A density stratification self-diffusion fermentation method
By using a density-stratified self-flowing reactor and an optimized fermentation method, the problems of long oil fermentation cycle and severe bubbling were solved, resulting in a shorter fermentation cycle, smaller equipment size, and cost savings, while improving fermentation rate and strain performance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2026-03-24
AI Technical Summary
Existing fermentation equipment for producing oils suffers from problems such as long fermentation cycles, large fermentation volumes, and severe foaming in the middle and later stages of fermentation, which affect the fermentation performance of the strains and the cost.
A density-stratified self-flooding reactor is adopted, which is connected by a connecting pipe from the first to the fourth fermenter. Taking advantage of the principle that strain density can easily float on the surface, strains that accumulate oil rapidly are transferred to the next fermenter for continuous fermentation. An inclined ring trough and connecting valve are designed to control the flow of the culture medium, and the culture medium formula and feeding process are optimized.
It shortens the fermentation cycle by 10-30%, reduces the volume of fermentation equipment, improves the fermentation rate and strain performance, reduces the phenomenon of bubbles in the middle and late stages of fermentation, and lowers production costs.
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Figure CN120192827B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fermentation technology, specifically to a density-stratified self-flooding fermentation method. Background Technology
[0002] Oils and fats are fundamental raw materials for food, bioenergy, functional materials, and pharmaceuticals, with their main chemical component being long-chain fatty acid triglycerides. Currently, global annual oil and fat production is approximately 250 million tons. As an important resource, oils and fats not only support traditional light chemical industries such as detergent and surfactant production, but are also the ideal raw material for developing sustainable aviation fuels. my country faces a shortage of oil and fat resources and has long relied on imports. To overcome this predicament, microbial oils have emerged. Microbial oils are lipids stored in the form of triglycerides within microorganisms. Microbial oils possess advantages such as short growth cycles, high yields, low production costs, and independence from geographical and environmental factors, thus demonstrating promising development prospects and becoming a current hot topic.
[0003] There are many shortcomings in using existing fermentation equipment to produce oils, including long fermentation cycles, large fermentation volumes that affect the fermentation performance of strains, and severe foaming in the middle and late stages of fermentation. To solve the difficulties in the fermentation process, improve the fermentation rate, and save costs, it is imperative to design new fermentation equipment suitable for oil fermentation. Summary of the Invention
[0004] The problem this invention aims to solve is to provide a density-stratified self-flooding fermentation method that shortens the production time per unit of product, reduces the fermentation cycle of the second, third, and fourth stages of fermentation by 10-30%, reduces the volume of fermentation equipment, improves the fermentation performance of the strain, reduces the phenomenon of bubbles in the middle and late stages of fermentation, increases the fermentation rate, and saves costs.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a density-stratified self-flooding reactor, comprising a primary fermenter, a secondary fermenter, a tertiary fermenter, and a quaternary fermenter with progressively decreasing installation heights. The primary and secondary fermenters are connected by a first connecting pipe, the secondary and tertiary fermenters are connected by a second connecting pipe, and the tertiary and quaternary fermenters are connected by a third connecting pipe. Each of the first, second, and third connecting pipes is equipped with one or two connecting valves. The inner walls of the primary, secondary, and tertiary fermenters each have inclined annular grooves. The horizontal height of the bottom of the inclined annular groove is lower than the horizontal height of the opening. The lowest point of the inclined annular groove is connected to a connecting pipe. The connection position of each connecting pipe with its upper-level fermenter is higher than its connection position with its lower-level fermenter.
[0006] Preferably, the bottom of each upstream fermenter is located at 3 / 5 of the height of the downstream fermenter.
[0007] Preferably, the connection point of each connecting pipe to its upstream fermenter is located at 2 / 3 of the tank height.
[0008] Preferably, the highest point of the inclined annular groove is located at 4 / 5 of the height of the fermenter body.
[0009] Preferably, each fermentation tank is covered with a jacket, and each fermentation tank has a feeding port and a replenishing port at the top. The sides of the tank have a transfer pipe, a sampling port and a sensor. The bottom of the tank has a drain port. The tank has an agitator inside, and the top of the agitator is connected to a motor. The tank has an air delivery pipe extending from the top of the tank to the bottom of the tank. The air delivery pipe is connected to a steam filter and an air fine filter.
[0010] The present invention also provides a density-stratified self-flooding fermentation method, using the density-stratified self-flooding reactor described above, comprising the following steps:
[0011] After sterilizing the culture medium in the primary fermenter, the seed is inoculated. When the OD value of the culture medium in the primary fermenter reaches 130-180, the connecting valve on the first connecting pipe is opened, so that the culture medium in the inclined annular groove in the primary fermenter flows into the secondary fermenter, which has been sterilized and cooled to the specified fermentation temperature, through the first connecting pipe under the action of gravity.
[0012] When 10% of the culture medium in the primary fermenter flows into the secondary fermenter, close the connecting valve on the first connecting pipe to replenish the culture medium in the primary fermenter to the initial volume of the culture medium in the primary fermenter.
[0013] When the OD value of the culture medium in the secondary fermenter reaches 130-180, open the connecting valve on the second connecting pipe, so that the culture medium in the inclined annular groove in the secondary fermenter flows into the tertiary fermenter, which has been eliminated and cooled to the specified fermentation temperature, under the action of gravity.
[0014] When 20% of the culture medium in the secondary fermenter flows into the tertiary fermenter, close the connecting valve on the second connecting pipe to replenish the culture medium to the secondary fermenter. The volume of the replenished culture medium is equal to the volume of the culture medium flowing from the secondary fermenter into the tertiary fermenter.
[0015] When the OD value of the culture medium in the tertiary fermenter reaches 130-180, open the connecting valve on the third connecting pipe to allow the culture medium in the inclined annular groove of the tertiary fermenter to flow into the tertiary fermenter, which has been eliminated and cooled to the specified fermentation temperature, through the third connecting pipe under the action of gravity.
[0016] When 30% of the culture medium in the tertiary fermenter flows into the quaternary fermenter, close the connecting valve on the third connecting pipe to replenish the culture medium in the tertiary fermenter. The volume of the replenished culture medium is equal to the volume of the culture medium flowing from the tertiary fermenter into the quaternary fermenter.
[0017] The primary, secondary, tertiary, and quaternary fermenters all reach the fermentation endpoint simultaneously.
[0018] Preferably, in step b, the culture medium supplemented to the primary fermenter is the same as the initial culture medium in the primary fermenter.
[0019] Preferably, in step d, the supplemental culture medium for the secondary fermenter is the same as the initial culture medium for the secondary fermenter.
[0020] Preferably, in step f, the culture medium supplemented to the tertiary fermenter is the same as the initial culture medium of the tertiary fermenter.
[0021] Preferably, in step g, the fermentation endpoint refers to the oil yield in the fermenter reaching 22-50%.
[0022] Preferably, the initial fermentation volumes differ; preferably, they decrease sequentially; preferably, the initial culture medium volume of the secondary fermenter is 90% of the initial culture medium volume of the primary fermenter; the initial culture medium volume of the tertiary fermenter is 80% of the initial culture medium volume of the primary fermenter; and the initial culture medium volume of the quaternary fermenter is 70% of the initial culture medium volume of the primary fermenter. The primary fermenter is prepared to its full volume, the secondary fermenter is prepared to 90% of its volume, the tertiary fermenter is prepared to 80% of its volume, and the quaternary fermenter is prepared to 70% of its volume.
[0023] Preferably, the initial culture medium components in the primary fermenter, secondary fermenter, tertiary fermenter, and quaternary fermenter have the same mass.
[0024] Preferably, the culture medium formulations of each fermenter are different, and the nitrogen source concentrations in each culture medium are different. Preferably, the nitrogen source concentration in the first-stage fermenter is 0.3-0.6%, the nitrogen source concentration in the second-stage fermenter is 0.6-0.9%, the nitrogen source concentration in the third-stage fermenter is 0.9-1.2%, and the nitrogen source concentration in the fourth-stage fermenter is 0.9-1.2%.
[0025] Preferably, the fermentation time of each fermenter is different and decreases sequentially; preferably, the culture medium in the first fermenter flows into the second fermenter through the first connecting pipe for 0-48 hours; the culture medium in the second fermenter flows into the third fermenter through the second connecting pipe for 0-48 hours; and the culture medium in the third fermenter flows into the fourth fermenter through the third connecting pipe for 0-48 hours.
[0026] Preferably, the fermentation medium contains 1-3% organic nitrogen source, 1-3% inorganic nitrogen source, 5-10% carbon source, 0.4-1.2% phosphate, 0.01-0.25% magnesium salt, 5-200 ppm vitamins, 24-40% pH adjuster, pH 3.5-5.5, and 1 ppm-120 ppm trace elements.
[0027] Preferably, the organic nitrogen source is selected from one or more of yeast extract, tryptone, corn extract, and soybean peptone.
[0028] Preferably, the inorganic nitrogen source is selected from one or more of ammonium sulfate, ammonium chloride, and urea.
[0029] Preferably, the carbon source is selected from one or more of glucose, sodium acetate, butyric acid, glycerol, cellulose sugar, and kitchen waste oil, and the phosphate is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.
[0030] Preferably, the magnesium salt is selected from magnesium sulfate or magnesium chloride.
[0031] Preferably, the vitamin is selected from one or more of niacin, vitamin B1, vitamin B6, pantothenic acid, biotin, inositol, and riboflavin.
[0032] Preferably, the trace elements are derived from one or more of boric acid, potassium iodide, sodium molybdate, cobalt chloride, copper sulfate, calcium chloride, magnesium sulfate, ferrous sulfate, and manganese sulfate.
[0033] The advantages and positive effects of this invention are as follows: Utilizing the principle that oil-absorbing bacteria are lightweight and easily float on the surface, bacteria that rapidly accumulate oil are transferred to a lower-level fermenter as seed culture for continuous fermentation. This reduces the frequency of cultivation in shake flasks, primary seed tanks, and secondary seed tanks, thereby lowering production costs. Simultaneously, it maintains the culture volume and dissolved oxygen levels at each fermentation stage, improving yield and solving the problem of excessive foaming in the later stages of fermentation. Attached Figure Description
[0034] Figure 1 This is a schematic diagram of the overall structure of a specific embodiment of the present invention;
[0035] Figure 2 yes Figure 1 A partial structural diagram of the groove structure inside the intermediate-stage fermenter;
[0036] Figure 3 yes Figure 1 Schematic diagram of a four-stage fermenter;
[0037] Figure 4 It is a diagram simulating the kinetic changes during fermentation;
[0038] Figure 5 This is a lipid yield distribution map simulating the fermentation endpoint;
[0039] Figure 6 It is a graph showing the biomass and lipid yields at the simulated fermentation endpoint;
[0040] In the diagram: 1. Primary fermenter; 1-1. Inclined annular trough; 1-2. Bottom position of the lowest point of the annular trough; 1-3. Opening position of the lowest point of the annular trough; 2. Secondary fermenter; 3. Tertiary fermenter; 4. Quaternary fermenter; 4-1. Motor; 4-2. Feed inlet; 4-3. Air delivery pipe; 4-4. Upper interface of the jacket; 4-5. Agitator; 4-6. Jacket; 4-7. Lower interface of the jacket; 4-8. Feed port; 4-9. Transfer pipe; 4-10. Sensor; 4-11. Sampling port; 4-12. Drainage port; 5. First connecting pipe; 6. Second connecting pipe; 7. Third connecting pipe; 8. Connecting valve. Detailed Implementation
[0041] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In the description of the present invention, it should be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. In the description of the present invention, it should be noted that unless otherwise explicitly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in the present invention through specific circumstances.
[0042] like Figure 1As shown, this invention provides a density-stratified self-flooding reactor, comprising a primary fermenter 1, a secondary fermenter 2, a tertiary fermenter 3, and a quaternary fermenter 4 with progressively decreasing installation heights. The primary fermenter 1 and the secondary fermenter 2 are connected by a first connecting pipe 5, the secondary fermenter 2 and the tertiary fermenter 3 are connected by a second connecting pipe 6, and the tertiary fermenter 3 and the quaternary fermenter 4 are connected by a third connecting pipe 7. Two connecting valves 8 are installed on each of the first connecting pipe 5, the second connecting pipe 6, and the third connecting pipe 7. The inner walls of the primary fermenter 1, the secondary fermenter 2, and the tertiary fermenter 3 all have inclined annular grooves 1-1, such as... Figure 2 As shown, the horizontal height of the bottom of the inclined annular trough 1-1 is lower than the horizontal height of the opening. The lowest point of the inclined annular trough 1-1 is connected to the connecting pipe. The connection position of each connecting pipe with its upper-level fermenter is higher than the connection position with its lower-level fermenter.
[0043] The bottom of each upstream fermenter is located at 3 / 5 of the height of the downstream fermenter. Each connecting pipe is positioned at 2 / 3 of its height from its upstream fermenter. The highest point of the inclined annular groove is located at 4 / 5 of the height of its corresponding fermenter.
[0044] like Figure 3 As shown, each fermentation tank is covered with a jacket. The upper part of each fermentation tank has a feeding port 4-8 and a replenishing port 4-2. The side of the tank has a transfer pipe 4-9, a sampling port 4-11 and a sensor 4-10. The bottom of the tank has a drain port 4-12. The tank has an agitator 4-5 inside. The top of the agitator 4-5 is connected to a motor 4-1. The tank has an air delivery pipe 4-3 extending from the upper part of the tank to the bottom of the tank. The air delivery pipe 4-3 is connected to a steam filter and an air fine filter.
[0045] Example 1
[0046] A density-stratified self-flooding fermentation method using the above-mentioned density-stratified self-flooding reactor includes the following steps:
[0047] After sterilizing the culture medium in the primary fermenter, the seed is inoculated. When the OD value of the culture medium in the primary fermenter reaches 130-180, the connecting valve on the first connecting pipe is opened, so that the culture medium in the inclined annular groove in the primary fermenter flows into the secondary fermenter, which has been sterilized and cooled to the specified fermentation temperature, through the first connecting pipe under the action of gravity.
[0048] b. When 10% of the culture medium in the primary fermenter flows into the secondary fermenter, the connecting valve on the first connecting pipe is closed. This process lasts for 0-48 hours from the start of the flow from the primary fermenter into the secondary fermenter, replenishing the culture medium in the primary fermenter to the initial volume of the culture medium in the primary fermenter. Preferably, the culture medium replenished to the primary fermenter is the same as the initial culture medium in the primary fermenter.
[0049] c. When the OD value of the culture medium in the secondary fermenter reaches 130-180, open the connecting valve on the second connecting pipe, so that the culture medium in the inclined annular groove in the secondary fermenter flows into the tertiary fermenter, which has been eliminated and cooled to the specified fermentation temperature, through the second connecting pipe under the action of gravity.
[0050] d. When 20% of the culture medium in the secondary fermenter flows into the tertiary fermenter, the connecting valve on the second connecting pipe is closed. This process lasts for 0-48 hours from the start of the flow into the tertiary fermenter. The volume of culture medium added to the secondary fermenter is equal to the volume of culture medium flowing into the tertiary fermenter from the secondary fermenter. Preferably, the culture medium added to the secondary fermenter is the same as the initial culture medium in the secondary fermenter.
[0051] When the OD value of the culture medium in the tertiary fermenter reaches 130-180, open the connecting valve on the third connecting pipe, so that the culture medium in the inclined annular groove in the tertiary fermenter flows into the tertiary fermenter, which has been eliminated and cooled to the specified fermentation temperature, through the third connecting pipe under the action of gravity.
[0052] When 30% of the culture medium in the tertiary fermenter flows into the quaternary fermenter, the connecting valve on the third connecting pipe is closed. This process lasts for 0-48 hours. The volume of culture medium replenished to the tertiary fermenter is equal to the volume of culture medium flowing from the tertiary fermenter into the quaternary fermenter. Preferably, the culture medium replenished to the tertiary fermenter is the same as the initial culture medium in the tertiary fermenter.
[0053] The primary, secondary, tertiary, and quaternary fermenters simultaneously reach the fermentation endpoint; the fermentation endpoint refers to an oil yield of 22-50% in the fermenter.
[0054] Preferably, the initial culture medium volume of the secondary fermenter is 90% of the initial culture medium volume of the primary fermenter; the initial culture medium volume of the tertiary fermenter is 80% of the initial culture medium volume of the primary fermenter; and the initial culture medium volume of the quaternary fermenter is 70% of the initial culture medium volume of the primary fermenter. The primary fermenter is prepared according to its full volume, the secondary fermenters are prepared to 90% of their volume, the tertiary fermenters are prepared to 80% of their volume, and the quaternary fermenters are prepared to 70% of their volume.
[0055] In a preferred embodiment, the components of the culture medium in the primary, secondary, tertiary, and quaternary fermenters are of the same mass.
[0056] Another preferred approach is to use different culture medium formulations for each fermentation stage, namely, a nitrogen source concentration of 0.3-0.6% for the first-stage fermentation stage, 0.6-0.9% for the second-stage fermentation stage, 0.9-1.2% for the third-stage fermentation stage, and 0.9-1.2% for the fourth-stage fermentation stage, as well as differences in the initial fermentation volume and the culture time for each fermentation stage, in a progressively decreasing manner.
[0057] The fermentation broth collected in the above fermentation method is formed by the accumulation of oil in the microbial cells, which reduces the density and causes them to float on the surface of the fermentation broth. The broth then flows along the wall of the fermenter into an inclined annular trough, and is then moved into the next fermenter by gravity. This process has a certain duration and needs to last for several or more than ten hours. The cultivation time of each fermenter is different and decreases in sequence.
[0058] Example 2
[0059] A density-stratified self-flooding fermentation method using the aforementioned density-stratified self-flooding reactor, wherein the fermentation strain is Yersinia lipolyticis.
[0060] Fermentation conditions are: temperature 28-32℃, dissolved oxygen controlled at 5%-20%, aeration rate 0.3-1.2 VVM, and turbine rotation speed controlled at 50-1000 rpm. The process includes the following steps:
[0061] After sterilizing the culture medium in the primary fermenter, the seed is inoculated. When the OD value of the culture medium in the primary fermenter reaches 130-180 (preferably 150), the connecting valve on the first connecting pipe is opened, so that the culture medium in the inclined annular groove in the primary fermenter flows into the secondary fermenter, which has been sterilized and cooled to the specified fermentation temperature, through the first connecting pipe under the action of gravity.
[0062] b. When 10% of the culture medium in the primary fermenter flows into the secondary fermenter, close the connecting valve on the first connecting pipe. The duration of this process is 0-48 hours from the start of the flow from the primary fermenter into the secondary fermenter. This process replenishes the culture medium in the primary fermenter to the initial volume of the culture medium in the primary fermenter. The culture medium replenished to the primary fermenter is the same as the initial culture medium in the primary fermenter.
[0063] c. When the OD value of the culture medium in the secondary fermenter reaches 130-180 (preferably 150), open the connecting valve on the second connecting pipe, so that the culture medium in the inclined annular groove in the secondary fermenter flows into the tertiary fermenter, which has been eliminated and cooled to the specified fermentation temperature, through the second connecting pipe under the action of gravity.
[0064] d When 20% of the culture medium in the secondary fermenter flows into the tertiary fermenter, close the connecting valve on the second connecting pipe. This process lasts for 0-48 hours from the start of the flow into the tertiary fermenter. The volume of culture medium added to the secondary fermenter is equal to the volume of culture medium flowing into the tertiary fermenter from the secondary fermenter. The culture medium added to the secondary fermenter is the same as the initial culture medium in the secondary fermenter.
[0065] When the OD value of the culture medium in the tertiary fermenter reaches 130-180 (preferably 150), open the connecting valve on the third connecting pipe so that the culture medium in the inclined annular groove in the tertiary fermenter flows into the tertiary fermenter, which has been eliminated and cooled to the specified fermentation temperature, through the third connecting pipe under the action of gravity.
[0066] f When 30% of the culture medium in the tertiary fermenter flows into the quaternary fermenter, the connecting valve on the third connecting pipe is closed. This process lasts for 0-48 hours. The volume of culture medium added to the tertiary fermenter is equal to the volume of culture medium flowing from the tertiary fermenter into the quaternary fermenter. The culture medium added to the tertiary fermenter is the same as the initial culture medium in the tertiary fermenter.
[0067] The primary, secondary, tertiary, and quaternary fermenters simultaneously reach the fermentation endpoint, which is defined as an oil yield of 22-50%.
[0068] The initial culture medium volume of the secondary fermenter is 90% of that of the primary fermenter; the initial culture medium volume of the tertiary fermenter is 80% of that of the primary fermenter; and the initial culture medium volume of the quaternary fermenter is 70% of that of the primary fermenter. All components of the culture medium are of equal mass. The primary fermenter is prepared to its full volume, the secondary fermenter is prepared to 90% of its volume, the tertiary fermenter is prepared to 80% of its volume, and the quaternary fermenter is prepared to 70% of its volume.
[0069] The fermentation broth in the above fermentation method is collected by the accumulation of oil in the cells, which reduces the density and causes them to float on the surface of the fermentation broth. The broth is then moved along the wall of the fermenter into an inclined ring trough, and then transferred to the next fermenter by gravity. This process has a certain duration and needs to last for several or more than ten hours. The initial fermentation volume of each fermenter is different, and the cultivation time of each fermenter is different, decreasing in a sequential manner.
[0070] The fermentation medium contained the following components: yeast extract 1 g / L, tryptone 2 g / L, glucose 80 g / L, ammonium sulfate 5.0 g / L, biotin 2.0 μg, calcium pantothenate 400.0 μg, folic acid 2.0 μg, inositol 2000 μg, niacin 400.0 μg, para-aminobenzoic acid 200.0 μg, pyridoxine hydrochloride 400.0 μg, riboflavin 200.0 μg, thiamine hydrochloride 400.0 μg, boric acid 500.0 μg, copper sulfate 40.0 μg, potassium iodide 100.0 μg, ferric chloride 200.0 μg, manganese sulfate 400.0 μg, sodium molybdate 200.0 μg, and zinc sulfate 400.0 μg. The culture medium for the bacterial strain contained yeast extract 1 g / L, tryptone 10 g / L, and glucose 20 g / L.
[0071] Beneficial effects of the present invention
[0072] 1. Based on the different cell density, this invention separates the strains that accumulate oil quickly and in large quantities in the upper layer and introduces them into the next fermentation tank for cultivation. The culture medium and feeding process are different in each fermentation tank, and the cultivation time decreases sequentially. The time for cultivating one fermentation tank is used to cultivate four fermentation tanks, which saves labor costs, reduces the cultivation frequency of shake flask seed, primary seed tank and secondary seed tank, and saves production costs.
[0073] 2. In traditional oil fermentation, the fermentation volume is large in the middle and later stages, which easily leads to overflow. To avoid this situation, this invention discharges the fermentation liquid into the next fermentation tank before the oil yield reaches its maximum value and when the fermentation volume reaches a certain level, thereby improving the yield.
[0074] 3. In traditional oil fermentation, the large fermentation volume in the middle and later stages leads to low dissolved oxygen in the fermentation system, which reduces oil yield. In this invention, dissolved oxygen can be maintained within the required range in the middle and later stages of fermentation, thereby increasing oil yield, improving sugar conversion rate, and saving costs.
[0075] 4. In traditional oil fermentation, severe foaming occurs in the middle and later stages of fermentation, leading to overflow or material runoff and causing production accidents. This invention reduces the fermentation volume of each fermentation tank, eliminates overflow and material runoff, reduces the occurrence of accidents, and at the same time increases the dissolved oxygen in the fermentation system to a certain extent, thereby increasing oil yield and sugar conversion rate.
[0076] To further demonstrate the technical effects of the density-stratified self-irrigation reactor and fermentation method in this invention, a single-cell model was used to simulate the fermentation process of the density-stratified self-irrigation reactor:
[0077] This model uses the Gillespie stochastic algorithm to simulate cell growth, division, and lipid synthesis events at the single-cell level, reflecting the heterogeneous distribution of lipid content.
[0078] Simulated fermentation process:
[0079] After the seed culture medium is inoculated into the primary fermenter, when the OD value of the medium in the primary fermenter reaches 150, the upper 10% of the medium flows into the secondary fermenter through the connecting valve on the first connecting pipe, subsequently replenishing the primary fermenter with medium to its initial volume. When the OD value of the medium in the secondary fermenter reaches 150, the upper 20% of the medium flows into the tertiary fermenter through the connecting valve on the second connecting pipe, subsequently replenishing the secondary fermenter with medium to its initial volume. When the OD value of the medium in the tertiary fermenter reaches 150, the upper 30% of the medium flows into the quaternary fermenter through the connecting valve on the third connecting pipe, subsequently replenishing the tertiary fermenter with medium to its initial volume. Because lipid bacteria are lightweight and easily float on the surface, it is assumed that the strains flowing from the upper fermenter into the lower fermenter have a high lipid content. To simplify the simulation, it is assumed that the process of replenishing the upper fermenter with medium is instantaneous. The initial culture medium volume of the secondary fermenter is 90% of that of the primary fermenter; the initial culture medium volume of the tertiary fermenter is 80% of that of the primary fermenter; and the initial culture medium volume of the quaternary fermenter is 70% of that of the primary fermenter. All components of the culture medium have the same mass, containing 80 g / L of glucose (carbon source) and 5.0 g / L of ammonium sulfate (nitrogen source).
[0080] Simulation results:
[0081] Figure 4 To simulate the kinetic changes during fermentation, when the OD value of the culture medium in the upper fermenter reached 150, the next fermenter was inoculated (red arrow), and the same volume of initial culture medium was added. The simulation terminated when all carbon sources in the fermenters were depleted. When the nitrogen source was depleted, lipid accumulation accelerated.
[0082] Figure 5 To simulate the lipid yield distribution at the fermentation endpoint, Figure 6 This diagram illustrates the biomass and lipid yields at the end of the fermentation process. At the end of fermentation, the distribution of cellular lipid yields was similar across the four fermenters, with most cells exhibiting lipid content exceeding 20%. Even with different fermentation times in each fermenter, similar biomass and lipid yields were ultimately achieved.
[0083] The embodiments of the present invention have been described in detail above, but the content is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made in accordance with the claims of the present invention should still fall within the patent coverage of the present invention.
Claims
1. A density-stratified self-flooding fermentation method, using a density-stratified self-flooding reactor, the density-stratified self-flooding reactor comprising a primary fermenter, a secondary fermenter, a tertiary fermenter, and a quaternary fermenter with progressively decreasing installation heights; the primary fermenter and the secondary fermenter are connected by a first connecting pipe, the secondary fermenter and the tertiary fermenter are connected by a second connecting pipe, and the tertiary fermenter and the quaternary fermenter are connected by a third connecting pipe; each of the first, second, and third connecting pipes is equipped with one or two connecting valves; the inner walls of the primary, secondary, and tertiary fermenters each have inclined annular grooves, the bottom of the inclined annular grooves being lower than the opening of the grooves, the lowest point of the inclined annular grooves being connected to the connecting pipes; and the connection point of each connecting pipe to its upper-level fermenter being higher than its connection point to its lower-level fermenter. The bottom of each upstream fermenter is located at 3 / 5 of the height of the downstream fermenter; the connection point of each connecting pipe to its upstream fermenter is located at 2 / 3 of the tank height; the highest point of the inclined annular groove is located at 4 / 5 of the height of its respective fermenter. Each fermentation tank is covered with a jacket. Each fermentation tank has a feeding port and a replenishing port at the top. The sides of the tank have a transfer pipe, a sampling port and a sensor. The bottom of the tank has a drain port. The tank contains an agitator, and the top of the agitator is connected to a motor. The tank contains an air delivery pipe that extends from the top of the tank to the bottom of the tank. The air delivery pipe is connected to a steam filter and an air fine filter. Its features Includes the following steps: After sterilizing the culture medium in the primary fermenter, the seed is inoculated. When the OD value of the culture medium in the primary fermenter reaches 130-180, the connecting valve on the first connecting pipe is opened, so that the culture medium in the inclined annular groove in the primary fermenter flows into the secondary fermenter, which has been sterilized and cooled to the specified fermentation temperature, through the first connecting pipe under the action of gravity. When 10% of the culture medium in the primary fermenter flows into the secondary fermenter, close the connecting valve on the first connecting pipe to replenish the culture medium in the primary fermenter to the initial volume of the culture medium in the primary fermenter. When the OD value of the culture medium in the secondary fermenter reaches 130-180, open the connecting valve on the second connecting pipe, so that the culture medium in the inclined annular groove in the secondary fermenter flows into the tertiary fermenter, which has been eliminated and cooled to the specified fermentation temperature, under the action of gravity. When 20% of the culture medium in the secondary fermenter flows into the tertiary fermenter, close the connecting valve on the second connecting pipe to replenish the culture medium to the secondary fermenter. The volume of the replenished culture medium is equal to the volume of the culture medium flowing from the secondary fermenter into the tertiary fermenter. When the OD value of the culture medium in the tertiary fermenter reaches 130-180, open the connecting valve on the third connecting pipe to allow the culture medium in the inclined annular groove of the tertiary fermenter to flow into the tertiary fermenter, which has been eliminated and cooled to the specified fermentation temperature, through the third connecting pipe under the action of gravity. When 30% of the culture medium in the tertiary fermenter flows into the quaternary fermenter, close the connecting valve on the third connecting pipe to replenish the culture medium in the tertiary fermenter. The volume of the replenished culture medium is equal to the volume of the culture medium flowing from the tertiary fermenter into the quaternary fermenter. The primary, secondary, tertiary, and quaternary fermenters all reach the fermentation endpoint simultaneously.
2. The density-stratified self-flooding fermentation method according to claim 1, characterized in that: The initial culture medium volume of the secondary fermenter is 90% of the initial culture medium volume of the primary fermenter; the initial culture medium volume of the tertiary fermenter is 80% of the initial culture medium volume of the primary fermenter; and the initial culture medium volume of the quaternary fermenter is 70% of the initial culture medium volume of the primary fermenter. The primary fermenter is prepared to its full volume, the secondary fermenter is prepared to 90% of its volume, the tertiary fermenter is prepared to 80% of its volume, and the quaternary fermenter is prepared to 70% of its volume.
3. The density-stratified self-flooding fermentation method according to claim 1, characterized in that: The culture medium in the primary fermenter flows into the secondary fermenter through the first connecting pipe for 0-48 hours; the culture medium in the secondary fermenter flows into the tertiary fermenter through the second connecting pipe for 0-48 hours; and the culture medium in the tertiary fermenter flows into the quaternary fermenter through the third connecting pipe for 0-48 hours.
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