Binary solvent extraction of intracellular lipids from Rhodotorula toruloides for cell recycling

Binary solvent extraction of intracellular lipids from Rhodotorula toruloides for cell recycling | 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 Method Article Binary solvent extraction of intracellular lipids from Rhodotorula toruloides for cell recycling Jingyi Song, Rasool Kamal, yadong chu, shiyu liang, Zongbao Zhao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5891876/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 May, 2025 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted 7 You are reading this latest preprint version Abstract Background: Microbial lipid extraction is a critical process in the production of biofuels and other valuable chemicals from oleaginous microorganisms. The process involves the separation of lipids from microbial cells. Given the complexity of microbial cell walls and the demand for efficient and environmentally friendly extraction methods, further research is still needed in this area. This study aims to pursue the extraction of intracellular lipids from oleaginous yeasts using inexpensive solvents, without disrupting the cells and even maintaining a certain level of cell viability. Results: The study used fresh fermentation broth of Rhodotorula toruloides as the lipid extraction target and employed a binary solvent of methyl tert-butyl ether (MTBE) and n-hexane for lipid extraction. The effects of extraction time and solvent ratio on cell viability, lipid extraction efficiency, and fatty acid composition were analyzed. Conditions that balanced lipid yield and cell survival were selected for lipid extraction. Specifically, using a binary solvent (with 40% MTBE) to extract an equal volume of R. toruloides fermentation broth achieved a total lipid extraction rate of 60%, while maintaining a 5% cell survival rate (the surviving cells served as the seed for the second round of lipid production). After separating the solvent phase and supplementing the Lipid-extracted cells with carbon sources and a small amount of nitrogen sources, the cells gradually regained biomass and produced lipids. Repeating this "gentle" extraction on surviving and regrown cells and adding carbon and nitrogen sources can enable a second round of growth and lipid production in these cells. Conclusions: This is an interesting finding that may potentially encompass the extraction mechanisms of polar/non-polar solvents and the phenomenon of yeast autophagy. This method does not require the destruction of the cell wall of oleaginous yeast. The separation after extraction is simple, and both the cells and solvents can be recycled. It provides a possible approach for simultaneous fermentation and lipid extraction oleaginous microorganisms lipid extraction MBTE cell recycling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Oleaginous microorganisms are an alternative source of oils, distinct from plants, animals, and petroleum. They produce what is known as single cell oils (SCO), which can be used in biofuels, pharmaceuticals, and other industries. These microorganisms include certain yeasts, bacteria, and algae [ 1 ]. With their short production cycles, high lipid yields, genetic tractability, and industrial scalability, oleaginous microorganisms are considered ideal substitutes for fossil fuels [ 2 ]. Rhodotorula toruloides is renowned for its broad substrate spectrum, strong stress resistance, and high lipid yield. These attributes make it an ideal candidate for the production of lipids, carotenoids, and other valuable chemicals [ 3 ]. Research has shown that under nitrogen-limited conditions, R. toruloides undergoes lipid remodeling, where phospholipids are converted into storage lipids. This process enhances the accumulation of both lipids and carotenoids [ 4 , 5 ]. However, the unique cellular structures of oleaginous microorganisms present significant challenges for lipid extraction. Given the minuscule size of these microbial cells, traditional mechanical pressing methods are ineffective for recovering intracellular lipids. Moreover, the cell walls of these oleaginous microorganisms, particularly those of oleaginous yeasts, are rich in chitin and mannans. These components endow the cell walls with high mechanical strength, thickness, and density, further complicating the extraction of lipids from microbial cells [ 6 , 7 ]. Therefore, prior to solvent extraction, pretreatment or cell disruption methods, such as mechanical (puffing, bead milling, ultrasonication, homogenization, microwave) and non-mechanical (enzymatic, acid, alkaline digestion, osmotic shock) approaches, are crucial for the effective recovery of lipids from oleaginous microorganisms [ 8 ]. After pretreatment, the increased accessibility of intracellular lipids makes the subsequent extraction of lipids from the pretreated material using organic solvents much more efficient and straightforward. However, inevitably, the disrupted cells can introduce impurities into the solvent phase, and the pretreatment process also involves a significant amount of energy consumption. Current lipid extraction technologies have also seen the development of novel green solvent - based methods. For example, the use of dimethyl carbonate (DMC) and supercritical CO₂, combined with deep eutectic solvents (DESs) and microwave pretreatment, has been shown to enhance lipid extraction [ 9 ]. Subcritical dimethyl ether (DME) [ 10 ] and switchable polarity solvents, such as dipropylamine (DPA) after CO₂ treatment [ 11 , 12 ], enable direct lipid extraction from wet biomass without the need for pretreatment. Meanwhile, highly economical extraction methods have also been explored, such as milk lipids without affecting the growth of the microalgae Botryococcus brauni i using n – hexane [ 13 ]. This study presents a method for lipid extraction from R. fermentation broth using a binary solvent system of methyl tert - butyl ether (MTBE) and n - hexane. The method involves separating the viable cells that remain after lipid extraction, replenishing the medium, and then conducting lipid production and extraction again, repeating this process for two cycles. By investigating the solvent ratio and extraction duration, the optimal extraction strategy was determined. This approach provides a practical method for lipid recovery and resource recycling, which can help reduce the cost of microbial lipid production. 2 Materials and methods 2.1 Microorganism and solvents R. toruloides CGMCC 2.1389 was obtained from the China General Microbiological Culture Collection Center. Methyl tert-butyl ether (MTBE), n-hexane and chloroform were purchased locally and were of analytical reagent grade. 2.2 Cultivation conditions The initial seed (fresh culture of R. toruloides ) was inoculated and enriched in yeast extract peptone dextrose (YEPD) medium, cultured at 30°C with shaking at 200 rpm for 24 hours. The enriched culture was inoculated into nitrogen-limited medium at a 10% (v/v) inoculum size for lipid production and cultured at 30°C with shaking at 200 rpm until complete glucose consumption. YEPD medium: glucose, 20 g/L; yeast extract, 10 g/L; and peptone, 20 g/L. Nitrogen-limited medium: glucose, 50 g/L; (NH 4 ) 2 SO 4 , 0.1 g/L; yeast extract, 0.75 g/L; MgSO 4 ·7H 2 O, 1.5 g/L; and trace elements solution, 10 mL/L. Trace elements solution: CaCl 2 ·2H 2 O 4 g/L, FeSO 4 ·7H 2 O 0.055 g/L, Citric acid monohydrate 0.52 g/L, ZnSO 4 ·7H 2 O 0.1 g/L, MnSO 4 ·H 2 O 0.076 g/L, 18 M H 2 SO 4 100 µL/L. pH adjustmen: 2-(N-Morpholino) ethanesulfonic acid hydrate (MES) buffer was added at 50 mmol/L to maintain the pH at 6.0. Take 50 mL of fermentation broth for binary solvent lipid extraction. After extraction, take 30 mL of the lower layer of the spent broth and supplement nutrients according to the following strategy: Nitrogen-limited (Nl) supplemented: 250 g/L glucose solution, 10 mL; 39 g/L MES buffer solution, 5 mL; sugar-free nitrogen-limited medium solution (yeast extract, 7.5 g/L; (NH 4 ) 2 SO 4 , 1 g/L; MgSO 4 ·7H 2 O, 6 g/L; 40 mL/L trace element solution), 5 mL; total 50 mL system. Nitrogen-rich (Nr) supplemented: An additional 5 mL of YEPD medium and other nutrient supplements consistent with Nl were added; a total of 55 mL of the system was used. 2.3 Lipid Extraction Method 2.3.1 Extraction of Total Lipids The total lipids were extracted by acid‒heating extraction (AHE) method. For each gram of dry cells, 6 mL of 4 M HCl was added and hydrolyzed at 78°C for 1 hour. After cooling, an equal volume of chloroform and methanol was added, vortexed for 3 minutes, and then centrifuged at 8000 r/min for 5 minutes to separate the organic phase. An equal volume of chloroform was added again for a second extraction, and the organic phases were separated and combined. The combined organic phase was washed with an equal volume of 0.1% NaCl solution, and the resulting extract was purified and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the lipids were dried to constant weight at 105°C and weighed. The lipid content was expressed as grams of lipids per liter of fermentation broth and was used as the standard for evaluating the lipid extraction efficiency of other methods. 2.3.2 Optimization of Binary Solvent Conditions 15 mL of fermentation broth was taken at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. Then, 15 mL of MTBE/hexane binary mixture containing 0%, 20%, 40%, 60%, 80%, and 100% MTBE was added to each sample. The extraction was carried out at 200 rpm for 30 min, 60 min, and 90 min, respectively. The solvent phase was separated by centrifugation, and moisture and protein impurities were removed using anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the lipids were dried to constant weight. Three replicates were set for each condition. 2.3.3 Lipid Extraction During the Re-fermentation Process of Lipid-extracted cells Lipid extraction was carried out using the MTBE-hexane binary solvent (40% MTBE), following the extraction and purification procedures described in Section 2.3.2. 2.4 Analytical methods 2.4.1 Determination of culture parameters The cell mass was determined via the dry weight method. The glucose content of the culture broth was measured via a biosensing analyser. The OD 600 value was obtained by measuring the absorbance at 600 nm via a UV‒Vis spectrophotometer. 2.4.2 Determination of total lipid content and analysis of fatty acid composition The fatty acid composition of lipids was analyzed using gas chromatography–mass spectrometry (GC–MS) according to a previously described method [ 14 ]. 2.5 Staining and observation methods 2.5.1 Examination of intracellular lipid droplets 9-(diethylamino) benzo[α]phenoxazin-5(5H)-one (Nile red), a lipophilic dye, can stain lipid droplets inside cells red. Nile red staining solution at a final concentration of 1 µg/mL was added to the cultures, which were subsequently stained in the dark for 5 min [ 15 ] and then observed in the RFP mode of the microscopic cell imaging system. 2.5.2 Examination of cell survival and the cell membrane Propidium iodide (PI), a DNA dye, can stain cells that have lost membrane integrity and can also be used to assess cell viability. PI staining solution at a final concentration of 1 µg/mL was added to the cultures, which were then stained in the dark for 15 min and observed in the RFP mode of the microscopic cell imaging system. In addition, the streak plate method and spread plate method were employed to assess the viability of the cells. 3 Results and discussion 3.1 Results and Mechanism of Solvent Treatment on R. toruloides Chloroform is commonly used for lipid extraction from oleaginous yeast cells, but it is somewhat toxic. MTBE is a greener solvent that has been used for lipid extraction in lipidomics analysis [ 16 ]. Additionally, n - hexane has been used to extract lipids during the cultivation of microalgal cells [ 13 ]. To test whether R. toruloides cells could tolerate the extraction process with these solvents, cells treated with the three solvents (with ethanol as a polar solvent control) were collected and stained with PI and Nile Red. The results showed that cells treated with ethanol, MTBE, and chloroform were all stained by PI, indicating cell death, while cells in the n-hexane group remained viable. The light field images clearly showed that chloroform and MTBE extracted the lipids from R. toruloides cells, leaving them as “empty shells,” whereas the polar solvent ethanol failed to extract intracellular lipids. Nile Red staining revealed that only the n-hexane group retained intact lipid droplet structures, while other groups showed organic solvents mixed with intracellular lipids being stained. Chloroform exhibited significant lipid extraction efficiency. Ethanol is miscible with water, while chloroform and MTBE are sparingly soluble in water (with solubilities of 0.8 g/L and 15 g/L, respectively). In contrast, n-hexane has an extremely low solubility in water and is virtually insoluble. The extraction mechanisms of these three types of solvents can be illustrated in Fig. 2 . The pathway for solvent mass transfer to intracellular lipids can be summarized as follows: solvent phase → cell wall → cytoplasmic membrane → cytoplasmic matrix → lipid droplet membrane → lipids. For polar solvents that are soluble in water, the mass transfer through the cell wall, cytoplasmic membrane, cytoplasmic matrix, and lipid droplet membrane is relatively straightforward, with the main resistance being the dissolution of lipids. For weak-polar solvents, both the cell membrane and intracellular lipids can be dissolved, facilitating the extraction of lipids from within the cells. However, different solvents will exhibit varying dissolution capabilities. For non-polar solvents, the water in the cell wall pores, the cytoplasmic membrane with its hydrophilic ends facing outward, the cytoplasmic matrix, and the lipid droplet membrane all pose significant barriers to mass transfer. As a result, even if non - polar solvents have good compatibility with lipids, they are unable to extract intracellular lipids effectively. 3.2 Exploration of Lipid Extraction Methods Balancing Lipid Recovery and Cell Viability Based on the above results, MTBE can be attempted as a bridge for the entry and exit of cells, like Figure S1 , while n-hexane can serve as a storage solvent to accommodate lipids. Generally, the oil-producing cultivation of R. toruloides reaches the fermentation endpoint at 96–120 hours. However, there are differences in lipid content, cell wall composition, and thickness at different cultivation time [ 17 ]. Therefore, fermentation broths of R. toruloides cultured for different durations were used as extraction materials, and binary solvents of MTBE/n-hexane at different ratios were employed as oil-extraction solvents to investigate the effects of various factors on lipid extraction. As shown in Fig. 3 , except for the cultivation duration of 24 hours, the lipid extraction capacity was significantly influenced by the MTBE ratio during cultivation durations of 48–120 hours, with an increased MTBE ratio enhancing the lipid extraction capacity. Additionally, as the cultivation time increased, the intracellular oil content rose, and the lipid extraction efficiency significantly improved. The accumulation of lipids enlarged the cell volume and narrowed the gap between the lipid droplets and the cell wall, thereby shortening the mass transfer pathway of the solvent. As shown in Figure S2, the microscopic images of intracellular lipids at 48 hours and 120 hours correspond to lipid contents of 37% and 65%, respectively, indicating that the higher the intracellular lipid content, the better the extraction effect. The extraction efficiency of the binary solvent increased with the rise in MTBE content, while a high content of n-hexane tended to play a “cell-protecting” role. As illustrated in Figure S3, when using a binary solvent with a 40% MTBE ratio, some cells survived after extraction. It can be inferred that cells in good condition with lower lipid content might be less affected by the binary solvent, becoming “survivors” after lipid extraction and resuming growth through self-repair. The lipid extraction data were summarized in Fig. 4 , which shows that during oil extraction from the 120-hour fermentation broth, the most significant change in oil extraction capacity occurred when the MTBE ratio was increased from 20–40%. Compared with the methods in Table S1 , the lipid recovery rate and process complexity have been reduced. Therefore, the optimal solvent ratio was selected as a binary mixture of 40% MTBE/n-hexane. The bacterial suspensions before and after lipid extraction were counted, and the survival rate of the bacterial cells was calculated using the following formula resulting in a survival rate of 5.34%. $$\:\varvec{s}\varvec{u}\varvec{e}\varvec{v}\varvec{i}\varvec{v}\varvec{a}\varvec{l}\varvec{\%}=\frac{{\varvec{N}}_{1}\times\:{\varvec{D}}_{1}÷100\varvec{\mu\:}\varvec{L}}{{\varvec{N}}_{2}\times\:{\varvec{D}}_{2}÷100\varvec{\mu\:}\varvec{L}}$$ N 1 : Number of strains growing after lipid extraction N 2 : Number of strains growing before lipid extraction D: Dilution times Within the same cultivation period, the latter was smaller than the former (as shown in Figure S4), indicating that the growth rate of the strain had slowed down after lipid extraction and required a certain period of recovery. Figure 5 shows the fatty acid composition of lipids extracted using different ratios of binary solvents. Except for the lipids extracted with pure n-hexane (which had an extremely low lipid extraction rate), the fatty acid composition ratio of the lipids extracted using binary solvents was comparable to that of AHE. Methyl tert-butyl ether (MTBE) plays a major role in the lipid extraction process and does not affect the composition of the harvested lipids. 3.3 Recycling of surviving cells after lipid extraction Based on the above observations, we designed a new lipid production process in which a carbon source is added to the spent cultures of R. toruloides after in situ lipid extraction with a 40% MTBE/n-hexane solvent for the subsequent production of lipids. Figure 6 shows the differences in cell growth, sugar consumption, and lipid production under nitrogen-limited (Nl) and nitrogen-rich (Nr) conditions due to the recycling of the spent cultures. The average cell growth rate of the initial culture over 96 hours was 12.75 OD/d, and the sugar consumption rate was 11.38 g/(L·d). Since a large amount of lipids was removed and only 60% of the spent culture was transferred to initiate a new round of cultivation after the addition of glucose and nitrogen sources, the OD value of the culture broth decreased at the beginning of each round. However, cell growth and glucose consumption resumed within 1 day (Fig. 6 a, b). For the Nl trials, a small amount of nitrogen source was added, and cell growth and glucose utilization increased rapidly after the lag phase. The average cell growth rate was 5.98 OD/d, and the sugar consumption rate was 7.18 g/(L·d) in the first round of lipid production. In the second round, the average rates of cell growth and sugar consumption decreased to 4.06 OD/d and 6.00 g/(L·d), respectively. For the Nr trials with additional YEPD supplementation, cell growth and glucose utilization were faster than those in the Nl trials, with average cell growth and sugar consumption rates of 6.55 OD/d and 7.94 g/(L·d) in the first round and 6.05 OD/d and 7.80 g/(L·d), respectively, in the second round. Although cell growth and sugar consumption were faster in the Nr trials, the final lipid yield was 0.108 g/g, which was lower than the 0.131 g/g obtained in the Nl trials (Fig. 6 c). The process shown in Fig. 6 is different from the previous processes used for lipid production. In the repeated fed-batch fermentation process, a small portion of the mature culture containing lipid-rich cells is left in the reactor as an inoculum to initiate new lipid production upon the addition of fresh media [ 18 ], and the proportion of dead cells has decreased. (as shown in Figure S5). Notably, the spent cell mass after lipid extraction has been chemically hydrolyzed, and the resulting hydrolysates have been used as nutrients for lipid production [ 19 ]. In this study, most of the culture broth (including cells) was recycled for the next round of cultivation. The surviving cells were used as seeds, and the spent cells were used as slow-release nutrients, retaining both organic matter and mineral nutrients. This likely achieved in situ utilization of waste and provided a material basis for lipid synthesis after the replenishment of carbon sources. Dead cells can be broken down by hydrolytic enzymes and taken up by living cells as nutrients for growth during yeast cultivation [ 20 ]. Therefore, this study provides a relatively more convenient process for recycling wastewater, nutrients, and cell mass in microbial lipid technology, which is more conducive to continuous operation in the future. 4 Conclusions We have demonstrated that under mild conditions, a mixture of MTBE and n-hexane can effectively extract lipids directly from the fermentation broth of R. toruloides without cell disruption, achieving a 60% lipid extraction rate while maintaining a 5% cell survival rate. The surviving cells present in the spent fermentation broth regrown and produced lipids upon supplementation with fresh carbon and nitrogen sources which enables a cyclic process of lipid extraction and cell regrowth. By combining polar and non-polar solvents for lipid extraction, this new approach exempts energy-intensive thermochemical cell disruption, reduces waste generation by recycling fermentation broth and solvents, and allows semi-continuous lipid production. The findings provide a promising foundation for further optimization and scaling of simultaneous fermentation and lipid extraction processes. Declarations Author contributions Jingyi Song designed and performed this research; Rasool Kamal and Shiyu Liang helped Jing yi Song perform this research; Chu Yadong analyzed data; Qitian Huang and Zongbao Zhao gave funding support and advices. Funding The work was financially supported by the National Key Research and Development Program of China (No. 2021YFA0910603), National Natural Science Foundation of China (No. 22238010 and 22208340), Science and Technology Bureau of Dalian City (No. 2021RT04), the Natural Science Foundation of Liaoning Province (2024-BSBA-29). Availability of data and materials The data collected upon which this article is based upon are all included in this manuscript and the additional files associated with it. Ethics approval and consent to participate No animals or human subjects were used in the above research. Consent for publication Our manuscript does not contain any individual data in any form. Competing interests The authors declare no competing interests References Meng X, Yang J, Xu X, Zhang L, Nie Q, Xian M. Biodiesel production from oleaginous microorganisms. Renewable Energy. 2009;34:1–5. Othman MF, Adam A, Najafi G, Mamat R. Green fuel as alternative fuel for diesel engine: A review. Renewable and Sustainable Energy Reviews. 2017;80:694–709. Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides : Insights for Future Biorefinery - PubMed. https://pubmed.ncbi.nlm.nih.gov/32974324/. Accessed 19 Mar 2025. Mishra S, Deewan A, Zhao H, Rao CV. Nitrogen starvation causes lipid remodeling in Rhodotorula toruloides . Microb Cell Fact. 2024;23:1–14. Compositional profiles of Rhodosporidium toruloides cells under nutrient limitation | Applied Microbiology and Biotechnology. https://link.springer.com/article/10.1007/s00253-017-8157-0. Accessed 19 Mar 2025. Khot M, Raut G, Ghosh D, Alarcón-Vivero M, Contreras D, Ravikumar A. Lipid recovery from oleaginous yeasts: Perspectives and challenges for industrial applications. Fuel. 2020;259:116292. Jaussaud A, Lupette J, Salvaing J, Jouhet J, Bastien O, Gromova M, et al. Stepwise Biogenesis of Subpopulations of Lipid Droplets in Nitrogen Starved Phaeodactylum tricornutum Cells. Front Plant Sci. 2020;11:48. Zainuddin MF, Fai CK, Ariff AB, Rios-Solis L, Halim M. Current Pretreatment/Cell Disruption and Extraction Methods Used to Improve Intracellular Lipid Recovery from Oleaginous Yeasts. Microorganisms. 2021;9:251. Tommasi E, Cravotto G, Galletti P, Grillo G, Mazzotti M, Sacchetti G, et al. Enhanced and Selective Lipid Extraction from the Microalga P. tricornutum by Dimethyl Carbonate and Supercritical CO2 Using Deep Eutectic Solvents and Microwaves as Pretreatment. ACS Sustainable Chem Eng. 2017;5:8316–22. Wang Q, Oshita K, Takaoka M. Effective lipid extraction from undewatered microalgae liquid using subcritical dimethyl ether. Biotechnol Biofuels. 2021;14:17. Yook SD, Kim J, Woo HM, Um Y, Lee S-M. Efficient lipid extraction from the oleaginous yeast Yarrowia lipolytica using switchable solvents. Renewable Energy. 2019;132:61–7. Choi OK, Lee JW. CO 2 -triggered switchable solvent for lipid extraction from microalgal biomass. Science of The Total Environment. 2022;819:153084. Jackson BA, Bahri PA, Moheimani NR. Repetitive non-destructive milking of hydrocarbons from Botryococcus braunii . Renewable and Sustainable Energy Reviews. 2017;79:1229–40. Kamal R, Huang Q, Li Q, Chu Y, Yu X, Limtong S, et al. Conversion of Arthrospira platensis Biomass into Microbial Lipids by the Oleaginous Yeast Cryptococcus curvatus . ACS Sustainable Chem Eng. 2021;9:11011–21. Chung T-Y, Kuo C-Y, Lin W-J, Wang W-L, Chou J-Y. Indole-3-acetic-acid-induced phenotypic plasticity in Desmodesmus algae . Sci Rep. 2018;8:10270. Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. Journal of Lipid Research. 2008;49:1137. Li Y. Lipid production by Rhodosporidium toruloides . Dalian University of Technology; 2007. Zhao X, Hu C, Wu S, Shen H, Zhao ZK. Lipid production by Rhodosporidium toruloides Y4 using different substrate feeding strategies. J Ind Microbiol Biotechnol. 2011;38:627–32. Yang X, Jin G, Gong Z, Shen H, Bai F, Zhao ZK. Recycling microbial lipid production wastes to cultivate oleaginous yeasts. Bioresource Technology. 2015;175:91–6. Carmona-Gutierrez D, Ruckenstuhl C, Bauer MA, Eisenberg T, Büttner S, Madeo F. Cell death in yeast: growing applications of a dying buddy. Cell Death Differ. 2010;17:733–4. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Published Journal Publication published 12 May, 2025 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted Editorial decision: Revision requested 16 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviewers agreed at journal 10 Apr, 2025 Reviewers agreed at journal 08 Apr, 2025 Reviewers invited by journal 08 Apr, 2025 Submission checks completed at journal 04 Apr, 2025 First submitted to journal 03 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5891876","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":440118717,"identity":"7d22c191-afc7-46b9-9c78-628ceade976f","order_by":0,"name":"Jingyi Song","email":"","orcid":"","institution":"Dalian Institute of Chemical Physics","correspondingAuthor":false,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Song","suffix":""},{"id":440118718,"identity":"fd5a572b-7922-4942-bb46-6bec61867c22","order_by":1,"name":"Rasool Kamal","email":"","orcid":"","institution":"Dalian Institute of Chemical Physics","correspondingAuthor":false,"prefix":"","firstName":"Rasool","middleName":"","lastName":"Kamal","suffix":""},{"id":440118719,"identity":"3432cce9-5085-4678-9c70-fb6c6f40b5d8","order_by":2,"name":"yadong chu","email":"","orcid":"","institution":"Dalian Institute of Chemical Physics","correspondingAuthor":false,"prefix":"","firstName":"yadong","middleName":"","lastName":"chu","suffix":""},{"id":440118720,"identity":"6da25229-ca8c-48b6-a17e-3da94697af35","order_by":3,"name":"shiyu liang","email":"","orcid":"","institution":"Dalian University of Technology","correspondingAuthor":false,"prefix":"","firstName":"shiyu","middleName":"","lastName":"liang","suffix":""},{"id":440118721,"identity":"80da8e37-2153-42f3-b83b-0dfb9155b8f9","order_by":4,"name":"Zongbao Zhao","email":"","orcid":"","institution":"Dalian Institute of Chemical Physics","correspondingAuthor":false,"prefix":"","firstName":"Zongbao","middleName":"","lastName":"Zhao","suffix":""},{"id":440118722,"identity":"b0dcb0ff-bb14-42a3-b6d8-6376ee9f5dfc","order_by":5,"name":"qitian huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYFACHjApx8BMtA42iBZj0rUkNhCtg+9+7zGJnztq0zccZ37A8KOGQd6ckBbJY3xpkr1njuduOMxmwNhzjMFwJyH7DI7xmEnwth0DagG6kLeBIcHgABFaJP+2HUs3AGph/EusFmnetpoEkBZmomyRPJZjbC3bdsBwJtAvh2WOSRhuIKSF7/AZw5tv2+rk+c4ffvjwTY2NPEFbGCAKDjMoHACzJQiph2upY5BvIELxKBgFo2AUjEwAAI59PtteQePWAAAAAElFTkSuQmCC","orcid":"","institution":"Dalian Institute of Chemical Physics","correspondingAuthor":true,"prefix":"","firstName":"qitian","middleName":"","lastName":"huang","suffix":""}],"badges":[],"createdAt":"2025-01-24 02:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5891876/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5891876/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13068-025-02655-0","type":"published","date":"2025-05-12T15:58:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80283356,"identity":"a6c2ae46-6d84-43ca-a670-dd9444fab13a","added_by":"auto","created_at":"2025-04-10 06:22:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":191368,"visible":true,"origin":"","legend":"\u003cp\u003eStaining of cells after lipid extraction with PI and Nile Red using solvents (the figure for the light field view, light and dark field superimposed view, dark field view)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5891876/v1/883c3321ed417b467a45df5c.png"},{"id":80283357,"identity":"f99147df-c5d7-4fb9-8ee0-6cc14b04a885","added_by":"auto","created_at":"2025-04-10 06:22:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67710,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of oil extraction with three types of solvents\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5891876/v1/df4a457b65a2b8bf3d5aa173.png"},{"id":80283363,"identity":"f03f455d-f898-4b39-9085-b4b18a22a81a","added_by":"auto","created_at":"2025-04-10 06:22:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":267199,"visible":true,"origin":"","legend":"\u003cp\u003eLipid extraction with different ratios of MTBE/n-hexane binary solvents at different periods ((a): 24 h, (b): 48 h, (c): 72 h, (d): 96 h, (e): 120 h; (f) the lipid content during fermentation)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5891876/v1/ed8e040bdb72294a5b4705f4.png"},{"id":80283359,"identity":"9250c946-ac6f-48a3-97b2-9f1bfb08e3e4","added_by":"auto","created_at":"2025-04-10 06:22:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68634,"visible":true,"origin":"","legend":"\u003cp\u003eLipid extraction rates of different ratios of MTBE/hexane binary solvents at different incubation times\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5891876/v1/cd41fcea1931cb3d5d7cc165.png"},{"id":80284296,"identity":"df30eaec-a496-4e81-9a71-c0e7b10c9616","added_by":"auto","created_at":"2025-04-10 06:30:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105961,"visible":true,"origin":"","legend":"\u003cp\u003eFatty acid composition of lipids extracted using different ratios of combined solvents (120 h fermentation culture;0%~100% represents the percentage of MTBE in the combined solvent)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5891876/v1/4bbebb618456d26be2d0e5dc.png"},{"id":80283370,"identity":"9b5d8cfe-167e-4611-b5d7-adb64d4e0d4b","added_by":"auto","created_at":"2025-04-10 06:22:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":374389,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the cultivation-extraction-recultivation process in which \u003cem\u003eR. toruloides\u003c/em\u003e was used as the lipid producer. ((a) Cell growth profiles; (b) glucoseconsumption profiles; (c) massflow of the cultivation-extraction-recultivation process; blue: Nl feeding; red: Nr feeding))\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5891876/v1/5f0ce1989a301b8948a8c3b3.png"},{"id":83068118,"identity":"ff3dd3dd-b812-4d2c-afa0-e39adf911200","added_by":"auto","created_at":"2025-05-19 16:10:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1845825,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5891876/v1/6545671d-6cbd-4fa9-b8cb-6bb3d212c064.pdf"},{"id":80284293,"identity":"f17c2fbf-cac8-4279-acf1-a660ba37d034","added_by":"auto","created_at":"2025-04-10 06:30:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1731893,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5891876/v1/443c175236b0d6afc7f6815b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Binary solvent extraction of intracellular lipids from Rhodotorula toruloides for cell recycling","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eOleaginous microorganisms are an alternative source of oils, distinct from plants, animals, and petroleum. They produce what is known as single cell oils (SCO), which can be used in biofuels, pharmaceuticals, and other industries. These microorganisms include certain yeasts, bacteria, and algae [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. With their short production cycles, high lipid yields, genetic tractability, and industrial scalability, oleaginous microorganisms are considered ideal substitutes for fossil fuels [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eRhodotorula toruloides\u003c/em\u003e is renowned for its broad substrate spectrum, strong stress resistance, and high lipid yield. These attributes make it an ideal candidate for the production of lipids, carotenoids, and other valuable chemicals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Research has shown that under nitrogen-limited conditions, \u003cem\u003eR. toruloides\u003c/em\u003e undergoes lipid remodeling, where phospholipids are converted into storage lipids. This process enhances the accumulation of both lipids and carotenoids [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, the unique cellular structures of oleaginous microorganisms present significant challenges for lipid extraction. Given the minuscule size of these microbial cells, traditional mechanical pressing methods are ineffective for recovering intracellular lipids. Moreover, the cell walls of these oleaginous microorganisms, particularly those of oleaginous yeasts, are rich in chitin and mannans. These components endow the cell walls with high mechanical strength, thickness, and density, further complicating the extraction of lipids from microbial cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, prior to solvent extraction, pretreatment or cell disruption methods, such as mechanical (puffing, bead milling, ultrasonication, homogenization, microwave) and non-mechanical (enzymatic, acid, alkaline digestion, osmotic shock) approaches, are crucial for the effective recovery of lipids from oleaginous microorganisms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. After pretreatment, the increased accessibility of intracellular lipids makes the subsequent extraction of lipids from the pretreated material using organic solvents much more efficient and straightforward. However, inevitably, the disrupted cells can introduce impurities into the solvent phase, and the pretreatment process also involves a significant amount of energy consumption.\u003c/p\u003e \u003cp\u003eCurrent lipid extraction technologies have also seen the development of novel green solvent - based methods. For example, the use of dimethyl carbonate (DMC) and supercritical CO₂, combined with deep eutectic solvents (DESs) and microwave pretreatment, has been shown to enhance lipid extraction [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Subcritical dimethyl ether (DME) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and switchable polarity solvents, such as dipropylamine (DPA) after CO₂ treatment [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], enable direct lipid extraction from wet biomass without the need for pretreatment. Meanwhile, highly economical extraction methods have also been explored, such as milk lipids without affecting the growth of the microalgae \u003cem\u003eBotryococcus brauni\u003c/em\u003ei using n \u0026ndash; hexane [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study presents a method for lipid extraction from R. fermentation broth using a binary solvent system of methyl tert - butyl ether (MTBE) and n - hexane. The method involves separating the viable cells that remain after lipid extraction, replenishing the medium, and then conducting lipid production and extraction again, repeating this process for two cycles. By investigating the solvent ratio and extraction duration, the optimal extraction strategy was determined. This approach provides a practical method for lipid recovery and resource recycling, which can help reduce the cost of microbial lipid production.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Microorganism and solvents\u003c/h2\u003e \u003cp\u003e \u003cem\u003eR. toruloides\u003c/em\u003e CGMCC 2.1389 was obtained from the China General Microbiological Culture Collection Center. Methyl tert-butyl ether (MTBE), n-hexane and chloroform were purchased locally and were of analytical reagent grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cultivation conditions\u003c/h2\u003e \u003cp\u003eThe initial seed (fresh culture of \u003cem\u003eR. toruloides\u003c/em\u003e) was inoculated and enriched in yeast extract peptone dextrose (YEPD) medium, cultured at 30\u0026deg;C with shaking at 200 rpm for 24 hours. The enriched culture was inoculated into nitrogen-limited medium at a 10% (v/v) inoculum size for lipid production and cultured at 30\u0026deg;C with shaking at 200 rpm until complete glucose consumption.\u003c/p\u003e \u003cp\u003eYEPD medium: glucose, 20 g/L; yeast extract, 10 g/L; and peptone, 20 g/L.\u003c/p\u003e \u003cp\u003eNitrogen-limited medium: glucose, 50 g/L; (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 0.1 g/L; yeast extract, 0.75 g/L; MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 1.5 g/L; and trace elements solution, 10 mL/L.\u003c/p\u003e \u003cp\u003eTrace elements solution: CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO 4 g/L, FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO 0.055 g/L, Citric acid monohydrate 0.52 g/L, ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO 0.1 g/L, MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO 0.076 g/L, 18 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 100 \u0026micro;L/L.\u003c/p\u003e \u003cp\u003epH adjustmen: 2-(N-Morpholino) ethanesulfonic acid hydrate (MES) buffer was added at 50 mmol/L to maintain the pH at 6.0.\u003c/p\u003e \u003cp\u003eTake 50 mL of fermentation broth for binary solvent lipid extraction. After extraction, take 30 mL of the lower layer of the spent broth and supplement nutrients according to the following strategy:\u003c/p\u003e \u003cp\u003eNitrogen-limited (Nl) supplemented: 250 g/L glucose solution, 10 mL; 39 g/L MES buffer solution, 5 mL; sugar-free nitrogen-limited medium solution (yeast extract, 7.5 g/L; (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 1 g/L; MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 6 g/L; 40 mL/L trace element solution), 5 mL; total 50 mL system.\u003c/p\u003e \u003cp\u003eNitrogen-rich (Nr) supplemented: An additional 5 mL of YEPD medium and other nutrient supplements consistent with Nl were added; a total of 55 mL of the system was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Lipid Extraction Method\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Extraction of Total Lipids\u003c/h2\u003e \u003cp\u003eThe total lipids were extracted by acid‒heating extraction (AHE) method. For each gram of dry cells, 6 mL of 4 M HCl was added and hydrolyzed at 78\u0026deg;C for 1 hour. After cooling, an equal volume of chloroform and methanol was added, vortexed for 3 minutes, and then centrifuged at 8000 r/min for 5 minutes to separate the organic phase. An equal volume of chloroform was added again for a second extraction, and the organic phases were separated and combined. The combined organic phase was washed with an equal volume of 0.1% NaCl solution, and the resulting extract was purified and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the lipids were dried to constant weight at 105\u0026deg;C and weighed. The lipid content was expressed as grams of lipids per liter of fermentation broth and was used as the standard for evaluating the lipid extraction efficiency of other methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Optimization of Binary Solvent Conditions\u003c/h2\u003e \u003cp\u003e15 mL of fermentation broth was taken at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. Then, 15 mL of MTBE/hexane binary mixture containing 0%, 20%, 40%, 60%, 80%, and 100% MTBE was added to each sample. The extraction was carried out at 200 rpm for 30 min, 60 min, and 90 min, respectively. The solvent phase was separated by centrifugation, and moisture and protein impurities were removed using anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the lipids were dried to constant weight. Three replicates were set for each condition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 \u003cem\u003eLipid Extraction During the Re-fermentation Process of Lipid-extracted cells\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eLipid extraction was carried out using the MTBE-hexane binary solvent (40% MTBE), following the extraction and purification procedures described in Section 2.3.2.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analytical methods\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Determination of culture parameters\u003c/h2\u003e \u003cp\u003eThe cell mass was determined via the dry weight method. The glucose content of the culture broth was measured via a biosensing analyser. The OD\u003csub\u003e600\u003c/sub\u003e value was obtained by measuring the absorbance at 600 nm via a UV‒Vis spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Determination of total lipid content and analysis of fatty acid composition\u003c/h2\u003e \u003cp\u003eThe fatty acid composition of lipids was analyzed using gas chromatography\u0026ndash;mass spectrometry (GC\u0026ndash;MS) according to a previously described method [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Staining and observation methods\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Examination of intracellular lipid droplets\u003c/h2\u003e \u003cp\u003e9-(diethylamino) benzo[α]phenoxazin-5(5H)-one (Nile red), a lipophilic dye, can stain lipid droplets inside cells red. Nile red staining solution at a final concentration of 1 \u0026micro;g/mL was added to the cultures, which were subsequently stained in the dark for 5 min [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and then observed in the RFP mode of the microscopic cell imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Examination of cell survival and the cell membrane\u003c/h2\u003e \u003cp\u003ePropidium iodide (PI), a DNA dye, can stain cells that have lost membrane integrity and can also be used to assess cell viability. PI staining solution at a final concentration of 1 \u0026micro;g/mL was added to the cultures, which were then stained in the dark for 15 min and observed in the RFP mode of the microscopic cell imaging system.\u003c/p\u003e \u003cp\u003eIn addition, the streak plate method and spread plate method were employed to assess the viability of the cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Results and Mechanism of Solvent Treatment on R. toruloides\u003c/h2\u003e \u003cp\u003eChloroform is commonly used for lipid extraction from oleaginous yeast cells, but it is somewhat toxic. MTBE is a greener solvent that has been used for lipid extraction in lipidomics analysis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, n - hexane has been used to extract lipids during the cultivation of microalgal cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To test whether \u003cem\u003eR. toruloides\u003c/em\u003e cells could tolerate the extraction process with these solvents, cells treated with the three solvents (with ethanol as a polar solvent control) were collected and stained with PI and Nile Red.\u003c/p\u003e \u003cp\u003eThe results showed that cells treated with ethanol, MTBE, and chloroform were all stained by PI, indicating cell death, while cells in the n-hexane group remained viable. The light field images clearly showed that chloroform and MTBE extracted the lipids from \u003cem\u003eR. toruloides\u003c/em\u003e cells, leaving them as \u0026ldquo;empty shells,\u0026rdquo; whereas the polar solvent ethanol failed to extract intracellular lipids. Nile Red staining revealed that only the n-hexane group retained intact lipid droplet structures, while other groups showed organic solvents mixed with intracellular lipids being stained. Chloroform exhibited significant lipid extraction efficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEthanol is miscible with water, while chloroform and MTBE are sparingly soluble in water (with solubilities of 0.8 g/L and 15 g/L, respectively). In contrast, n-hexane has an extremely low solubility in water and is virtually insoluble. The extraction mechanisms of these three types of solvents can be illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The pathway for solvent mass transfer to intracellular lipids can be summarized as follows: solvent phase \u0026rarr; cell wall \u0026rarr; cytoplasmic membrane \u0026rarr; cytoplasmic matrix \u0026rarr; lipid droplet membrane \u0026rarr; lipids. For polar solvents that are soluble in water, the mass transfer through the cell wall, cytoplasmic membrane, cytoplasmic matrix, and lipid droplet membrane is relatively straightforward, with the main resistance being the dissolution of lipids. For weak-polar solvents, both the cell membrane and intracellular lipids can be dissolved, facilitating the extraction of lipids from within the cells. However, different solvents will exhibit varying dissolution capabilities. For non-polar solvents, the water in the cell wall pores, the cytoplasmic membrane with its hydrophilic ends facing outward, the cytoplasmic matrix, and the lipid droplet membrane all pose significant barriers to mass transfer. As a result, even if non - polar solvents have good compatibility with lipids, they are unable to extract intracellular lipids effectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Exploration of Lipid Extraction Methods Balancing Lipid Recovery and Cell Viability\u003c/h2\u003e \u003cp\u003eBased on the above results, MTBE can be attempted as a bridge for the entry and exit of cells, like Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, while n-hexane can serve as a storage solvent to accommodate lipids. Generally, the oil-producing cultivation of \u003cem\u003eR. toruloides\u003c/em\u003e reaches the fermentation endpoint at 96\u0026ndash;120 hours. However, there are differences in lipid content, cell wall composition, and thickness at different cultivation time [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, fermentation broths of \u003cem\u003eR. toruloides\u003c/em\u003e cultured for different durations were used as extraction materials, and binary solvents of MTBE/n-hexane at different ratios were employed as oil-extraction solvents to investigate the effects of various factors on lipid extraction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, except for the cultivation duration of 24 hours, the lipid extraction capacity was significantly influenced by the MTBE ratio during cultivation durations of 48\u0026ndash;120 hours, with an increased MTBE ratio enhancing the lipid extraction capacity.\u003c/p\u003e \u003cp\u003eAdditionally, as the cultivation time increased, the intracellular oil content rose, and the lipid extraction efficiency significantly improved. The accumulation of lipids enlarged the cell volume and narrowed the gap between the lipid droplets and the cell wall, thereby shortening the mass transfer pathway of the solvent. As shown in Figure S2, the microscopic images of intracellular lipids at 48 hours and 120 hours correspond to lipid contents of 37% and 65%, respectively, indicating that the higher the intracellular lipid content, the better the extraction effect. The extraction efficiency of the binary solvent increased with the rise in MTBE content, while a high content of n-hexane tended to play a \u0026ldquo;cell-protecting\u0026rdquo; role.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs illustrated in Figure S3, when using a binary solvent with a 40% MTBE ratio, some cells survived after extraction. It can be inferred that cells in good condition with lower lipid content might be less affected by the binary solvent, becoming \u0026ldquo;survivors\u0026rdquo; after lipid extraction and resuming growth through self-repair. The lipid extraction data were summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, which shows that during oil extraction from the 120-hour fermentation broth, the most significant change in oil extraction capacity occurred when the MTBE ratio was increased from 20\u0026ndash;40%. Compared with the methods in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, the lipid recovery rate and process complexity have been reduced. Therefore, the optimal solvent ratio was selected as a binary mixture of 40% MTBE/n-hexane.\u003c/p\u003e \u003cp\u003eThe bacterial suspensions before and after lipid extraction were counted, and the survival rate of the bacterial cells was calculated using the following formula resulting in a survival rate of 5.34%.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{s}\\varvec{u}\\varvec{e}\\varvec{v}\\varvec{i}\\varvec{v}\\varvec{a}\\varvec{l}\\varvec{\\%}=\\frac{{\\varvec{N}}_{1}\\times\\:{\\varvec{D}}_{1}\u0026divide;100\\varvec{\\mu\\:}\\varvec{L}}{{\\varvec{N}}_{2}\\times\\:{\\varvec{D}}_{2}\u0026divide;100\\varvec{\\mu\\:}\\varvec{L}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eN\u003csub\u003e1\u003c/sub\u003e: Number of strains growing after lipid extraction\u003c/p\u003e \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003e: Number of strains growing before lipid extraction\u003c/p\u003e \u003cp\u003eD: Dilution times\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eWithin the same cultivation period, the latter was smaller than the former (as shown in Figure S4), indicating that the growth rate of the strain had slowed down after lipid extraction and required a certain period of recovery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the fatty acid composition of lipids extracted using different ratios of binary solvents. Except for the lipids extracted with pure n-hexane (which had an extremely low lipid extraction rate), the fatty acid composition ratio of the lipids extracted using binary solvents was comparable to that of AHE. Methyl tert-butyl ether (MTBE) plays a major role in the lipid extraction process and does not affect the composition of the harvested lipids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Recycling of surviving cells after lipid extraction\u003c/h2\u003e \u003cp\u003eBased on the above observations, we designed a new lipid production process in which a carbon source is added to the spent cultures of \u003cem\u003eR. toruloides\u003c/em\u003e after in situ lipid extraction with a 40% MTBE/n-hexane solvent for the subsequent production of lipids. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the differences in cell growth, sugar consumption, and lipid production under nitrogen-limited (Nl) and nitrogen-rich (Nr) conditions due to the recycling of the spent cultures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe average cell growth rate of the initial culture over 96 hours was 12.75 OD/d, and the sugar consumption rate was 11.38 g/(L\u0026middot;d). Since a large amount of lipids was removed and only 60% of the spent culture was transferred to initiate a new round of cultivation after the addition of glucose and nitrogen sources, the OD value of the culture broth decreased at the beginning of each round. However, cell growth and glucose consumption resumed within 1 day (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). For the Nl trials, a small amount of nitrogen source was added, and cell growth and glucose utilization increased rapidly after the lag phase. The average cell growth rate was 5.98 OD/d, and the sugar consumption rate was 7.18 g/(L\u0026middot;d) in the first round of lipid production. In the second round, the average rates of cell growth and sugar consumption decreased to 4.06 OD/d and 6.00 g/(L\u0026middot;d), respectively. For the Nr trials with additional YEPD supplementation, cell growth and glucose utilization were faster than those in the Nl trials, with average cell growth and sugar consumption rates of 6.55 OD/d and 7.94 g/(L\u0026middot;d) in the first round and 6.05 OD/d and 7.80 g/(L\u0026middot;d), respectively, in the second round. Although cell growth and sugar consumption were faster in the Nr trials, the final lipid yield was 0.108 g/g, which was lower than the 0.131 g/g obtained in the Nl trials (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThe process shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e is different from the previous processes used for lipid production. In the repeated fed-batch fermentation process, a small portion of the mature culture containing lipid-rich cells is left in the reactor as an inoculum to initiate new lipid production upon the addition of fresh media [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and the proportion of dead cells has decreased. (as shown in Figure S5). Notably, the spent cell mass after lipid extraction has been chemically hydrolyzed, and the resulting hydrolysates have been used as nutrients for lipid production [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this study, most of the culture broth (including cells) was recycled for the next round of cultivation. The surviving cells were used as seeds, and the spent cells were used as slow-release nutrients, retaining both organic matter and mineral nutrients. This likely achieved in situ utilization of waste and provided a material basis for lipid synthesis after the replenishment of carbon sources. Dead cells can be broken down by hydrolytic enzymes and taken up by living cells as nutrients for growth during yeast cultivation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, this study provides a relatively more convenient process for recycling wastewater, nutrients, and cell mass in microbial lipid technology, which is more conducive to continuous operation in the future.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eWe have demonstrated that under mild conditions, a mixture of MTBE and n-hexane can effectively extract lipids directly from the fermentation broth of \u003cem\u003eR. toruloides\u003c/em\u003e without cell disruption, achieving a 60% lipid extraction rate while maintaining a 5% cell survival rate. The surviving cells present in the spent fermentation broth regrown and produced lipids upon supplementation with fresh carbon and nitrogen sources which enables a cyclic process of lipid extraction and cell regrowth. By combining polar and non-polar solvents for lipid extraction, this new approach exempts energy-intensive thermochemical cell disruption, reduces waste generation by recycling fermentation broth and solvents, and allows semi-continuous lipid production. The findings provide a promising foundation for further optimization and scaling of simultaneous fermentation and lipid extraction processes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJingyi Song designed and performed this research; Rasool Kamal\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand Shiyu Liang\u003csup\u003e\u0026nbsp;\u003c/sup\u003ehelped Jing yi Song perform this research; Chu Yadong analyzed data;\u003csup\u003e\u0026nbsp;\u003c/sup\u003eQitian Huang and Zongbao Zhao gave funding support and advices.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was financially supported by the National Key Research and Development Program of China (No. 2021YFA0910603), National Natural Science Foundation of China (No. 22238010 and 22208340), Science and Technology Bureau of Dalian City (No. 2021RT04), the Natural Science Foundation of Liaoning Province (2024-BSBA-29).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data collected upon which this article is based upon are all included in this manuscript and the additional files associated with it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo animals or human subjects were used in the above research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur manuscript does not contain any individual data in any form.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMeng X, Yang J, Xu X, Zhang L, Nie Q, Xian M. Biodiesel production from oleaginous microorganisms. Renewable Energy. 2009;34:1\u0026ndash;5.\u003c/li\u003e\n\u003cli\u003eOthman MF, Adam A, Najafi G, Mamat R. Green fuel as alternative fuel for diesel engine: A review. Renewable and Sustainable Energy Reviews. 2017;80:694\u0026ndash;709.\u003c/li\u003e\n\u003cli\u003eXylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in \u003cem\u003eRhodotorula toruloides\u003c/em\u003e: Insights for Future Biorefinery - PubMed. https://pubmed.ncbi.nlm.nih.gov/32974324/. Accessed 19 Mar 2025.\u003c/li\u003e\n\u003cli\u003eMishra S, Deewan A, Zhao H, Rao CV. Nitrogen starvation causes lipid remodeling in \u003cem\u003eRhodotorula toruloides\u003c/em\u003e. Microb Cell Fact. 2024;23:1\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eCompositional profiles of \u003cem\u003eRhodosporidium toruloides\u003c/em\u003e cells under nutrient limitation | Applied Microbiology and Biotechnology. https://link.springer.com/article/10.1007/s00253-017-8157-0. Accessed 19 Mar 2025.\u003c/li\u003e\n\u003cli\u003eKhot M, Raut G, Ghosh D, Alarc\u0026oacute;n-Vivero M, Contreras D, Ravikumar A. Lipid recovery from oleaginous yeasts: Perspectives and challenges for industrial applications. Fuel. 2020;259:116292.\u003c/li\u003e\n\u003cli\u003eJaussaud A, Lupette J, Salvaing J, Jouhet J, Bastien O, Gromova M, et al. Stepwise Biogenesis of Subpopulations of Lipid Droplets in Nitrogen Starved \u003cem\u003ePhaeodactylum tricornutum\u003c/em\u003e Cells. Front Plant Sci. 2020;11:48.\u003c/li\u003e\n\u003cli\u003eZainuddin MF, Fai CK, Ariff AB, Rios-Solis L, Halim M. Current Pretreatment/Cell Disruption and Extraction Methods Used to Improve Intracellular Lipid Recovery from Oleaginous Yeasts. Microorganisms. 2021;9:251.\u003c/li\u003e\n\u003cli\u003eTommasi E, Cravotto G, Galletti P, Grillo G, Mazzotti M, Sacchetti G, et al. Enhanced and Selective Lipid Extraction from the Microalga \u003cem\u003eP. tricornutum\u003c/em\u003e by Dimethyl Carbonate and Supercritical CO2 Using Deep Eutectic Solvents and Microwaves as Pretreatment. ACS Sustainable Chem Eng. 2017;5:8316\u0026ndash;22.\u003c/li\u003e\n\u003cli\u003eWang Q, Oshita K, Takaoka M. Effective lipid extraction from undewatered microalgae liquid using subcritical dimethyl ether. Biotechnol Biofuels. 2021;14:17.\u003c/li\u003e\n\u003cli\u003eYook SD, Kim J, Woo HM, Um Y, Lee S-M. Efficient lipid extraction from the oleaginous yeast \u003cem\u003eYarrowia lipolytica\u003c/em\u003e using switchable solvents. Renewable Energy. 2019;132:61\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eChoi OK, Lee JW. CO\u003csub\u003e2\u003c/sub\u003e-triggered switchable solvent for lipid extraction from microalgal biomass. Science of The Total Environment. 2022;819:153084.\u003c/li\u003e\n\u003cli\u003eJackson BA, Bahri PA, Moheimani NR. Repetitive non-destructive milking of hydrocarbons from \u003cem\u003eBotryococcus braunii\u003c/em\u003e. Renewable and Sustainable Energy Reviews. 2017;79:1229\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eKamal R, Huang Q, Li Q, Chu Y, Yu X, Limtong S, et al. Conversion of \u003cem\u003eArthrospira platensis\u003c/em\u003e Biomass into Microbial Lipids by the Oleaginous Yeast \u003cem\u003eCryptococcus curvatus\u003c/em\u003e. ACS Sustainable Chem Eng. 2021;9:11011\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eChung T-Y, Kuo C-Y, Lin W-J, Wang W-L, Chou J-Y. Indole-3-acetic-acid-induced phenotypic plasticity in \u003cem\u003eDesmodesmus algae\u003c/em\u003e. Sci Rep. 2018;8:10270.\u003c/li\u003e\n\u003cli\u003eMatyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. Journal of Lipid Research. 2008;49:1137.\u003c/li\u003e\n\u003cli\u003eLi Y. Lipid production by\u003cem\u003e Rhodosporidium toruloides\u003c/em\u003e. Dalian University of Technology; 2007.\u003c/li\u003e\n\u003cli\u003eZhao X, Hu C, Wu S, Shen H, Zhao ZK. Lipid production by \u003cem\u003eRhodosporidium toruloides \u003c/em\u003eY4 using different substrate feeding strategies. J Ind Microbiol Biotechnol. 2011;38:627\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eYang X, Jin G, Gong Z, Shen H, Bai F, Zhao ZK. Recycling microbial lipid production wastes to cultivate oleaginous yeasts. Bioresource Technology. 2015;175:91\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eCarmona-Gutierrez D, Ruckenstuhl C, Bauer MA, Eisenberg T, B\u0026uuml;ttner S, Madeo F. Cell death in yeast: growing applications of a dying buddy. Cell Death Differ. 2010;17:733\u0026ndash;4.\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":"oleaginous microorganisms, lipid extraction, MBTE, cell recycling","lastPublishedDoi":"10.21203/rs.3.rs-5891876/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5891876/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Microbial lipid extraction is a critical process in the production of biofuels and other valuable chemicals from oleaginous microorganisms. The process involves the separation of lipids from microbial cells. Given the complexity of microbial cell walls and the demand for efficient and environmentally friendly extraction methods, further research is still needed in this area. This study aims to pursue the extraction of intracellular lipids from oleaginous yeasts using inexpensive solvents, without disrupting the cells and even maintaining a certain level of cell viability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe study used fresh fermentation broth of \u003cem\u003eRhodotorula toruloides\u003c/em\u003e as the lipid extraction target and employed a binary solvent of methyl tert-butyl ether (MTBE) and n-hexane for lipid extraction. The effects of extraction time and solvent ratio on cell viability, lipid extraction efficiency, and fatty acid composition were analyzed. Conditions that balanced lipid yield and cell survival were selected for lipid extraction.\u003c/p\u003e\n\u003cp\u003eSpecifically, using a binary solvent (with 40% MTBE) to extract an equal volume of \u003cem\u003eR. toruloides\u003c/em\u003e fermentation broth achieved a total lipid extraction rate of 60%, while maintaining a 5% cell survival rate (the surviving cells served as the seed for the second round of lipid production). After separating the solvent phase and supplementing the Lipid-extracted cells with carbon sources and a small amount of nitrogen sources, the cells gradually regained biomass and produced lipids. Repeating this \"gentle\" extraction on surviving and regrown cells and adding carbon and nitrogen sources can enable a second round of growth and lipid production in these cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis is an interesting finding that may potentially encompass the extraction mechanisms of polar/non-polar solvents and the phenomenon of yeast autophagy. This method does not require the destruction of the cell wall of oleaginous yeast. The separation after extraction is simple, and both the cells and solvents can be recycled. It provides a possible approach for simultaneous fermentation and lipid extraction\u003c/p\u003e","manuscriptTitle":"Binary solvent extraction of intracellular lipids from Rhodotorula toruloides for cell recycling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 06:22:51","doi":"10.21203/rs.3.rs-5891876/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-16T06:41:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T13:35:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"57453469996766153423637659466238486131","date":"2025-04-10T13:28:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335121967081730314424099860978328395771","date":"2025-04-08T13:16:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-08T13:08:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-04T07:26:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels and Bioproducts","date":"2025-04-03T08:46:00+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":"78c786db-a7ad-40bc-bc97-71d23b2b421c","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:07:43+00:00","versionOfRecord":{"articleIdentity":"rs-5891876","link":"https://doi.org/10.1186/s13068-025-02655-0","journal":{"identity":"biotechnology-for-biofuels-and-bioproducts","isVorOnly":false,"title":"Biotechnology for Biofuels and Bioproducts"},"publishedOn":"2025-05-12 15:58:13","publishedOnDateReadable":"May 12th, 2025"},"versionCreatedAt":"2025-04-10 06:22:51","video":"","vorDoi":"10.1186/s13068-025-02655-0","vorDoiUrl":"https://doi.org/10.1186/s13068-025-02655-0","workflowStages":[]},"version":"v1","identity":"rs-5891876","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5891876","identity":"rs-5891876","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}