction method, \u0026beta;-carotene was showing the highest proportion in \u003cem\u003eR. toruloides\u003c/em\u003e CBS 14 (8). However, we have argued that torulene and torularhodin were not detected most-likely caused by degradation during the saponification step employed (Table 2). The saponification step was crucial for eliminating unwanted lipids from the carotenoid extracts before UHPLC analyses. This resulted in a two-fold reduction of the total carotenoid content of the saponified carotenoid extract when compared to the unsaponified samples (8), strengthening our hypothesis that torulene and torularhodin were degraded, as those carotenoids have been found in \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003eearlier (38).\u003c/p\u003e\n\u003cp\u003eIn contrast, the SC-CO\u003csub\u003e2\u003c/sub\u003e method involved the extraction of lipids from the sample prior to carotenoid extraction. Consequently, the carotenoid extracts obtained were free from lipids, eliminating the need for saponification. These lipid-free extracts were directly injected into the UHPLC system. Torularhodin and torulene were observed in even higher concentrations than \u0026beta;-carotene, confirming our previous hypothesis that torularhodin and torulene were earlier degraded during saponification. This could also be seen in an increased amount of extracted total carotenoids with SC-CO\u003csub\u003e2\u003c/sub\u003e extraction. In a study conducted by Martinez \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e(16) similar results were obtained, where upon SC-CO\u003csub\u003e2\u003c/sub\u003e extraction of carotenoids from \u003cem\u003eRhodotorula glutinis\u003c/em\u003e with ethanol as the co-solvent, torularhodin was identified as the primary carotenoid. This finding is consistent with other studies that have also confirmed torularhodin as the most abundant carotenoid in various \u003cem\u003eRhodotorula\u003c/em\u003e species, accounting for approximately 60-70% of the total carotenoids extracted (50, 51). Also, Hosseini \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e(52) confirmed enhanced carotenoid recovery from \u003cem\u003eDunaliella salin\u003c/em\u003ea extracts using SC-CO\u003csub\u003e2\u003c/sub\u003e extraction, compared to the conventional extraction method (Soxhlet method). However, there might still be a variation in the content of these carotenoids, depending on strain and substrate as Qi \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e(38) found more than 10 times higher values of both total carotenoids in different strains of \u003cem\u003eR. toruloides\u003c/em\u003e, grown on tea waste. Zheng \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e(39) on the other hand found \u0026beta;-carotene as the major carotenoid in different \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003estrains grown on cane molasses.\u003c/p\u003e\n\u003cp\u003eAlthough some carotenoid loss into the lipid extract occurred with SC-CO\u003csub\u003e2\u003c/sub\u003e extraction, it was less substantial compared to the loss observed with acetone extraction. This suggests that SC-CO\u003csub\u003e2\u003c/sub\u003e extraction offers a more effective recovery of carotenoids than acetone extraction.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eSupercritical carbon-dioxide (SC-CO\u003csub\u003e2\u003c/sub\u003e) extraction emerged as preferable method to extract high amounts of carotenoids and increased proportion of UFA from \u003cem\u003eR. toruloides\u003c/em\u003e. Gas chromatography analysis showed that lipids extracted using SC-CO\u003csub\u003e2\u003c/sub\u003e exhibited higher levels of UFA compared to the lipids extracted using the Folch method. SC-CO\u003csub\u003e2\u003c/sub\u003e extraction revealed that torularhodin and torulene were the major carotenoids in \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003eCBS14. This finding can be of significance since these carotenoids are of high potential value since they have a higher antioxidant potential than \u0026beta;- and \u0026gamma;-carotene. Up to now, natural sources for their isolation are limited, thus, the established extraction may enable future biotechnological production of these carotenoids The SC-CO\u003csub\u003e2\u003c/sub\u003e extraction method demonstrated the ability to preserve the integrity of unsaturated lipids and abundance of carotenoids, resulting in high-quality extracts. However, it is important to note that despite the lower lipid recovery, the SC-CO\u003csub\u003e2\u003c/sub\u003e extraction method offers the advantage of extraction without any organic solvents, eliminating the need for potentially harmful and environmentally unfriendly solvents. These findings hold great potential for future industrial applications and can serve as a starting point for further research in this area.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eGC\u003c/strong\u003e Gas chromatography\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC\u003c/strong\u003e High pressure liquid chromatography\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDAD\u003c/strong\u003e Diode array detector\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePDA\u003c/strong\u003e Photodiode array\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePUFAs\u003c/strong\u003e Polyunsaturated fatty acids\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSFAs\u003c/strong\u003e Saturated fatty acids\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUFAs\u003c/strong\u003e Unsaturated fatty acids\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSC-CO\u003csub\u003e2\u003c/sub\u003e\u003c/strong\u003e Supercritical carbon-dioxide\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSFE\u003c/strong\u003e Supercritical fluid extraction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTAGs\u003c/strong\u003e Triacylglycerols\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUHPLC\u003c/strong\u003e Ultra-high pressure liquid chromatography\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was financially supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas) (Grant Number 2018-01877) and Nordforsk-SAFE/Swedish Research council for Environment, Agricultural Sciences and Spatial Planning (Formas), (Grant Number 2020-02637).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1.\u0026nbsp; \u0026nbsp;Department of Molecular Science, Swedish University of Agricultural Sciences, Uppsala BioCentre, P.O. Box 7051, SE-750 07 Uppsala, Sweden.\u003c/p\u003e\n\u003cp\u003eYashaswini Nagavara Nagaraj (YNN), Johanna Blomqvist (JB), Sabine Sampels (SaS), Jana Pickova (JP), Mats Sandgren (MS) and Volkmar Passoth (VP).\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp; \u0026nbsp;Institute of Biotechnology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37, Bratislava, Slovakia.\u003c/p\u003e\n\u003cp\u003eMilan Čertík (MC) and Peter Gajdoš (PG).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental work, methodology: YNN, JB, SaS and PG; Result evaluation: YNN, JB, MC, SaS, JP, and VP; Conceptualization: SaS, JP, and VP.; Writing - original draft: YNN; Writing review and editing: SaS, JP, JB, MS, VP and MC; Supervision: SaS, JP, JB, MS, VP.; Funding acquisition: MS and VP; Project administration: MS and VP. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Volkmar Passoth.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRosillo-Calle F, Pelkmans L, Walter A. 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The Journal of Supercritical Fluids. 2017;121:89-95.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"R. toruloides CBS 14, supercritical carbon dioxide extraction, lipids, carotenoids, Folch method, acetone extraction, saponification","lastPublishedDoi":"10.21203/rs.3.rs-5460903/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5460903/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted extractions using supercritical carbon dioxide (SC-CO\u003csub\u003e2\u003c/sub\u003e) and conventional solvent methods to extract lipids and carotenoids from \u003cem\u003eR. toruloides\u003c/em\u003e CBS 14 cells grown on wheat straw hydrolysate. The lipid extracts were analyzed using gas chromatography (GC), and the carotenoids were identified and quantified using ultra-high performance liquid chromatography (UHPLC).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour main carotenoids in the extracts from both extraction methods were identified including β-carotene, γ-carotene, torularhodin, and torulene. Interestingly, torularhodin was the major carotenoid extracted using SC-CO\u003csub\u003e2\u003c/sub\u003e extraction, followed by torulene. This was different from the conventional acetone extraction method, where β-carotene was the main carotenoid. After the conventional extraction, torularhodin and torulene underwent degradation due to the saponification step, which was necessary to remove lipids before UHPLC analysis. The total carotenoid concentration obtained from SC-CO\u003csub\u003e2\u003c/sub\u003e extraction was 332.09 ± 27.32 μg/g dry weight compared to 19.9 ± 2.74 μg/g dry weight in acetone extraction. A small amount of carotenoids was observed to be lost into the lipid extract, but this loss was not as substantial as that seen with acetone extraction. Additionally, the total lipid content in samples extracted using SC-CO\u003csub\u003e2\u003c/sub\u003e was significantly lower than that obtained using the conventional Folch method. GC analysis revealed that oleic acid was the major fatty acid in both lipid extracts, followed by palmitic acid and linoleic acid. Notably, the proportion of unsaturated fatty acids was higher in the extracts from the SC-CO\u003csub\u003e2\u003c/sub\u003e method compared to the conventional method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese findings indicate that the SC-CO\u003csub\u003e2\u003c/sub\u003e extraction method outperformed conventional methods by preserving the integrity of unsaturated lipids and retaining an abundance of carotenoids, resulting in high-quality extracts.\u003c/p\u003e","manuscriptTitle":"Supercritical carbon-dioxide (SC-CO2) extraction of lipids and carotenoids from Rhodotorula toruloides CBS 14 in comparison with conventional extraction methods","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-16 06:01:26","doi":"10.21203/rs.3.rs-5460903/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-01T08:43:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-18T10:26:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2440159630093451844958190347541516729","date":"2024-12-14T10:06:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13896259937519213862726998860423623806","date":"2024-12-08T14:02:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-07T23:11:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-16T06:19:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-16T06:18:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels and Bioproducts","date":"2024-11-15T13:32:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2734fb9b-b3d0-4e45-81d0-6d216f5faab1","owner":[],"postedDate":"December 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-24T16:05:25+00:00","versionOfRecord":{"articleIdentity":"rs-5460903","link":"https://doi.org/10.1186/s13068-025-02632-7","journal":{"identity":"biotechnology-for-biofuels-and-bioproducts","isVorOnly":false,"title":"Biotechnology for Biofuels and Bioproducts"},"publishedOn":"2025-03-21 15:57:21","publishedOnDateReadable":"March 21st, 2025"},"versionCreatedAt":"2024-12-16 06:01:26","video":"","vorDoi":"10.1186/s13068-025-02632-7","vorDoiUrl":"https://doi.org/10.1186/s13068-025-02632-7","workflowStages":[]},"version":"v1","identity":"rs-5460903","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5460903","identity":"rs-5460903","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}