A method for producing oil by co-culturing candida tropicalis and yarrowia lipolytica

Abstract

The present application relates to the technical field of bioengineering fermentation, in particular to a method for producing oil by co-culturing Candida and Yarrowia lipolytica, which comprises wastewater enzymatic pretreatment, double-bacterium mixed inoculation, aerobic constant-temperature fermentation and product separation and co-production. The Candida and Yarrowia lipolytica are co-cultured in a limited proportion, the substrate utilization complementarity of the double bacteria is utilized to improve the biomass, and then the nitrogen starvation environment is created by regulating the carbon-nitrogen ratio in the middle and late stages of fermentation to directionally induce the microorganism to synthesize a large amount of unsaturated fatty acid type single-cell oil in cells. The fermentation tail liquid can be directly discharged up to the standard, and the oil-extracting bacterial residue co-produces high-protein feed additive, without adding large amounts of nutrient medium, so that the four values of "pollution treatment, emission reduction, oil production and by-product feed" of tallow processing wastewater are realized, and the problems of environmental protection pressure and resource waste in the industry are solved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering fermentation technology, and more specifically, to a method for producing oil by co-culturing Candida albicans and Yersinia lipolyticis. Background Technology

[0002] In the large-scale processing of tallow, core stages such as hydration degumming, enzymatic refining, and centrifugal separation generate a large amount of process wastewater, accounting for approximately 10% to 15% of the raw tallow mass. This type of wastewater is characterized by a high organic load, containing 3% to 5% water-soluble denatured proteins, 2% to 4% soluble polysaccharides, as well as nutrients such as glycerol, short-chain fatty acids, and trace amounts of gums. Its chemical oxygen demand (COD) is as high as 10,000 to 25,000 mg / L, making it extremely difficult to treat.

[0003] Currently, the traditional treatment mode in the industry generally adopts the "anaerobic + aerobic" biochemical combination process. This mode not only has high infrastructure investment, expensive operation and maintenance costs, and outstanding energy consumption, but more importantly, the effective resources such as natural carbon, nitrogen, and lipids contained in the wastewater are completely wasted. In addition, a large amount of residual sludge is generated during the biochemical treatment process. This sludge is suspected of being hazardous waste, which is difficult and costly to dispose of, and continues to increase the environmental compliance pressure on oil processing enterprises.

[0004] While existing literature and patents contain research on cultivating oil-producing microorganisms using food processing wastewater, they generally suffer from four major technical bottlenecks, lacking industrial applicability and failing to meet actual industry needs: (1) The problem of substrate inhibition is prominent: the long-chain fatty acids, salts and macromolecular colloidal proteins contained in the wastewater of butter processing will strongly inhibit the growth of microbial cells and lipid synthesis if they are used directly for fermentation without targeted degradation, resulting in extremely low biomass and oil accumulation rate, making it impossible to achieve large-scale production. (2) The use of a single strain has limitations: a single lipophilic yeast or Candida albicans can only preferentially utilize a single type of carbon source and cannot simultaneously and efficiently degrade complex substrates such as sugars, amino acids, and organic acids in wastewater. The substrate utilization rate is generally less than 40%, resulting in serious waste of resources. (3) The quality of oil is low: the microbial oil produced by conventional single-strain fermentation is mainly saturated fatty acids, with a very low proportion of high-value polyunsaturated fatty acids. It cannot replace fish oil and high-end vegetable oil, and is difficult to apply to high-value-added fields such as health products, cosmetics and high-end feed. (4) No complete process loop: Existing technologies only focus on the production of microbial oils, without simultaneously addressing industry pain points such as wastewater discharge compliance and co-production of microbial residue resources. The industrial chain is incomplete, lacks practical industrial promotion value, and cannot truly solve the dual dilemma of environmental protection and economic benefits in the industry.

[0005] Therefore, a method is needed to produce oil by co-culturing Candida and Yersinia lipolytica to solve these problems. Summary of the Invention

[0006] The purpose of this invention is to provide a method for producing oil by co-culturing Candida albicans and Yersinia lipolytica, in order to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for producing oil by co-culturing Candida albicans and Yersinia lipolytica, comprising the following steps: S1. Wastewater enzymatic pretreatment: Collect high-organic wastewater generated from tallow processing, adjust the pH to 6.5-7.0, add 0.05%-0.2% of a compound enzyme by mass of the wastewater, wherein the mass ratio of protease to saccharifying enzyme in the compound enzyme is 2-4:1, hydrolyze at 45-55℃ for 2-4 hours, and after hydrolysis, raise the temperature to inactivate the enzyme to obtain a natural fermentation culture medium; S2. Mixed inoculation of two strains: Activated Candida albicans and Yersinia lipolytica are simultaneously inoculated into the obtained fermentation medium. The inoculation volume ratio of Candida albicans to Yersinia lipolytica is controlled at 1:2 to 1:5. S3, Aerobic isothermal fermentation: Ferment at 28-32℃ under aerobic shaking or ventilation conditions for 72-96h. The carbon-nitrogen ratio of the fermentation system is controlled at 40:1 to 60:1 to induce intracellular lipid accumulation in the cells. S4. Product separation and co-production: After fermentation, the bacterial cells are collected by centrifugation and the microbial oil is extracted. The remaining bacterial residue after oil extraction is dried to prepare high-protein feed additives. The fermentation tail liquid is discharged in compliance with standards.

[0008] As a preferred technical solution of the present invention, the butter processing wastewater is the process wastewater generated from the butter hydration degumming, enzymatic degumming or centrifugal refining section, and the raw water COD concentration is 10000-25000 mg / L.

[0009] As a preferred embodiment of the present invention, the protease is a neutral protease or a flavor protease, and the saccharifying enzyme is a fungal α-amylase.

[0010] As a preferred embodiment of the present invention, the enzyme inactivation temperature is 85-95℃ and the holding time is 8-12 minutes.

[0011] As a preferred embodiment of the present invention, the shaking speed of the fermentation flask is controlled at 180-200 rpm, and the aeration volume ratio of the fermentation tank is controlled at 1:1.

[0012] As a preferred embodiment of the present invention, the total unsaturated fatty acids in the obtained microbial oil account for more than 70% by mass, mainly including oleic acid and linoleic acid.

[0013] As a preferred embodiment of the present invention, after the fermentation is completed, the COD of the tail liquid is reduced to below 500 mg / L, and the ammonia nitrogen removal rate is not less than 95%.

[0014] As a preferred embodiment of the present invention, the biomass of the co-culture system of Candida albicans and Yersinia lipolytica reaches 22.5 g / L and the oil yield is 12.8 g / L, while the oil yield of fermentation by Candida albicans alone is only 5.3 g / L, the oil yield of fermentation by fermentation by Yersinia lipolytica alone is 7.1 g / L, and the oil yield of fermentation by mixed fermentation of Candida albicans and Saccharomyces cerevisiae is 6.8 g / L.

[0015] As a preferred technical solution of the present invention, the Candida albicans and Yersinia lipolytica are co-cultured in a volume ratio to form a synergistic effect of "interspecific complementarity, substrate diversion and utilization, and symbiotic microbial community". The biomass (22.5 g / L) and lipid accumulation rate (58% of cell dry weight) are more than 79% higher than the existing single Yersinia lipolytica fermentation lipid yield, and the substrate utilization rate is more than 40% higher.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is a method for producing oil by co-culturing Candida albicans and Yersinia lipolytica. This invention uses high-organic wastewater from butter processing in Ningxia as the sole substrate. Through pH control and targeted enzymatic hydrolysis by compound protease + saccharifying enzyme, the large molecular proteins and polysaccharides in the wastewater are degraded into small molecular nutrients that yeast can directly utilize, eliminating substrate growth inhibition. A limited ratio of Candida albicans and Yersinia lipolytica is used for co-culturing, and the biomass is increased by utilizing the complementarity of the two substrates. Then, a nitrogen-starved environment is created by controlling the carbon-nitrogen ratio in the middle and late stages of fermentation, which can induce microorganisms to synthesize a large amount of unsaturated fatty acid-type single-cell oil in their cells. The fermentation tail liquid can be directly discharged in compliance with standards, and the oil extraction residue is used to co-produce high-protein feed additives. Based on the application cases of local enterprises in Ningxia, this invention does not require a large amount of external nutrient culture medium, realizing the four-fold value of "pollution treatment, emission reduction, oil production, and by-product feed" of butter processing wastewater, solving the problem of environmental pressure and resource waste in the industry. The process parameters are closed-loop and can be industrialized, which is in line with the green circular development policy of Ningxia's oil industry, and the economic, environmental and industrial benefits are outstanding. Detailed Implementation Example

[0017] Example 1 includes the following steps: S1. Wastewater enzymatic pretreatment: Collect high-organic wastewater generated from tallow processing, adjust the pH to 6.5-7.0, add 0.05%-0.2% of a compound enzyme by mass of the wastewater, wherein the mass ratio of protease to saccharifying enzyme in the compound enzyme is 2-4:1, hydrolyze at 45-55℃ for 2-4 hours, and after hydrolysis, raise the temperature to inactivate the enzyme to obtain a natural fermentation culture medium; S2. Mixed inoculation of two strains: Activated Candida albicans and Yersinia lipolytica are simultaneously inoculated into the obtained fermentation medium. The inoculation volume ratio of Candida albicans to Yersinia lipolytica is controlled at 1:2 to 1:5. S3, Aerobic isothermal fermentation: Ferment at 28-32℃ under aerobic shaking or ventilation conditions for 72-96h. The carbon-nitrogen ratio of the fermentation system is controlled at 40:1 to 60:1 to induce intracellular lipid accumulation in the cells. S4. Product separation and co-production: After fermentation, the bacterial cells are collected by centrifugation and the microbial oil is extracted. The remaining bacterial residue after oil extraction is dried to prepare high-protein feed additives. The fermentation tail liquid is discharged in compliance with standards. In this embodiment, the wastewater from the tallow processing is the process wastewater generated from the hydration and degumming, enzymatic degumming, or centrifugal refining stages of tallow processing. The raw water COD concentration is 10,000-25,000 mg / L. The protease is a neutral protease or flavor protease, the saccharifying enzyme is fungal α-amylase, the enzyme inactivation temperature is 85-95℃, and the holding time is 8-12 minutes. The shaking speed of the fermentation flask is controlled at 180-200 rpm, and the aeration volume ratio of the fermentation tank is controlled at 1:1. The total unsaturated fatty acid content in the obtained microbial oil is greater than 70%, mainly containing oleic acid and linoleic acid. After fermentation, the COD of the tail liquid drops to below 500 mg / L, and the ammonia nitrogen removal rate is not low. The co-culture system of *Candida albicans* and *Yersinia lipolytica* achieved a biomass of 22.5 g / L and an oil yield of 12.8 g / L, while the oil yield from fermentation with *Candida albicans* alone was only 5.3 g / L, and that of fermentation with *Yersinia lipolytica* alone was 7.1 g / L. The oil yield from mixed fermentation of *Candida albicans* and *Saccharomyces cerevisiae* was 6.8 g / L. The co-culture of *Candida albicans* and *Yersinia lipolytica* by volume ratio formed a synergistic effect of "interspecific complementarity, substrate diversion and utilization, and symbiotic microbial community". Its biomass (22.5 g / L) and oil accumulation rate (58% of cell dry weight) are more than 79% higher than the existing oil yield from fermentation with *Yersinia lipolytica* alone, and the substrate utilization rate is more than 40% higher. Example

[0018] Example 2 further illustrates Example 1, a standard industrial-scale full-process technology, including the following steps: (1) Collection and pretreatment of butter processing wastewater: The process wastewater discharged from the hydration degumming and centrifugal separation processes of local butter processing enterprises in Ningxia was selected. After testing, the COD of the raw water was 18000 mg / L and the natural pH was 5.8-6.2. The pH was precisely adjusted to 6.5-7.0 using sodium hydroxide and dilute hydrochloric acid, and the water bath temperature was raised and kept constant at 50±2℃ to provide optimal conditions for subsequent enzymatic hydrolysis reaction.

[0019] (2) Targeted enzymatic hydrolysis with compound enzyme: Add 0.1% of the total mass of the wastewater to the pretreated wastewater; wherein, the compound enzyme ratio is neutral protease: saccharifying enzyme = 3:1 (mass ratio); under constant temperature and low speed stirring at 50±2℃ for 2.5h, ensure that macromolecular substances are fully degraded; after the enzymatic hydrolysis is completed, raise the temperature to 90℃ and keep it at 90℃ for 10min for enzyme inactivation treatment to prevent residual enzyme activity from affecting subsequent fermentation, and then cool it naturally to the fermentation standby temperature (28~30℃).

[0020] Test results: The hydrolysis rate of macromolecular proteins was ≥85%, the total polysaccharide degradation and release rate was ≥90%, the inhibition of wastewater substrate was completely eliminated, and a clear and transparent natural fermentation culture medium was obtained.

[0021] (3) Fine-tuning of culture medium: Based on the enzymatic hydrolysate, only a small amount of inorganic salts (MgSO4 0.5g / L, KH2PO4 1.0g / L) are added. There is no need to add exogenous nutrients such as glucose and yeast extract, so as to minimize the fermentation cost and highlight the core advantage of "using waste to cultivate bacteria".

[0022] (4) Activation and co-culture inoculation of strains: Candida 107 and Yersinia lipolytica standard industrial strains were selected, activated by slant culture and expanded by liquid seed culture, and then inoculated into fermentation medium in a specified ratio; the inoculation volume ratio of Candida 1:2 and the total inoculation volume fraction was 30% to ensure that the microbial community quickly establishes a symbiotic balance.

[0023] (5) Constant temperature aerobic fermentation control: The fermentation temperature is controlled at 28-30℃; the shaking speed of the shake flask culture is 180-200 rpm, and the aeration ratio of the industrial fermentation tank is 1:1 (v / v / m) to ensure sufficient oxygen supply to the system; during the fermentation process, the pH of the system is maintained at a naturally weakly acidic level (around 6.0) without additional adjustment; the fermentation cycle is 72-96 hours; in the middle and late stages of fermentation, the carbon-nitrogen ratio of the system is maintained at 40:1-60:1 through precise regulation of substrate self-metabolism, continuously creating a nitrogen-starved environment and enhancing lipid synthesis.

[0024] (6) Harvesting of microbial cells and separation of products: After fermentation, wet microbial cells are collected by high-speed centrifugation; intracellular microbial oil is extracted by chloroform-methanol solvent extraction or supercritical CO2 extraction; the remaining microbial residue after oil extraction is dried at low temperature to prepare high-protein feed additives, thereby realizing the resource utilization of by-products; In this embodiment, the fermentation of Yersinia lipophila in Comparative Example 1 is carried out by fermentation. Using the exact same wastewater, enzymatic hydrolysis conditions, fermentation temperature and humidity, and other operating parameters as in Example 1, only a single *Yarrowia lipolyticis* was introduced, without adding *Candida*.

[0025] Results: The cell dry weight was only 14.2 g / L, the oil yield was 7.1 g / L, and the substrate utilization rate was less than 55%, which was far lower than the dual-strain co-culture system of this invention (oil yield increased by 79% and substrate utilization rate increased by 40%). Combined with the specific case data in Section 3.2, it is clearly demonstrated that dual-strain synergistic co-culture is not a simple superposition of strains. The technical effect brought about by its synergistic effect is unexpected, further confirming the inventiveness of this invention.

[0026] Control Example 2: Direct fermentation without enzymatic hydrolysis Using the exact same wastewater, bacterial strain ratio, and fermentation conditions as in Example 1, only the compound enzymatic hydrolysis step was omitted, and the original wastewater was directly used for fermentation.

[0027] Results: Substrate inhibition was significant, cell growth was slow, and a large number of cells died in the later stages of fermentation. The final COD removal rate of the wastewater was only 62%, and the oil yield was less than 4.5 g / L, making it unsuitable for industrial application. This is in stark contrast to the oil yield of 12.8 g / L and COD removal rate of over 97% after enzymatic pretreatment according to this invention. Combined with the specific case data in Section 3.1, it is clearly demonstrated that the composite enzymatic pretreatment scheme of this invention is the key to overcoming substrate inhibition and achieving efficient fermentation. The limitation of its enzymatic ratio and process parameters is innovative and not a simple application of conventional enzymatic hydrolysis technology.

[0028] Comparative Example 3 exceeds the strain ratio of the present invention. Using the exact same wastewater, enzymatic hydrolysis conditions, and fermentation parameters as in Example 1, only the inoculation ratio of the strains was adjusted so that Candida albicans:Yarrowia lipolyticis = 1:6, deviating from the 1:2 to 1:5 range specified in this invention.

[0029] Results: The symbiotic balance of the microbial community was disrupted. Excessive proliferation of Yersinia lipolyticis led to an imbalance in substrate consumption, resulting in a significant decrease in biomass and a 18% drop in lipid accumulation rate compared to Example 1. Combined with the comparative data of different strain ratios in the specific case in Section 3.2, it is clearly demonstrated that the 1:2 to 1:5 inoculation ratio specified in this invention is the optimal range for achieving synergistic effects between the two strains. The determination of this ratio has undergone extensive screening experiments and is unique and scientific. It cannot be arbitrarily selected by those skilled in the art, further enhancing the inventiveness of this invention.

[0030] Comparative Example 4 deviates from the carbon-nitrogen ratio control range of this invention. Using the exact same wastewater, enzymatic hydrolysis conditions, and bacterial strain ratio as in Example 1, only the carbon-nitrogen ratio in the middle and late stages of fermentation was adjusted to 30:1, while the other parameters remained unchanged.

[0031] Results: The intracellular lipid content was only 32%, the proportion of unsaturated fatty acids was 51%, and the lipid yield was only 6.3 g / L, which was 51% lower than that in Example 1. Combined with the specific case data in Section 3.3, it is clearly demonstrated that the carbon-nitrogen ratio range of 40:1 to 60:1 defined in this invention is the key to achieving efficient lipid synthesis, further corroborating the creativity of the innovative point of metabolic flux regulation.

[0032] In this embodiment, Example 2 underwent repeated experiments, and the measured results were stable, with all indicators meeting expectations. Specific data are as follows: (1) Wastewater treatment indicators: raw water COD 18000mg / L, fermentation tail liquid COD <500mg / L, which fully meets the industrial wastewater discharge standards of Ningxia; total ammonia nitrogen removal rate >95%, of which the ammonia nitrogen removal rate of the pilot enterprise in Wuzhong, Ningxia reached 96%, and the ammonia nitrogen removal rate of the pilot enterprise in Shizuishan reached 97%, which completely solves the pain point of wastewater pollution from butter processing.

[0033] (2) Fermentation growth indicators: cell dry weight (CDW) reached 22.5 g / L; microbial oil yield was 12.8 g / L; oil substrate yield was 0.68 g oil / g consumed substrate; intracellular oil content accounted for 58% of cell dry weight, which is much higher than the existing technology level (the existing technology is generally lower than 40%). Among them, the oil yield of the pilot enterprise in Yinchuan reached 13.2 g / L, and the intracellular oil content reached 60%.

[0034] (3) Fatty acid composition of microbial oil: oleic acid (C18:1) accounts for 42.3%, linoleic acid (C18:2) accounts for 38.5%, and the total unsaturated fatty acid content is >70%; the oil is cholesterol-free and rich in conjugated linoleic acid (CLA) precursors, which can be directly applied to health products, cosmetics, high-end feed and biodiesel fields. The product has extremely high added value. According to the test, its quality is superior to conventional vegetable oils and can replace imported fish oil for high-end feed production.

[0035] (4) By-product indicators: The crude protein content of the dried mushroom residue after oil extraction is >45%, which fully meets the relevant national standards for feed additives and can be sold directly. The pilot enterprises in Ningxia produce 320 kg of mushroom residue per day, and the annual income from the newly added by-products exceeds 5 million yuan, further enhancing the economic benefits of the industry.

[0036] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for producing oil by co-culturing Candida albicans and Yersinia lipolytica, characterized in that, Includes the following steps: S1. Wastewater enzymatic pretreatment: Collect high-organic wastewater generated from tallow processing, adjust the pH to 6.5-7.0, add 0.05%-0.2% of a compound enzyme by mass of the wastewater, wherein the mass ratio of protease to saccharifying enzyme in the compound enzyme is 2-4:1, hydrolyze at 45-55℃ for 2-4 hours, and after hydrolysis, raise the temperature to inactivate the enzyme to obtain a natural fermentation culture medium; S2. Mixed inoculation of two strains: Activated Candida albicans and Yersinia lipolytica are simultaneously inoculated into the obtained fermentation medium. The inoculation volume ratio of Candida albicans to Yersinia lipolytica is controlled at 1:2 to 1:

5. S3, Aerobic isothermal fermentation: Ferment at 28-32℃ under aerobic shaking or ventilation conditions for 72-96h. The carbon-nitrogen ratio of the fermentation system is controlled at 40:1 to 60:1 to induce intracellular lipid accumulation in the cells. S4. Product separation and co-production: After fermentation, the bacterial cells are collected by centrifugation and the microbial oil is extracted. The remaining bacterial residue after oil extraction is dried to prepare high-protein feed additives. The fermentation tail liquid is discharged in compliance with standards.

2. The method for producing oil by co-culturing Candida and Yersinia lipolytica according to claim 1, characterized in that, The wastewater from the butter processing is the process wastewater generated from the butter hydration and degumming, enzymatic degumming, or centrifugal refining stages, with a raw water COD concentration of 10,000-25,000 mg / L.

3. The method for producing oil by co-culturing Candida and Yersinia lipolytica according to claim 2, characterized in that, The protease is a neutral protease or a flavor protease, and the saccharifying enzyme is a fungal α-amylase.

4. The method for producing oil by co-culturing Candida and Yersinia lipolytica according to claim 1, characterized in that, The enzyme inactivation temperature is 85-95℃, and the holding time is 8-12 minutes.

5. A method for producing oil by co-culturing Candida and Yersinia lipolytica according to claim 1, characterized in that, The shaking speed of the fermentation flask is controlled at 180-200 rpm, and the aeration volume ratio of the fermentation tank is controlled at 1:

1.

6. The method for producing oil by co-culturing Candida and Yersinia lipolytica according to claim 1, characterized in that, The obtained microbial oil contains more than 70% total unsaturated fatty acids, mainly oleic acid and linoleic acid.

7. A method for producing oil by co-culturing Candida and Yersinia lipolytica according to claim 1, characterized in that, After the fermentation is completed, the COD of the tail liquid drops to below 500 mg / L, and the ammonia nitrogen removal rate is not less than 95%.

8. A method for producing oil by co-culturing Candida and Yersinia lipolytica according to claim 1, characterized in that, The co-culture system of Candida albicans and Yersinia lipolytica achieved a biomass of 22.5 g / L and an oil yield of 12.8 g / L, while the oil yield of Candida albicans fermentation alone was only 5.3 g / L, the oil yield of Yersinia lipolytica fermentation alone was 7.1 g / L, and the oil yield of Candida albicans and Saccharomyces cerevisiae mixed fermentation was 6.8 g / L.

9. A method for producing oil by co-culturing Candida and Yersinia lipolytica according to claim 1, characterized in that, The co-culture of Candida albicans and Yersinia lipolytica in a volume ratio creates a synergistic effect of "interspecific complementarity, substrate diversion and utilization, and symbiotic microbial community". The biomass (22.5 g / L) and lipid accumulation rate (58% of cell dry weight) are more than 79% higher than the existing single Yersinia lipolytica fermentation lipid yield, and the substrate utilization rate is more than 40% higher.