Density layering self-flooding reactor and fermentation method using same

Abstract

The invention provides a density stratified self-flooding reactor and a fermentation method using the same, the density stratified self-flooding reactor comprises a primary fermentation tank, a secondary fermentation tank, a tertiary fermentation tank and a quaternary fermentation tank, the installation heights of the primary fermentation tank, the secondary fermentation tank, the tertiary fermentation tank and the quaternary fermentation tank are decreased step by step, and the primary fermentation tank is communicated with the secondary fermentation tank through a first communicating pipe; the first-stage fermentation tank is communicated with the first-stage fermentation tank through a first communicating pipe, the second-stage fermentation tank is communicated with the third-stage fermentation tank through a second communicating pipe, the third-stage fermentation tank is communicated with the fourth-stage fermentation tank through a third communicating pipe, the first communicating pipe, the second communicating pipe and the third communicating pipe are each provided with one or two communicating valves, and the inner wall of the first-stage fermentation tank, the inner wall of the second-stage fermentation tank and the inner wall of the third-stage fermentation tank are each provided with an inclined annular groove. The horizontal height of the groove bottom position of the inclined ring groove is lower than the horizontal height of the groove opening position, the lowest point position of the inclined ring groove is communicated with the communicating pipe, and the communicating position of each communicating pipe and the previous-stage fermentation tank is higher than the communicating position of the communicating pipe and the next-stage fermentation tank.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation, in particular to a density-stratified self-flooding reactor and a fermentation method using the device. Background Art

[0002] Oils and fats are the basic raw materials for food, bioenergy, functional materials and pharmaceutical chemicals. Their main chemical components are long-chain fatty acid triglycerides. The current global annual oil production is about 250 million tons. As an important resource, oils and fats not only support the production of traditional light chemical industries such as detergents and surfactants, but are also the most ideal raw materials for the development of sustainable aviation fuels. my country is short of oil and fat resources and has long relied on foreign imports. In order to get out of the predicament, microbial oils and fats came into being. Microbial oils and fats are lipids stored in the form of triglycerides in microorganisms. Microbial oils and fats have the advantages of short growth cycle, high oil yield, low production cost, and are not affected by geographical and environmental factors. Therefore, they have good development prospects and have become a hot spot for development today.

[0003] There are many shortcomings in the production of oil using existing fermentation equipment, such as long fermentation cycle, large fermentation volume, affecting the fermentation performance of the strain, severe foaming in the middle and late stages of fermentation, etc. In order to solve the difficulties in the fermentation process, increase 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 to be solved by the present invention is to provide a density-stratified self-flooding reactor and a fermentation method using the reactor, which shortens the time for producing a unit product, shortens the fermentation cycle of the second, third and fourth stage fermentations by 10-30%, reduces the volume of the fermentation equipment, improves the fermentation performance of the strain, reduces the bubble phenomenon in the middle and late stages of fermentation, increases the fermentation rate and saves costs.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present invention is: a density stratified self-flooding reactor, comprising a primary fermentation tank, a secondary fermentation tank, a tertiary fermentation tank and a quaternary fermentation tank with gradually decreasing installation heights, the primary fermentation tank is connected to the secondary fermentation tank through a first connecting pipe, the secondary fermentation tank is connected to the tertiary fermentation tank through a second connecting pipe, the tertiary fermentation tank is connected to the quaternary fermentation tank through a third connecting pipe, one or two connecting valves are arranged on the first connecting pipe, the second connecting pipe and the third connecting pipe, the primary fermentation tank, the secondary fermentation tank and the tertiary fermentation tank are provided with inclined annular grooves on their inner walls, the horizontal height of the groove bottom position of the inclined annular groove is lower than the horizontal height of the groove mouth position, the lowest point position of the inclined annular groove is connected to the connecting pipe, and the connecting position of each connecting pipe with the upper-level fermentation tank is higher than the connecting position with the lower-level fermentation tank.

[0006] Preferably, the bottom of each upper-stage fermentation tank is at a height of 3 / 5 of the height of the tank body of the lower-stage fermentation tank.

[0007] Preferably, the connection position of each connecting pipe to the fermentation tank at the previous level is located at the 2 / 3 position of the height of the tank body.

[0008] Preferably, the highest point of the inclined annular groove is located at the 4 / 5 position of the height of the fermentation tank body where it is located.

[0009] Preferably, each fermentation tank is covered with a jacket. The upper part of the tank body of each fermentation tank has a feeding port and a supplementary feeding port. The side of the tank body has a material transfer pipe, a sampling port and a sensor. The bottom of the tank body has a sewage discharge port. There is a stirring paddle in the tank body. The top of the stirring paddle is connected to a motor. There is an air delivery pipe extending from the upper part of the tank body into the bottom of the tank body in the tank body. The air delivery pipe is communicated with a steam filter and an air fine filter.

[0010] The present invention also provides a fermentation method, using the density stratification self-flooding reactor described in any one of the above, including the following steps:

[0011] a Sterilize the culture medium in the first-stage fermentation tank and then inoculate seeds. When the OD value of the culture medium in the first-stage fermentation tank reaches 130-180, open the connection valve on the first connecting pipe, so that the culture medium in the inclined annular groove in the first-stage fermentation tank flows into the second-stage fermentation tank that has been sterilized with the culture medium and cooled to the specified fermentation temperature under the action of gravity through the first connecting pipe;

[0012] b When 10% of the culture medium in the first-stage fermentation tank flows into the second-stage fermentation tank, close the connection valve on the first connecting pipe, and supplement the culture medium to the first-stage fermentation tank to the initial volume of the culture medium in the first-stage fermentation tank;

[0013] c When the OD value of the culture medium in the second-stage fermentation tank reaches 130-180, open the connection valve on the second connecting pipe, so that the culture medium in the inclined annular groove in the second-stage fermentation tank flows into the third-stage fermentation tank that has been sterilized with the culture medium and cooled to the specified fermentation temperature under the action of gravity through the second connecting pipe;

[0014] d When 20% of the culture medium in the second-stage fermentation tank flows into the third-stage fermentation tank, close the connection valve on the second connecting pipe, and supplement the culture medium to the second-stage fermentation tank. The volume of the supplemented culture medium is equal to the volume of the culture medium flowing from the second-stage fermentation tank into the third-stage fermentation tank;

[0015] e When the OD value of the culture medium in the third-stage fermentation tank reaches 130-180, open the connection valve on the third connecting pipe, so that the culture medium in the inclined annular groove in the third-stage fermentation tank flows into the fourth-stage fermentation tank that has been sterilized with the culture medium and cooled to the specified fermentation temperature under the action of gravity through the third connecting pipe;

[0016] f When 30% of the medium in the third-stage fermenter flows into the fourth-stage fermenter, close the connecting valve on the third connecting pipe, replenish the medium for the third-stage fermenter, and the volume of the replenished medium is equal to the volume of the medium flowing from the third-stage fermenter into the fourth-stage fermenter;

[0017] g The first-stage fermenter, the second-stage fermenter, the third-stage fermenter, and the fourth-stage fermenter all reach the fermentation end point simultaneously.

[0018] Preferably, in step b, the medium replenished for the first-stage fermenter is the same as the initial medium in the first-stage fermenter.

[0019] Preferably, in step d, the medium replenished for the second-stage fermenter is the same as the initial medium in the second-stage fermenter.

[0020] Preferably, in step f, the medium replenished for the third-stage fermenter is the same as the initial medium in the third-stage fermenter.

[0021] Preferably, in step g, the fermentation end point means that the oil production in the fermenter reaches 22 - 50%.

[0022] Preferably, there are differences in the initial fermentation volumes; preferably, in a decreasing order; preferably, the volume of the initial medium in the second-stage fermenter is 90% of the volume of the initial medium in the first-stage fermenter; the volume of the initial medium in the third-stage fermenter is 80% of the volume of the initial medium in the first-stage fermenter; the volume of the initial medium in the fourth-stage fermenter is 70% of the volume of the initial medium in the first-stage fermenter. The first-stage fermenter is prepared according to 100% of its volume, the second-stage fermenter is fixed-volume according to 90% of the volume, the third-stage fermenter is fixed-volume according to 80% of the volume, and the fourth-stage fermenter is fixed-volume according to 70% of the volume.

[0023] Preferably, the masses of the components of the initial media in the first-stage fermenter, the second-stage fermenter, the third-stage fermenter, and the fourth-stage fermenter are the same.

[0024] Preferably, the medium formulas of each stage of the fermenter are different. Preferably, the nitrogen source concentrations in each stage of the 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 culture times of each stage of the fermenter are different, in a decreasing order; preferably, the time for the medium in the first-stage fermenter to flow into the second-stage fermenter through the first connecting pipe lasts for 0 - 48 h; the time for the medium in the second-stage fermenter to flow into the third-stage fermenter through the second connecting pipe lasts for 0 - 48 h; the time for the medium in the third-stage fermenter to flow into the fourth-stage fermenter through the third connecting pipe lasts for 0 - 48 h.

[0026] Preferably, the components of the fermentation medium include 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 vitamin, 24-40% pH regulator, pH 3.5-5.5, and trace elements 1 ppm-120 ppm.

[0027] Preferably, the organic nitrogen source is selected from one or more of yeast extract powder, tryptone, corn steep powder, and soy 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, cellobiose, 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 one of magnesium sulfate or magnesium chloride.

[0031] Preferably, the vitamin is selected from one or more of nicotinic acid, vitamin B1, vitamin B6, pantothenic acid, biotin, inositol, and riboflavin.

[0032] Preferably, the trace elements are 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 the present invention are as follows: Utilizing the principle that oleaginous bacteria are light in specific gravity and easily float on the surface, the strains with rapid oil accumulation are transferred to the next-stage fermentation tank as seed liquid for continuous fermentation, reducing the culture frequencies of shake flask seeds, primary seed tanks, and secondary seed tanks, and reducing the production cost. At the same time, the culture volumes of each stage of fermentation are maintained, the dissolved oxygen of each stage of fermentation is maintained, the yield is increased, and the problem of excessive foam in the middle and late stages of fermentation is solved. Description of the Drawings

[0034] Figure 1 is the overall structural schematic diagram of the specific implementation manner of the present invention;

[0035] Figure 2 is Figure 1 the partial structural schematic diagram of the internal groove structure in the primary fermentation tank in;

[0036] Figure 3 is Figure 1 the structural schematic diagram of the fourth-stage fermentation tank in;

[0037] Figure 4 is the kinetic change process diagram of the simulated fermentation;

[0038] Figure 5 is the lipid production distribution map simulating the end point of fermentation;

[0039] Figure 6 is the biomass and lipid production map simulating the end point of fermentation;

[0040] In the figure: 1. Primary fermenter; 1-1. Tilted annular groove; 1-2. Bottom position of the lowest point of the annular groove; 1-3. Notch position of the lowest point of the annular groove; 2. Secondary fermenter; 3. Tertiary fermenter; 4. Quaternary fermenter; 4-1. Motor; 4-2. Feeding port; 4-3. Air delivery pipeline; 4-4. Upper interface of the jacket; 4-5. Agitator; 4-6. Jacket; 4-7. Lower interface of the jacket; 4-8. Charging port; 4-9. Transfer pipe; 4-10. Sensor; 4-11. Sampling port; 4-12. Drain port; 5. First connecting pipe; 6. Second connecting pipe; 7. Third connecting pipe; 8. Connecting valve. Specific embodiments

[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 only used to explain the present invention and are not used to limit the present 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", "outer", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be understood as a limitation to the present invention. In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood through specific situations.

[0042] Such as Figure 1As shown in the figure, the present invention provides a density-stratified self-flooding reactor, which includes a first-stage fermentation tank 1, a second-stage fermentation tank 2, a third-stage fermentation tank 3, and a fourth-stage fermentation tank 4 with gradually decreasing installation heights. The first-stage fermentation tank 1 is connected to the second-stage fermentation tank 2 through a first connecting pipe 5, the second-stage fermentation tank 2 is connected to the third-stage fermentation tank 3 through a second connecting pipe 6, and the third-stage fermentation tank 3 is connected to the fourth-stage fermentation tank 4 through a third connecting pipe 7. Two connecting valves 8 are provided on each of the first connecting pipe 5, the second connecting pipe 6, and the third connecting pipe 7. Inclined ring grooves 1-1 are provided on the inner walls of the first-stage fermentation tank 1, the second-stage fermentation tank 2, and the third-stage fermentation tank 3. As Figure 2 shown, the horizontal height of the bottom position of the inclined ring groove 1-1 is lower than the horizontal height of the notch position. The lowest point position of the inclined ring groove 1-1 is connected to the connecting pipe, and the connecting position of each connecting pipe with its upper-stage fermentation tank is higher than the connecting position with its lower-stage fermentation tank.

[0043] The height of the bottom of each upper-stage fermentation tank is located at the 3 / 5 position of the height of the body of its lower-stage fermentation tank. The connection position of each connecting pipe with its upper-stage fermentation tank is located at the 2 / 3 position of the height of the tank body. The highest point of the inclined ring groove is located at the 4 / 5 position of the height of the fermentation tank body where it is located.

[0044] As Figure 3 shown, jackets are provided outside each stage of fermentation tank. Each stage of fermentation tank has a feeding port 4-8 and a feeding supplement port 4-2 at the upper part of the tank body, a material transfer pipe 4-9, a sampling port 4-11, and a sensor 4-10 on the side of the tank body, a sewage discharge port 4-12 at the bottom of the tank body, a stirring paddle 4-5 inside the tank body, the top of the stirring paddle 4-5 is connected to a motor 4-1, and an air delivery pipe 4-3 extending from the upper part of the tank body to the bottom of the tank body is provided inside the tank body. The air delivery pipe 4-3 is connected to a steam filter and an air fine filter.

[0045] Example 1

[0046] A fermentation method using the above density-stratified self-flooding reactor includes the following steps:

[0047] a. After sterilizing the culture medium in the first-stage fermentation tank and inoculating seeds, when the OD value of the culture medium in the first-stage fermentation tank reaches 130-180, open the connecting valve on the first connecting pipe, so that the culture medium in the inclined ring groove in the first-stage fermentation tank flows into the second-stage fermentation tank that has been sterilized with the culture medium and cooled to the specified fermentation temperature under the action of gravity through the first connecting pipe;

[0048] b When 10% of the medium in the primary fermenter flows into the secondary fermenter, close the connection valve on the first connecting pipe. The duration of this process is 0 - 48 hours starting from the time when the primary fermenter begins to flow into the secondary fermenter. Supplement the medium to the primary fermenter to the initial volume of the medium in the primary fermenter. Preferably, the medium supplemented to the primary fermenter is the same as the initial medium in the primary fermenter.

[0049] c When the OD value of the medium in the secondary fermenter reaches 130 - 180, open the connection valve on the second connecting pipe, so that the medium in the inclined ring groove in the secondary fermenter flows into the tertiary fermenter that has been sterilized and cooled to the specified fermentation temperature through the second connecting pipe under the action of gravity.

[0050] d When 20% of the medium in the secondary fermenter flows into the tertiary fermenter, close the connection valve on the second connecting pipe. The duration of this process is 0 - 48 hours starting from the time when it begins to flow into the tertiary fermenter. Supplement the medium to the secondary fermenter, and the volume of the supplemented medium is equal to the volume of the medium that the secondary fermenter flows into the tertiary fermenter. Preferably, the medium supplemented to the secondary fermenter is the same as the initial medium in the secondary fermenter.

[0051] e When the OD value of the medium in the tertiary fermenter reaches 130 - 180, open the connection valve on the third connecting pipe, so that the medium in the inclined ring groove in the tertiary fermenter flows into the quaternary fermenter that has been sterilized and cooled to the specified fermentation temperature through the third connecting pipe under the action of gravity.

[0052] f When 30% of the medium in the tertiary fermenter flows into the quaternary fermenter, close the connection valve on the third connecting pipe. The duration of this process is 0 - 48 hours. Supplement the medium to the tertiary fermenter, and the volume of the supplemented medium is equal to the volume of the medium that the tertiary fermenter flows into the quaternary fermenter. Preferably, the medium supplemented to the tertiary fermenter is the same as the initial medium in the tertiary fermenter.

[0053] g The primary fermenter, secondary fermenter, tertiary fermenter, and quaternary fermenter reach the fermentation end point simultaneously. The fermentation end point means that the oil production in the fermenter reaches 22 - 50%.

[0054] Preferably, the initial volume of the medium in the secondary fermenter is 90% of the initial volume of the medium in the primary fermenter; the initial volume of the medium in the tertiary fermenter is 80% of the initial volume of the medium in the primary fermenter; the initial volume of the medium in the quaternary fermenter is 70% of the initial volume of the medium in the primary fermenter. The primary fermenter is prepared according to 100% of its volume, the second group of fermenters is fixed at 90% of the volume, the tertiary fermenter is fixed at 80% of the volume, and the quaternary fermenter is fixed at 70% of the volume.

[0055] In a preferred embodiment, the components of the culture medium in the first-stage fermenter, the second-stage fermenter, the third-stage fermenter, and the fourth-stage fermenter have the same mass.

[0056] In another preferred embodiment, the culture medium formulations of each stage of the fermenter are different. Specifically, 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%. There are also differences in the initial fermentation volume and the culture time of each stage of the fermenter, showing a decreasing trend in sequence.

[0057] The fermentation broth of the above fermentation method is collected as follows: after the cells accumulate oil, their density decreases, and they float on the surface of the fermentation broth. They enter the inclined ring groove along the wall of the fermenter and then move into the next fermenter according to natural gravity in the inclined ring groove. This process has a certain duration in time and needs to last for several or more than ten hours; the culture times of each stage of the fermenter are different, showing a decreasing trend in sequence.

[0058] Example 2

[0059] A fermentation method using the above density-stratified self-flooding reactor, and the strain used for fermentation is Yarrowia lipolytica.

[0060] The fermentation conditions are as follows: temperature 28 - 32°C, dissolved oxygen controlled at 5% - 20%, ventilation rate 0.3 - 1.2 VVM, and rotation speed controlled at 50 - 1000 rpm. The method includes the following steps:

[0061] a. After sterilizing the culture medium in the first-stage fermenter, inoculate the seeds. When the OD value of the culture medium in the first-stage fermenter reaches 130 - 180 (preferably 150), open the connection valve on the first connecting pipe, and let the culture medium in the inclined ring groove of the first-stage fermenter flow into the second-stage fermenter that 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 first-stage fermenter flows into the second-stage fermenter, close the connection valve on the first connecting pipe. The duration of this process is 0 - 48 h starting from the time when the first-stage fermenter starts to flow into the second-stage fermenter. Supplement the culture medium to the first-stage fermenter to its initial volume, and the supplemented culture medium is the same as the initial culture medium of the first-stage fermenter.

[0063] c. When the OD value of the culture medium in the second-stage fermenter reaches 130 - 180 (preferably 150), open the connection valve on the second connecting pipe, and let the culture medium in the inclined ring groove of the second-stage fermenter flow into the third-stage fermenter that has been sterilized 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 fermentation tank flows into the tertiary fermentation tank, close the connection valve on the second connecting pipe. The duration of this process is 0 - 48 hours starting from the time when the flow into the tertiary fermentation tank begins. The volume of the culture medium supplemented to the secondary fermentation tank is equal to the volume of the culture medium flowing from the secondary fermentation tank into the tertiary fermentation tank, and the supplemented culture medium is the same as the initial culture medium in the secondary fermentation tank;

[0065] e When the OD value of the culture medium in the tertiary fermentation tank reaches 130 - 180 (preferably 150), open the connection valve on the third connecting pipe, so that the culture medium in the inclined ring groove in the tertiary fermentation tank flows into the quaternary fermentation tank that has been sterilized and cooled to the specified fermentation temperature under the action of gravity through the third connecting pipe;

[0066] f When 30% of the culture medium in the tertiary fermentation tank flows into the quaternary fermentation tank, close the connection valve on the third connecting pipe. The duration of this process is 0 - 48 hours. The volume of the culture medium supplemented to the tertiary fermentation tank is equal to the volume of the culture medium flowing from the tertiary fermentation tank into the quaternary fermentation tank, and the supplemented culture medium is the same as the initial culture medium in the tertiary fermentation tank;

[0067] g The primary fermentation tank, secondary fermentation tank, tertiary fermentation tank, and quaternary fermentation tank all reach the fermentation end point simultaneously. The fermentation end point means that the oil yield reaches 22 - 50%.

[0068] Among them, the volume of the initial culture medium in the secondary fermentation tank is 90% of the volume of the initial culture medium in the primary fermentation tank; the volume of the initial culture medium in the tertiary fermentation tank is 80% of the volume of the initial culture medium in the primary fermentation tank; the volume of the initial culture medium in the quaternary fermentation tank is 70% of the volume of the initial culture medium in the primary fermentation tank. The mass of each component of the culture medium is the same. The primary fermentation tank is prepared according to 100% of its volume, the secondary fermentation tank is fixed-volume at 90% of the volume, the tertiary fermentation tank is fixed-volume at 80% of the volume, and the quaternary fermentation tank is fixed-volume at 70% of the volume.

[0069] The collection of the fermentation broth of the above fermentation method is that after the bacteria accumulate oil, the density becomes lower, floats on the surface of the fermentation broth, enters the inclined ring groove along the wall of the fermentation tank, and then moves into the next fermentation tank according to natural gravity in the inclined ring groove. This process has a certain continuity in time and needs to last for several or more than ten hours; the initial fermentation volumes of each stage of the fermentation tank are different, and the culture times of each stage of the fermentation tank are different, showing a decreasing manner in turn.

[0070] The components of the above fermentation medium are as follows: yeast extract powder 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, nicotinic acid 400.0 μg, p-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, zinc sulfate 400.0 μg. The components of the strain medium are: yeast extract powder 1 g / L, tryptone 10 g / L, glucose 20 g / L.

[0071] Advantages of the present invention

[0072] 1. According to the different cell densities of the present invention, the strains with fast and high oil accumulation located in the upper layer are separated and enter the next-stage fermenter for cultivation. The culture media of each stage of fermenter are different, the feeding process is different, and the culture time decreases successively. The time for culturing one fermenter is used to culture four fermenters, saving labor costs, reducing the cultivation frequency of shake flask seeds, primary seed tanks, and secondary seed tanks, and saving production costs.

[0073] 2. In traditional oil fermentation, in the middle and late stages of fermentation, the fermentation volume is huge and overflow is likely to occur. To avoid this situation, in the present invention, when the oil production has not reached the highest value and the fermentation volume reaches a certain level, the fermentation broth is discharged to the next-stage fermenter, improving the productivity.

[0074] 3. In traditional oil fermentation, in the middle and late stages of fermentation, the fermentation volume is huge, leading to too low dissolved oxygen in the fermentation system and reducing the oil production. In the present invention, the dissolved oxygen can be maintained within the required range in the middle and late stages of fermentation, increasing the oil production, improving the sugar conversion rate, and saving costs.

[0075] 4. In traditional oil fermentation, in the middle and late stages of fermentation, the foam is serious, resulting in overflow or material leakage, causing production accidents. The present invention reduces the fermentation volume of each stage of fermenter, eliminates overflow and material leakage, reduces the occurrence of accidents, and at the same time improves the dissolved oxygen in the fermentation system to a certain extent, increasing the oil production and improving the sugar conversion rate.

[0076] In order to further prove the technical effects produced by the density stratification self-irrigation reactor and fermentation method in the present invention, a single-cell model is used to simulate the fermentation process of the density stratification 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, and can reflect the heterogeneous distribution of lipid content.

[0078] Simulated fermentation process:

[0079] After the seeds are inoculated into the culture medium in the first-stage fermenter, when the OD value of the culture medium in the first-stage fermenter reaches 150, 10% of the upper-layer culture medium flows into the second-stage fermenter through the connection valve on the first connecting pipe, and then the culture medium is supplemented to the first-stage fermenter to the initial volume of the culture medium in the first-stage fermenter. When the OD value of the culture medium in the second-stage fermenter reaches 150, 20% of the upper-layer culture medium flows into the third-stage fermenter through the connection valve on the second connecting pipe, and then the culture medium is supplemented to the second-stage fermenter to the initial volume of the culture medium in the second-stage fermenter. When the OD value of the culture medium in the third-stage fermenter reaches 150, 30% of the upper-layer culture medium flows into the fourth-stage fermenter through the connection valve on the third connecting pipe, and then the culture medium is supplemented to the third-stage fermenter to the initial volume of the culture medium in the third-stage fermenter. Since the oleaginous bacteria are light in specific gravity and tend to float on the surface, it is assumed that the lipid content of the strains flowing from the upper-stage fermenter to the lower-stage fermenter is relatively high. For simplicity of simulation, it is assumed that the culture medium in the upper-stage fermenter flows into the lower-stage fermenter, and the process of supplementing the culture medium to the upper-stage fermenter is completed instantaneously. Among them, the initial volume of the culture medium in the second-stage fermenter is 90% of the initial volume of the culture medium in the first-stage fermenter; the initial volume of the culture medium in the third-stage fermenter is 80% of the initial volume of the culture medium in the first-stage fermenter; the initial volume of the culture medium in the fourth-stage fermenter is 70% of the initial volume of the culture medium in the first-stage fermenter. The quality of each component of the culture medium is the same, containing 80 g / L of glucose (carbon source) and 5.0 g / L of ammonium sulfate (nitrogen source).

[0080] Simulation results:

[0081] Figure 4 For the kinetic change process diagram of the simulated fermentation, when the OD value of the culture medium in the upper-stage fermenter reaches 150, it is inoculated into the lower-stage fermenter (red arrow), and the same volume of the initial culture medium is supplemented. When the carbon source in all fermenters is consumed, the simulation terminates. When the nitrogen source is consumed, lipid accumulation accelerates.

[0082] Figure 5 For the lipid yield distribution diagram at the end of the simulated fermentation, Figure 6 For the biomass and lipid yield diagram at the end of the simulated fermentation. When the fermentation ends, the lipid yield distributions of the cells in the four fermenters are similar, and the lipid content of most cells reaches more than 20%. Even though the fermentation times of each stage of the fermenter are different, similar biomass and lipid yields can be obtained finally.

[0083] The above has described the embodiments of the present invention in detail, but the content is only the preferred embodiment of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of the application of the present invention should still fall within the scope covered by the patent of the present invention.

Claims

1. A density-stratified self-flooding reactor, characterized in that: The invention comprises a primary fermentation tank, a secondary fermentation tank, a tertiary fermentation tank and a quaternary fermentation tank with gradually decreasing installation heights. The primary fermentation tank is connected to the secondary fermentation tank via a first connecting pipe, the secondary fermentation tank is connected to the tertiary fermentation tank via a second connecting pipe, the tertiary fermentation tank is connected to the quaternary fermentation tank via a third connecting pipe, one or two connecting valves are arranged on the first connecting pipe, the second connecting pipe and the third connecting pipe, the primary fermentation tank, the secondary fermentation tank and the tertiary fermentation tank are provided with inclined annular grooves on their inner walls, the horizontal height of the groove bottom position of the inclined annular groove is lower than the horizontal height of the groove mouth position, the lowest point position of the inclined annular groove is connected to the connecting pipe, and the connecting position of each connecting pipe with the upper-stage fermentation tank is higher than the connecting position with the lower-stage fermentation tank.

2. The density stratified self-flooding reactor according to claim 1, characterized in that: The bottom of each upper-level fermentation tank is located at 3 / 5 of the height of the tank body of the lower-level fermentation tank.

3. The density stratified self-flooding reactor according to claim 1, characterized in that: The connection position of each connecting pipe to the upper fermentation tank is located at 2 / 3 of the tank body height.

4. The density stratified self-flooding reactor according to claim 1, characterized in that: The highest point of the inclined ring groove is located at 4 / 5 of the height of the fermentation tank body.

5. A density stratified self-flooding reactor according to any one of claims 1 to 4, characterized in that: Each level of fermentation tank is covered with a jacket, and the upper part of the tank body of each level of fermentation tank is provided with a feeding port and a feeding port, the side of the tank body is provided with a material transfer pipe, a sampling port and a sensor, the bottom of the tank body is provided with a sewage outlet, the tank body is provided with a stirring paddle, the top of the stirring paddle is connected to a motor, the tank body is provided with an air delivery pipe extending from the upper part of the tank body to the bottom of the tank body, and the air delivery pipe is connected to a steam filter and an air fine filter.

6. A fermentation method using the density-stratified self-flooding reactor according to any one of claims 1 to 5, characterized in that The steps include: a. Sterilize the culture medium in the primary fermentation tank and then inoculate the seeds. When the OD value of the culture medium in the primary fermentation tank reaches 130-180, open the connecting valve on the first connecting pipe, so that the culture medium in the inclined ring groove in the primary fermentation tank flows into the secondary fermentation tank where the culture medium has been sterilized and cooled to the specified fermentation temperature through the first connecting pipe under the action of gravity; b. When 10% of the culture medium in the primary fermentation tank flows into the secondary fermentation tank, the connecting valve on the first connecting pipe is closed to replenish the culture medium for the primary fermentation tank to the initial volume of the culture medium in the primary fermentation tank; c. When the OD value of the culture medium in the secondary fermentation tank reaches 130-180, the connecting valve on the second connecting pipe is opened, so that the culture medium in the inclined ring groove in the secondary fermentation tank flows into the tertiary fermentation tank through the second connecting pipe under the action of gravity, where the culture medium has been eliminated and cooled to the specified fermentation temperature; d. When 20% of the culture medium in the secondary fermentation tank flows into the tertiary fermentation tank, close the connecting valve on the second connecting pipe to replenish the culture medium for the secondary fermentation tank, and the volume of the supplemented culture medium is equal to the volume of the culture medium flowing from the secondary fermentation tank into the tertiary fermentation tank; e. When the OD value of the culture medium in the third-stage fermentation tank reaches 130-180, the connecting valve on the third connecting pipe is opened, so that the culture medium in the inclined ring groove in the third-stage fermentation tank flows into the fourth-stage fermentation tank where the culture medium has been eliminated and cooled to the specified fermentation temperature through the third connecting pipe under the action of gravity; f. When 30% of the culture medium in the third-stage fermentation tank flows into the fourth-stage fermentation tank, the connecting valve on the third connecting pipe is closed to replenish the culture medium for the third-stage fermentation tank, and the volume of the supplemented culture medium is equal to the volume of the culture medium flowing from the third-stage fermentation tank into the fourth-stage fermentation tank; gThe primary fermentation tank, the secondary fermentation tank, the tertiary fermentation tank and the quaternary fermentation tank reach the fermentation endpoint at the same time.

7. The fermentation method according to claim 6, characterized in that: The volume of the initial culture medium of the secondary fermentation tank is 90% of the volume of the initial culture medium of the primary fermentation tank; the volume of the initial culture medium of the tertiary fermentation tank is 80% of the volume of the initial culture medium of the primary fermentation tank; the volume of the initial culture medium of the quaternary fermentation tank is 70% of the volume of the initial culture medium of the primary fermentation tank. The primary fermentation tank is prepared according to 100% of its volume, the second group of fermentations are prepared at 90% of the volume, the tertiary fermentation tank is prepared at 80% of the volume, and the quaternary fermentation tank is prepared at 70% of the volume.

8. The fermentation method according to claim 6, characterized in that: The culture medium in the primary fermentation tank flows into the secondary fermentation tank through the first connecting pipe for 0-48 hours; the culture medium in the secondary fermentation tank flows into the tertiary fermentation tank through the second connecting pipe for 0-48 hours; the culture medium in the tertiary fermentation tank flows into the quaternary fermentation tank through the third connecting pipe for 0-48 hours.

9. The fermentation method according to claim 6, characterized in that: The nitrogen source concentration in the primary fermentation tank is 0.3-0.6%, the nitrogen source concentration in the secondary fermentation tank is 0.6-0.9%, the nitrogen source concentration in the tertiary fermentation tank is 0.9-1.2%, and the nitrogen source concentration in the quaternary fermentation tank is 0.9-1.2%.

10. The fermentation method according to any one of claims 6 to 9, characterized in that: The components of the fermentation medium include 1-3% organic nitrogen source, 1-3% inorganic nitrogen source, 5-10% carbon source, 0.4-1.2% phosphate, 0.0.01-0.25% magnesium salt, 5-200ppm vitamins, 24-40% pH regulator, pH3.5-5.5, and trace elements 1ppm-120ppm.

Images