Rhodosporidium toruloides capable of producing microbial oil by using crude glycerine and application of rhodosporidium toruloides

Abstract

The invention belongs to the technical field of application of microorganisms, and relates to rhodosporidium toruloides capable of producing microbial oil by using crude glycerine and application of the rhodosporidium toruloides. The R.toruloides XR20 is obtained by carrying out long-period super centrifugal force stress adaptive evolution on wild R.toruloides CGMCC (China General Microbiological Culture Collection Center) 2.1389, and the R.toruloides XR20 is named as R.toruloides XR20. The cell volume is larger; the grease yield of batch fermentation of the crude glycerine is 19.8 g / L; the grease content is 63.6%; by means of fed-batch fermentation of the crude glycerol, 42.56 g / L of microbial oil can be produced at most, the oil content is 74.9%, the oil yield is 0.182 g / g, and the oil yield is 0.161 g / L / h. By utilizing the strain, a byproduct crude glycerol which is difficult to treat in biodiesel production can be effectively reformed into a grease raw material, and sustainable development of the biodiesel industry is facilitated.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and microbiology, and relates to a Rhodosporium toruloides yeast capable of utilizing crude glycerol and its application, a Rhodotorula toruloides yeast mutant and its application in producing microbial oils by fermenting crude glycerol waste. Background Art

[0002] Biodiesel fuel has properties very similar to fossil fuel components, making it a suitable alternative to fossil diesel. Transesterification of oils and fats is currently the primary biodiesel production process. During this process, one ton of crude glycerin is produced for every 10 tons of biodiesel produced. Crude glycerin contains numerous impurities, making direct refining to pure glycerin prohibitively expensive. Failure to properly handle crude glycerin can cause significant environmental damage, hindering the sustainable development of the biodiesel industry.

[0003] Oleaginous yeast is one of the best microbial oil cell factories. Using oleaginous yeast to reform crude glycerol into oil raw materials and further use it in biodiesel production can minimize waste emissions from the biodiesel industry. Currently, some studies have attempted to use crude glycerol to produce microbial oils. For example, Purdue et al. from MARA RENEWABLES CORPORATION used the Thraustochytrium ONC-T18 strain and crude glycerol as a carbon source to produce 116.96 g / L of oil (CA20162980679); Zhou Maohong et al. from Wenzhou University used the Cryptococcus curvatus CGMCC NO.8096 strain and crude glycerol as a carbon source to produce 66.89±3.15 g / L of microbial oil (CN201711318897.2); Wang Qunhui et al. from the University of Science and Technology Beijing used the Rhodotorula glutinis 32489 strain and crude glycerol as a carbon source to produce 12.89 g / L of microbial oil. However, most of these studies focused on the optimization of fermentation processes, with less focus on the directed evolution and transformation of strains.

[0004] Natural oleaginous yeasts, limited by their small intracellular space and weak oil production capacity, have always struggled to meet the requirements of industrial microbial oil production. Rhodosporidium toruloides is an oleaginous yeast capable of producing microbial oils from a variety of carbon sources. It can utilize glycerol and fatty acid methyl esters in crude glycerol and exhibits good tolerance to various impurities, making it an excellent platform for direct production of microbial oils from crude glycerol. However, effective methods are needed to significantly enhance the ability of wild-type Rhodosporidium toruloides to produce microbial oils from crude glycerol. Summary of the Invention

[0005] In order to solve the problem in the prior art that the yield of oil produced by Rhodosporidium toruloides using crude glycerol is low, the present invention obtains a Rhodosporidium toruloides XR20 that can use crude glycerol to produce high oil by adopting the method of ultracentrifugal adaptive evolution. The deposit number is CCTCC M20251198, the deposit address is China Center for Type Culture Collection, and the deposit date is May 27, 2025.

[0006] A method for obtaining a Rhodosporidium toruloides yeast capable of utilizing crude glycerol to produce microbial oil comprises the following steps:

[0007] (1) Preparation of nitrogen-limited culture medium containing crude glycerol;

[0008] (2) The starting strain of Rhodosporidium toruloides was cultured for ultracentrifugal adaptive evolution.

[0009] Furthermore, in the above step (1), the crude glycerol raw material used has a glycerol content of 400-900 g / L and a fatty acid methyl ester content of about 10-200 g / L, mainly including methyl myristate (C14:0), methyl palmitate (C16:0), methyl palmitoleate (C16:1), methyl stearate (C18:0), methyl oleate (C18:1), and methyl linoleate (C18:2), and the remaining impurities also include methanol, fatty acids, soaps, and inorganic salts.

[0010] Furthermore, in the above step (1), the crude glycerol content in the crude glycerol culture medium used for evolution is 50-200 g / L, and the nutrient salts include 0.1-5 g / L corn steep liquor, 0.1-5 g / L potassium dihydrogen phosphate, 0.1-5 g / L magnesium sulfate heptahydrate and 0.1-5 g / L ammonium sulfate.

[0011] Furthermore, in step (2) above, the strain culture is performed by inoculating the activated original strain of R. toruloides CGMCC 2.1389 (purchased from the China General Microbiological Culture Collection Center) into a crude glycerol nitrogen-limited culture medium at a 5-20% (v / v) inoculum. The culture conditions are: a rotation speed of 50-750 rpm (depending on the volume of the reactor and the liquid filling equipment), a culture temperature of 15-37°C, and a culture broth obtained after 96-240 hours of culture.

[0012] Furthermore, in step (2), continuous subculture, centrifugal stimulation, and screening are performed: after the culture is completed, the bacterial solution is obtained and placed in a centrifuge for centrifugal stress and screening, with the centrifugation time gradually increased to 15 minutes. After the centrifugation, the supernatant cell mass is taken as the seed for the next generation of subculture, and the inoculum volume is 5-20% (v / v). The continuous subculture, centrifugal stimulation, and screening are performed 20 times or more.

[0013] Furthermore, in the above step (2), the mutant was isolated and its stability was evaluated: the final bacterial solution was streaked on a crude glycerol medium plate to obtain a single colony, which was preserved after continuous passage and named R. toruloides XR20, with a preservation number of CCTCC M20251198, a preservation address of China Center for Type Culture Collection, and a preservation date of May 27, 2025.

[0014] The present invention provides a Rhodosporidium toruloides yeast capable of utilizing crude glycerol to produce microbial oil, the strain being named Rhodosporidium toruloides XR20, having a deposit number of CCTCC M20251198, a deposit address of China Center for Type Culture Collection, and a deposit date of May 27, 2025.

[0015] The present invention also provides an application of a Rhodosporidium toruloides strain capable of utilizing crude glycerol to produce microbial oils:

[0016] The application uses the above-obtained R. toruloides XR20 strain as a fermentation strain to further evaluate its potential for producing microbial oils using crude glycerol, including the following steps:

[0017] 1. R. toruloides XR20 uses crude glycerol for batch fermentation to produce microbial oil;

[0018] 2. R. toruloides XR20 uses crude glycerol for fed-batch fermentation to produce microbial oil.

[0019] Furthermore, in the above step 1, the crude glycerol content in the fermentation medium is 50-200 g / L, and the nutrient salts include 0.1-5 g / L corn steep liquor, 0.1-5 g / L potassium dihydrogen phosphate, 0.1-5 g / L magnesium sulfate heptahydrate, and 0.1-5 g / L ammonium sulfate.

[0020] Furthermore, in step 1, during seed culture, the Rhodosporidium toruloides XR20 strain is activated, inoculated with a crude glycerol content of 50-200 g / L, nutrient salts including 0.1-5 g / L corn steep liquor, 0.1-5 g / L potassium dihydrogen phosphate, 0.1-5 g / L magnesium sulfate heptahydrate, and 0.1-5 g / L ammonium sulfate, and cultured for 96-240 hours. The inoculum is then introduced into a fermentation apparatus at a 5-20% (v / v) inoculum size. Depending on the volume of the reactor and the liquid filling apparatus, the liquid filling volume is 20-50%, the rotation speed is 50-750 rpm, and the culture temperature is 15-37° C. The culture is carried out for a total of 96-240 hours, with sampling every 6-36 hours.

[0021] Furthermore, in step 1 above, the extraction and determination of oil is as follows: the bacterial solution is diluted and applied to a solid culture medium, and then cultured at 15-37°C for 24-72 hours and the CFU is calculated by colony count. The bacterial solution is diluted and the glycerol concentration is determined by high-performance liquid chromatography. The bacterial solution is shaken and 20 mL is centrifuged. The supernatant is discarded and the cells are washed. The cells are then resuspended in 3-10 mL of ultrapure water and dried and weighed. 5 mL of 3-6 M HCl is added to the dried cells, and acid hydrolyzed at room temperature for 4-6 hours. The liquid is collected, boiled for 5-15 minutes, and cooled. After extraction with 20 mL of a mixture of chloroform and methanol for 1-5 hours, the chloroform layer is evaporated to obtain the oil and weigh it. After calculation, parameters such as the oil content and yield of the oily cells can be obtained.

[0022] Furthermore, in the above step 1, the fermentation medium configuration, seed culture, fermentation conditions, and oil extraction and determination methods are consistent with step (1).

[0023] Furthermore, in the fed-batch fermentation in step 2, the fermentation medium configuration, seed culture, fermentation conditions, and oil extraction and determination methods are the same as those in step 1. The number of feedings is 1-5, and the feeding interval is 24-72 hours. The strain is cultured in batches until the glycerol in the last feeding is completely consumed. The feed medium contains 50-200g of crude glycerol, 0.1-5g of corn steep liquor, 0.1-5g of potassium dihydrogen phosphate, 0.1-5g of magnesium sulfate heptahydrate, and 0.1-5g of ammonium sulfate. The mother liquor is prepared with an equal amount of ultrapure water based on the volume of the culture medium reduced by sampling.

[0024] The Rhodosporidium toruloides yeast obtained by the present invention, which is capable of producing microbial oils using crude glycerol, is named R. toruloides XR20. This mutant strain has a larger cell volume, 3.6 times that of the original strain; the oil yield in batch fermentation using crude glycerol is 19.8g / L, a 3.4-fold increase over the original strain; the oil content is 63.6%, twice that of the original strain; and batch fed-batch fermentation using crude glycerol can produce up to 42.56g / L of microbial oil, with an oil content of 74.9%, an oil yield of 0.182g / g, and an oil productivity of 0.161g / L / h. This strain can effectively reform crude glycerol, a difficult-to-process byproduct in biodiesel production, into an oil feedstock, contributing to the sustainable development of the biodiesel industry.

[0025] The Rhodosporidium toruloides XR20 yeast, which is capable of producing microbial oils from crude glycerol, is obtained in the present invention. Compared with the prior art, the present invention has significant advantages, including the following:

[0026] (1) Using the method of ultracentrifugal adaptive evolution, the Rhodosporidium toruloides CGMCC2.1389 was continuously passaged, centrifuged and screened, and finally an evolved strain R. toruloides XR20 was obtained that can efficiently utilize crude glycerol to synthesize oil.

[0027] (2) Batch fermentation experiments demonstrated that the oil yield of the strain R. toruloides XR20 obtained by the above method in crude glycerol medium was significantly higher than that of the original strain, and its lipid titer reached 24.5 g / L, an increase of 40.8% compared with the original strain.

[0028] (3) When crude glycerol was used as the sole carbon source, a fed-batch fermentation strategy was adopted to further optimize the oil synthesis performance of strain R. toruloides XR20, and the final yield was increased to 42.56 g / L, an increase of 73.7% compared with the batch fermentation mode, showing the potential for industrial production.

[0029] Classification name R.toruloides XR20:

[0030] The accession number is CCTCC M20251198, the deposit address is China Center for Type Culture Collection, and the deposit date is May 27, 2025. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Adaptive evolution diagram for centrifugal selection to improve oil production of Rhodosporidium toruloides in crude glycerol system.

[0032] Figure 2 Comparison of microscope and SEM images of R. toruloides XR20 and the original strain.

[0033] Figure 3 This is a batch fermentation diagram of R. toruloides XR20 and the original strain using crude glycerol to produce microbial oil.

[0034] Figure 4 Figure 1. Fed-batch fermentation of Rhodosporidium toruloides XR20 using crude glycerol to produce microbial lipids. DETAILED DESCRIPTION

[0035] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following implementation methods are, unless otherwise specified, conventional methods. The experimental materials used in the following examples are, unless otherwise specified, purchased from conventional biochemical reagent stores. The glycerol content of the crude glycerol raw material used in the following examples is 853 g / L, and the fatty acid methyl ester content is about 100 g / L, mainly comprising methyl myristate (C14:0), methyl palmitate (C16:0), methyl palmitoleate (C16:1), methyl stearate (C18:0), methyl oleate (C18:1), methyl linoleate (C18:2), and all the other impurities also include methanol, fatty acids, soaps, and inorganic salts.

[0036] Example 1: Acquisition and characterization of R. toruloides XR20 strain

[0037] Activated R. toruloides CGMCC 2.1389 was inoculated into ultracentrifugal adaptive evolution medium (118.0 g / L crude glycerol (containing 100.0 g / L glycerol), 1.0 g / L corn steep liquor, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, 1.0 g / L ammonium sulfate, 50 mL per 500 mL conical flask) and incubated at 30°C, 200 rpm in a shaker for 144 hours before passage. The culture was then divided into two 50 mL centrifuge tubes (20 mL for screening and 20 mL for parameter analysis). One of the tubes was then ultracentrifuged at varying centrifugal forces, increasing the centrifugation force to 53,000 g for 15 minutes. During transfer, the cell clumps at the top of the tube were gently dispersed with a pipette tip, and 5 mL of the upper layer of cells was aspirated to serve as seed for the next round of adaptive evolution.

[0038] At the 13th generation, due to the high crude glycerol content in the original culture medium, which may have restricted the growth of the strain, the glycerol and ammonium sulfate concentrations were reduced. The culture medium used for subsequent evolution was 95.0 g / L crude glycerol (containing 80.0 g / L glycerol), 1.0 g / L corn steep liquor, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, and 0.475 g / L ammonium sulfate. The highest centrifugal force was maintained for 15 minutes and evolution continued. When the oil production of Rhodosporidium toruloides fermentation was high and relatively stable, the culture was stopped. This evolution process was carried out for a total of 36 generations ( Figure 1The final evolved strain was streaked and single colonies were frozen and named R. toruloides XR20 (CCTCCM20251198). Compared with the original strain R. toruloides CGMCC2.1389, the cell volume of the evolved strain R. toruloides XR20 gradually increased, and the lipid droplets inside the cell also continued to increase. Subsequently, the intracellular lipid droplets changed from two small lipid droplets at both ends of the cell to large lipid droplets that almost filled the entire cell, and the proportion of intracellular lipids increased significantly ( Figure 2 The cell volume of the evolved strain increased by about 3.6 times compared to the original strain, and its oil content was 63.6%, twice that of the original strain. Its oil yield in crude glycerol nitrogen-limited medium increased from 4.5g / L to 19.8g / L, an increase of more than 3.4 times. The content of glucan, mannan, and chitin per unit surface area of ​​the cell wall decreased.

[0039] Example 2: Batch fermentation of Rhodosporidium toruloides XR20 using crude glycerol as a carbon source to produce microbial oil

[0040] The activated Rhodosporidium toruloides CGMCC2.1389 and R. toruloides XR20 were inoculated into a seed culture medium (95.0 g / L crude glycerol (containing approximately 80.0 g / L glycerol), 1.0 g / L magnesium sulfate heptahydrate, 0.415 g / L corn steep liquor, 1.25 g / L ammonium sulfate, and 2.0 g / L potassium dihydrogen phosphate) and cultured for 144 h. The culture was then inoculated into a 3 L fermentor at a 10% (v / v) inoculum size. Batch fermentation medium contained 95.0 g / L of crude glycerol (containing approximately 80.0 g / L of glycerol), 1.0 g / L of magnesium sulfate heptahydrate, 0.415 g / L of corn steep liquor, 1.25 g / L of ammonium sulfate, 2.0 g / L of potassium dihydrogen phosphate, and a 1 L liquid volume. The fermentation temperature was 30°C, the ventilation rate was 1.0 vvm, the rotation speed was 600 rpm, the pH was 5.6 (automatically adjusted with 3 M HCl and 3 M NaOH), and 5 mL of defoamer was added at the start of fermentation. Samples were taken at fermentation 0 h for initial sugar concentration, and after fermentation began, samples were taken every 24 h to measure the bacterial culture volume, residual sugar concentration, and oil yield.

[0041] Batch fermentation culture of R. toruloides XR20 was carried out for 144 hours. At the end of the fermentation, R. toruloides XR20 produced 24.5 g / L oil, which was about 40.8% higher than the oil yield of the original strain (17.4 g / L). The oil content was 65.3%, the oil yield was 0.32 g / g, and the oil productivity was 0.17 g / L / h. Figure 3The strain R. toruloides XR20 was able to utilize a small amount of fatty acid methyl esters contained in the crude glycerol culture medium. The fatty acids produced by the strain were mostly oleic acid and linoleic acid, accounting for more than 57% of the total.

[0042] Example 3: Fed-batch fermentation of microbial oil production by R. toruloides XR20 using crude glycerol as a carbon source

[0043] The culture conditions and method were the same as those in Example 2. 47.0 g of crude glycerol (containing approximately 40.0 g of glycerol), 0.725 g of ammonium sulfate, 1.0 g of potassium dihydrogen phosphate, 0.208 g of corn steep liquor, and 0.5 g of magnesium sulfate heptahydrate were weighed, diluted to 80 mL with ultrapure water, and sterilized for use as the feed solution for fed-batch fermentation. After activation, the strain R. toruloides XR20 was cultured as a fermentation seed and inoculated into a 3 L fermentor. The feed solution was added when half of the glycerol was consumed. Feeds were added on days 3, 6, and 8 of the culture, and the culture was continued for 11 days, at which point the glycerol in the culture medium was completely consumed. During the fed-batch fermentation, a total of 234.0 g / L crude glycerol was added to the culture system (containing about 200.0 g / L glycerol), and 42.56 g / L oil was produced, with an oil content of 74.9%, an oil yield of 0.182 g / g, and an oil productivity of 0.161 g / L / h. The oil yield was 73.79% higher than that of the batch fermentation ( Figure 4 ).

Claims

1. A Rhodosporidium toruloides yeast capable of utilizing crude glycerol to produce microbial oil, characterized in that: It is obtained by the following method: (1) Preparation of nitrogen-limited culture medium containing crude glycerol; (2) The starting strain of Rhodosporidium toruloides was cultured for ultracentrifugal adaptive evolution.

2. The Rhodosporidium toruloides strain capable of utilizing crude glycerol to produce microbial oil according to claim 1, characterized in that: In step (1), the crude glycerol raw material used has a glycerol content of 400-900 g / L and a fatty acid methyl ester content of about 10-200 g / L, mainly including methyl myristate, methyl palmitate, methyl palmitoleate, methyl stearate, methyl oleate, and methyl linoleate, and other impurities also include methanol, fatty acids, soaps, and inorganic salts.

3. The Rhodosporidium toruloides strain capable of producing microbial oils from crude glycerol according to claim 1, characterized in that: In step (1), the crude glycerol content in the crude glycerol culture medium used for the evolution is 50-200 g / L, and the nutrient salts include 0.1-5 g / L corn steep liquor, 0.1-5 g / L potassium dihydrogen phosphate, 0.1-5 g / L magnesium sulfate heptahydrate, and 0.1-5 g / L ammonium sulfate.

4. The Rhodosporidium toruloides strain capable of producing microbial oils from crude glycerol according to claim 1 is characterized in that: In step (2), the ultracentrifugal force adaptive evolution culture of Rhodosporidium toruloides is carried out according to the following steps: (1) Strain culture: The activated original strain of R. toruloides CGMCC 2.1389 was inoculated into a crude glycerol nitrogen-limited medium at a 5-20% (v / v) inoculum; culture conditions: a rotation speed of 50-750 rpm, a culture temperature of 15-37°C, and the culture liquid was collected after 96-240 hours of culture; (2) Continuous subculture, centrifugal stimulation, and screening: The bacterial solution was stressed and screened in a centrifuge, and the supernatant cell pellet was collected after each centrifugation for subculture; the centrifugal force was gradually increased from 0 g to 53,000 g, the centrifugation time was 0-15 min, and the inoculum size was 5-20% (v / v); continuous subculture, centrifugal stimulation, and screening were performed 20 times or more; (3) Isolation and stability evaluation of mutants: The final bacterial liquid was streaked on a crude glycerol medium plate to obtain a single colony, which was preserved after continuous subculture and named R. toruloides XR20, with the accession number CCTCC M 20251198. The deposit address was China Center for Type Culture Collection, and the deposit date was May 27, 2025.

5. The Rhodosporidium toruloides strain capable of utilizing crude glycerol to produce microbial oil according to claim 1, wherein in step (2), the stable genetic Rhodosporidium toruloides strain finally evolved is named R. toruloides XR20.

6. A strain of Rhodosporidium toruloides that can utilize crude glycerol to produce microbial oil, characterized in that: It is named R. toruloides XR20, the deposit number is CCTCC M 20251198, the deposit address is China Center for Type Culture Collection, and the deposit date is May 27, 2025.

7. Use of the Rhodosporidium toruloides strain capable of producing microbial oils from crude glycerol according to any one of claims 1 to 6, characterized in that: Fermentation of microbial lipids by mutant Rhodosporidium toruloides strains using crude glycerol.

8. The use of the Rhodosporidium toruloides strain capable of producing microbial oils from crude glycerol according to claim 7, characterized in that: The steps of batch fermentation of the strain using crude glycerol fermentation are as follows: (1) Culture medium configuration: crude glycerol content is 50-200 g / L, nutrient salts include corn steep liquor 0.1-5 g / L, potassium dihydrogen phosphate 0.1-5 g / L, magnesium sulfate heptahydrate 0.1-5 g / L, and ammonium sulfate 0.1-5 g / L; (2) Seed culture: After the Rhodosporidium toruloides XR20 strain is activated, it is inoculated with a crude glycerol content of 50-200 g / L, nutrient salts including corn steep liquor 0.1-5 g / L, potassium dihydrogen phosphate 0.1-5 g / L, magnesium sulfate heptahydrate 0.1-5 g / L, and ammonium sulfate 0.1-5 g / L, and cultured for 96-240 hours, and then inoculated with a 5-20% (v / v) inoculum into a fermentation equipment; (3) Culture conditions: Depending on the volume of the reactor and the liquid filling equipment, the liquid filling volume is 20-50%, the rotation speed is 50-750 rpm, the culture temperature is 15-37°C, and the total culture time is 96-240 h, with sampling every 6-36 h; (4) Extraction and determination of oil: dilute the bacterial solution and spread it on solid culture medium. After incubation at 15-37℃ for 24-72h, calculate the CFU by colony count; dilute the bacterial solution and determine the glycerol concentration by high performance liquid chromatography; shake the bacterial solution and take 20mL of it for centrifugation. After discarding the supernatant and washing the bacteria, resuspend the bacteria in 3-10mL of ultrapure water and dry and weigh; add 5mL of 3-6M HCl to the dry bacteria, acid hydrolyze at room temperature for 4-6h, collect the liquid, boil it for 5-15min and cool it, add 20mL of chloroform and methanol mixture and extract it for 1-5h. Take the chloroform layer and evaporate the chloroform to obtain oil and weigh it. After calculation, the oil content and yield of the oily bacteria can be obtained.

9. The use of a strain of Rhodosporidium toruloides capable of producing microbial oils from crude glycerol according to claim 7, characterized in that: The steps of using crude glycerol fermentation to carry out fed-batch fermentation of the strain are as follows: Steps (1) to (4) are consistent with claim 7, the number of feeding times is 1-5 times, the feeding interval is 24-72 hours, and the strain culture is stopped when the glycerol of the last feeding is completely consumed.

10. The use of the Rhodosporidium toruloides strain capable of utilizing crude glycerol to produce microbial oil according to claim 9, wherein the feed medium contains 50-200 g of crude glycerol, 0.1-5 g of corn steep liquor, 0.1-5 g of potassium dihydrogen phosphate, 0.1-5 g of magnesium sulfate heptahydrate, and 0.1-5 g of ammonium sulfate, and a small amount of ultrapure water is used to prepare a mother liquor.

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