CN105018539A - Method for cultivating high-yield DHA (docosahexaenoic acid) through schizochytrium - Google Patents
Method for cultivating high-yield DHA (docosahexaenoic acid) through schizochytrium Download PDFInfo
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Abstract
The invention discloses a method for cultivating high-yield DHA (docosahexaenoic acid) through schizochytrium, and belongs to the field of microbial fermentation. The method comprises steps as follows: a schizochytrium seed solution with the dry cell weight being 18-22 g/L after culturing in a shake flask is inoculated into a seed tank and then inoculated into a fermentation tank after being cultured to reach an inoculation transfer level, high-density culture is performed at the high temperature being 22 DEG C-33 DEG C in an early stage, induction culture is performed in a threatening manner for 110-130 hours at the low temperature being 18 DEG C-22 DEG C in a late stage, and glucose and total nitrogen are kept in a certain concentration and pH (potential of hydrogen) is kept ranging from 5.5 to 6.6 through feeding of a glucose solution, a nitrogen source solution and an alkaline solution in the fermentation process. The dry cell weight of a fermentation solution obtained with the method reaches 120g/L-150 g/L, the density of the DHA reaches 40g/L-50 g/L and is 1.2-1.5 times higher than reported DHA productivity, and the method is very suitable for industrial production of DHA.
Description
Technical field
The invention belongs to field of microbial fermentation, relate to the production of DHA, be specifically related to a kind of method of cultivating schizochytrium limacinum high yield DHA.
Background technology
Docosahexenoic acid (22:6n-3, docosahexaenoic acid, DHA) be a kind of important serial polyunsaturated fatty acid of ω-3 (ω-3polyunsaturated fatty acids), it can either promote infant's intelligence and visual acuity, has again the effect such as prevention the elderly cardiovascular disorder, anticancer, anti-inflammatory, strengthening immunity.The method of traditional extraction polyunsaturated fatty acid obtains from fish oil, deficient gradually along with environmental pollution and marine fishery resources, from bathypelagic fish, extract unsaturated fatty acids manifest and deposit problems, such as quality instability, exogenous pollution, smell are bad, purifying high in cost of production, and the EPA contained in fish oil and cholesterol also make it be unsuitable for being added in pregnant baby's food.On the contrary, along with the continuous maturation of microbial fermentation technology, and the further investigation of fermentable grease technology, from microorganism, obtain grease not only easily realize scale operation, but also the problem that traditional fish oil ubiquity is subject to region and weather conditionality can be overcome, application prospect is boundless.
Schizochytrium limacinum (Schizochytrium) is one of desirable species of current suitability for industrialized production DHA, it belongs to a class marine microalgae of thraustochytriale section, cultivated by high density fermentation, significant quantities of fat can be accumulated in its body, and 70% exists as a triglyceride, wherein DHA can account for 45 ~ 55% of total fatty acids.
Schizochytrium limacinum belongs to heterotroph microorganism, and glucose, fructose and glycerine are the good sources of carbon that schizochytrium limacinum fermentation produces DHA, and wherein glucose is the suitableeest carbon source.Traditional Industrialization is produced algae oil DHA and is mainly used glucose as carbon source, but produce 1 ton of DHA grease and approximately need consumption 5 tons of glucose, this conversion is inefficient, in addition need in production process to input large energy, investment in equipment and maintenance costly, makes the production cost of current algae oil DHA very high.
Summary of the invention
The object of the invention is to overcome the shortcoming of prior art and deficiency, a kind of method of cultivating schizochytrium limacinum high yield DHA is provided.
Object of the present invention is achieved through the following technical solutions:
Cultivate a method of schizochytrium limacinum high yield DHA, comprise the steps:
(1) by schizochytrium limacinum strain inoculation in the shaking flask that substratum is housed, in 25 ~ 35 DEG C of shaking table, carry out the permanent rotating speed of constant temperature with 150 ~ 250rpm rotating speed cultivate 20 ~ 35h.
(2) seed liquor that step (1) obtains is transferred to is equipped with in the shaking flask of substratum, inoculation volume is 5 ~ 10% of substratum liquid amount, in 25 ~ 30 DEG C, carry out the permanent rotating speed of constant temperature under 150 ~ 250rpm condition and cultivate 16 ~ 20h, measure the dry cell weight of gained seed liquor, if dry cell weight reaches 18 ~ 22g/L in the seed liquor obtained, then directly carry out step (4).
(3) if dry cell weight does not reach 18 ~ 22g/L in the seed liquor that obtains of step (2), then it is transferred to further and is equipped with in the shaking flask of substratum, cultivate according to the method in step (2); Repeat this step one or many extremely to cultivate dry cell weight in the seed liquor obtained and reach 18 ~ 22g/L.
(4) seed liquor above-mentioned dry cell weight being reached 18 ~ 22g/L is inoculated in the seeding tank that substratum is housed with the inoculum size of 5 ~ 10% (V/V) carries out fermentation culture; Controlling fermentation pH by ammoniacal liquor or liquid caustic soda maintains between 5.5 ~ 6.5, and culture temperature controls at 25 ~ 35 DEG C, and air flow controls at 0.3 ~ 0.5vvm, is cultured to glucose in fermented liquid and exhausts, and stops cultivating;
(5) by the fermented liquid in step (4) seeding tank with 5 ~ 10% inoculum size be inoculated in the fermentor tank that substratum is housed and carry out fermentation culture; Culture temperature controls at 22 ~ 33 DEG C, air flow controls at 0.3 ~ 0.5vvm, add glucose concn in glucose solution control fermented liquid by stream and maintain 30 ~ 50g/L, the concentration being added total nitrogen in mixed nitrogen solution control fermenting process by stream maintains 15 ~ 25g/L, comprises one or more in yeast extract, Dried Corn Steep Liquor Powder, Sodium Glutamate, peptone in this mixed nitrogen; Control fermenting process pH by Feeding ammonia water or liquid caustic soda and maintain 5.5 ~ 6.5; Carry out low temperature stress cultivation after fermentation culture to 70 ~ 100h, temperature is reduced to 18 ~ 22 DEG C, all the other conditions are constant, continue cultivation 30 ~ 50h.
Above-mentioned steps (1), (2) formula of the substratum and in (3) is: glucose 40 ~ 50g/L, yeast extract 10 ~ 25g/L, sodium sulfate 5.0 ~ 10.0g/L, Repone K 0.6 ~ 0.8g/L, magnesium sulfate 1.8 ~ 2.2g/L, potassium primary phosphate 1.9 ~ 2.lg/L, calcium chloride 0.14 ~ 0.16g/L, ammonium sulfate 0.8 ~ 1.2g/L, tetrahydrate manganese chloride 1.68 ~ 1.72mg/L, Zinc Sulphate Heptahydrate 2.8 ~ 3.0mg/L, cupric sulfate pentahydrate 1.4 ~ 1.6mg/L, Sodium Molybdate Dihydrate 0 ~ 0.05mg/L, CoCL2 6H2O 0 ~ 0.05mg/L.
The formula of the substratum in above-mentioned steps (4) and (5) is: glucose 75.0 ~ 85.0g/L, Dried Corn Steep Liquor Powder 15 ~ 20g/L, yeast extract 2.9 ~ 3.lg/L, Sodium Glutamate 5.0 ~ 15.0g/L, sodium sulfate 5.0 ~ 10.0g/L, Repone K 0.6 ~ 0.8g/L, magnesium sulfate 1.8 ~ 2.2g/L, potassium primary phosphate 1.9 ~ 2.lg/L, calcium chloride 0.14 ~ 0.16g/L, ammonium sulfate 0.8 ~ 1.2g/L, tetrahydrate manganese chloride 1.68 ~ 1.72mg/L, Zinc Sulphate Heptahydrate 2.8 ~ 3.0mg/L, cupric sulfate pentahydrate 1.4 ~ 1.6mg/L, Sodium Molybdate Dihydrate 0 ~ 0.05mg/L, CoCL2 6H2O 0 ~ 0.05mg/L.
Schizochytrium limacinum bacterial classification described in step (1) is preferably schizochytrium limacinum ATCC20888.Preferred, step (1) is: by the schizochytrium limacinum ATCC20888 strain inoculation of preserving with glycerine in the 250m shaking flask that 50mL substratum is housed, and carries out the permanent rotating speed of constant temperature cultivate 25 ~ 35h in 25 ~ 30 DEG C of shaking tables with 150 ~ 250rpm rotating speed.
The concentration of step (4) and the ammoniacal liquor described in (5) is preferably 28 ~ 30% (V/V), and described liquid paper mill wastewater is preferably 30 ~ 40% (W/V).
The concentration of the glucose solution described in step (5) is preferably 700 ~ 750g/L, and the concentration of described mixed nitrogen solution is preferably 200 ~ 500g/L.
The volume of the seeding tank described in step (4) is preferably 200L, and tank body liquid amount is preferably 150L; The volume of the fermentor tank described in step (5) is preferably 3000L, and tank body liquid amount is preferably 1500L.
The present invention is by being optimized the substratum of schizochytrium limacinum fermentative production DHA and culture condition, the traditional aerobic fermentation method of Bian, relatively high temperature is adopted to carry out high-density culture in the 3000L ferment tank prometaphase, the fermentation later stage carries out low temperature stress cultivation, this cultural method both reduced fermentation costs, reduce again the control difficulty of fermenting process, the dry cell weight of fermentation ends secondary fermentation liquid reaches 120 ~ 150g/L, DHA reaches 40 ~ 50g/L, exceed 1.2 ~ 1.5 times than reported DHA productivity, be applicable to very much the suitability for industrialized production being applied to DHA.The method that the present invention cultivates schizochytrium limacinum high yield DHA has following advantage:
(1) adopt prometaphase high-temperature cultivation, the low temperature stress inducing culture of short period of time in later stage, compared with traditional low temperature fermentation, energy consumption at least reduces by 30%, greatly reduces the demand of fermenting process to energy, and the requirement to refrigeration equipment.
(2) in the present invention, used medium formula is compared to traditional zymotic, and production level is higher, and cost significantly reduces, and can reduce fermentation risk while additional income.
(3) by high-density aerobic fermentation method cultivate schizochytrium limacinum produce the polyunsaturated fatty acid such as DHA (PuFA) Ke Minus is few cause because the market requirement is excessive fish for the environmental influence brought in a large number; therefore the protection of this invention to environmental resources has potential meaning; simultaneously by products such as Production by Microorganism Fermentation docosahexenoic acids; the three wastes produced in production process are less, and construction and the cost of investment of factory are relatively low.
(4) although the lipid content of the high-density culture schizochytrium limacinum fermentation production found at present is usually low than traditional fish oil, but the grease that fermentable is produced contains necessary lipid acid in a large number, and form fairly simple by the lipid acid that microbe fermentation method obtains, the content of polyunsaturated fatty acid is very high, enrichment is also than extracting much easier from traditional fish oil, this high purity is very favourable to subsequent extracted technique, can purification process be simplified, greatly can reduce production cost in the industrial production.
(5) do not limited by the factor of season and climate change by the technique of microbe fermentation method acquisition polyunsaturated fatty acid, can produce the whole year, and be not subject to the restriction in raw material and the place of production, add the handiness that polyunsaturated fatty acid is produced.
(6) extract by microbe fermentation method the grease obtained and contain more cholesterol unlike fish oil goods, add the purity of product and the applicable surface of product, make the quality of product more guaranteed.
(7) producing DHA by Schizochytrium in high-density culture through fermentation, its bacterial classification schizochytrium limacinum rich in proteins, trace element, VITAMIN, antioxidant etc., can as the source of macronutrient and micro-nutrients in food prescription.
(8) quality that not only can improve product of fermenting is carried out by microorganism, and can genetic engineering means be utilized and in conjunction with the pathways metabolism of production by biological polyunsaturated fatty acid, utilize the method for metabolic regulation to the composition of control PuFA, thus the Artificial Control of products production can be realized.
Embodiment
Below in conjunction with embodiment, further detailed description is done to the present invention, but embodiments of the present invention are not limited thereto.
Embodiment 1
(1) substratum is prepared
The compound method of Shake flask medium is as follows: after the component tetrahydrate manganese chloride in formula, Zinc Sulphate Heptahydrate, cupric sulfate pentahydrate, Sodium Molybdate Dihydrate, CoCL2 6H2O being weighed, fully dissolve with sterilized water, be prepared into mother liquor, under acidic conditions, 121 DEG C of high pressure steam sterilization 20min; After other compositions weigh, divide after fully dissolving with sterilized water and be filled to shaking flask, 121 DEG C of high pressure steam sterilization 20min.
Shake-flask culture based formulas is as follows: glucose 45g/L, yeast extract 10g/L, sodium sulfate 8.0g/L, Repone K 0.7g/L, magnesium sulfate 2.0g/L, potassium primary phosphate 2.0g/L, calcium chloride 0.15g/L, ammonium sulfate 1.0g/L, tetrahydrate manganese chloride 1.7mg/L, Zinc Sulphate Heptahydrate 3.0mg/L, cupric sulfate pentahydrate 1.5mg/L, Sodium Molybdate Dihydrate 0.05mg/L, CoCL2 6H2O 0.05mg/L.
The compound method of fermentation tank culture medium is as follows: after being weighed in proportion by all components in formula, fully dissolve, 121 DEG C of high pressure steam sterilization 20min with sterilized water, for subsequent use after cooling.
Fermentor cultivation based formulas is as follows: glucose 80.0g/L, Dried Corn Steep Liquor Powder 15g/L, yeast extract 3.0g/L, Sodium Glutamate 10.0g/L, sodium sulfate 8.0g/L, Repone K 0.7g/L, magnesium sulfate 2.0g/L, potassium primary phosphate 2.0g/L, calcium chloride 0.15g/L, ammonium sulfate 1.0g/L, tetrahydrate manganese chloride 1.7mg/L, Zinc Sulphate Heptahydrate 3.0mg/L, cupric sulfate pentahydrate 1.5mg/L, Sodium Molybdate Dihydrate 0.05mg/L, CoCL2 6H2O 0.05mg/L.
(2) go bail for and be hidden in the glycerine pipe schizochytrium limacinum ATCC20888 bacterial classification of Ultralow Temperature Freezer, be seeded to 2 and be equipped with in the 250mL Oscillating bottle of 50mL Shake flask medium, with the rotating speed of 150rpm in 25 DEG C Oscillating beds, cultivate 24h; Then be seeded to 300 with the inoculum size of 5% Secondary Culture in the 250mL Oscillating bottle of 50mL substratum is housed, with the rotating speed of 150rpm in 25 DEG C Oscillating beds, cultivate 19.5h, the dry cell weight measured in seed liquor is 18.7g/L, collects seed liquor.
(3) Jiang Oscillating bottle seed liquor according to 10% in the 200L first class seed pot that 150L fermentation tank culture medium is housed, carry out fermentation culture by planting by kind amount, culture temperature is 28 DEG C, air flow is 0.5vvm, whole fermenting process utilizes 30% ammoniacal liquor to control the pH of fermented liquid between 5.5 ~ 6.0, when glucose content is lower than 5g/L in fermented liquid during fermentation to 35 ~ 45h, stop fermentation.
(4) the seed liquor 150L in first class seed pot is all seeded in the 3000L fermentor tank that 1500L fermentation tank culture medium is housed and carries out fermentation culture, culture temperature is 28 DEG C, air flow is 0.5vvm, and whole fermenting process utilizes 30% ammoniacal liquor to control the pH of fermented liquid between 5.5 ~ 6.0.Fermentation adds the glucose solution of 750g/L and the mixed nitrogen solution (this solution comprises 30g/L yeast extract, 100g/L Sodium Glutamate, 100g/L Dried Corn Steep Liquor Powder) of 230g/L to starting stream during 20h, glucose and total nitrogen content in fermented liquid is detected every 3h sampling, by in stream rate of acceleration control fermented liquid, glucose concn maintains 30 ~ 50g/L all the time, total nitrogen content maintains 15 ~ 25g/L, when fermentation culture is to 115h, terminate fermentation.By lyophilize detect put tank time fermented liquid in dry cell weight be 121.9g/L; Detect compound lard content by organic solvent extractionprocess and account for 61.02% of dry cell weight, gas phase analysis detects DHA content in compound lard and accounts for 51.12% of compound lard dry weight, and in fermented liquid, the content of DHA can reach 38.02g/L.After this Batch fermentation, the component analysis of gained compound lard is in table 1:
Table 1
| Fatty acid species | Percent mass composition (%) |
| C12:0 | 0.06 |
| C14:0 | 0.57 |
| C16:0 | 32.16 |
| C16:1 | 0.06 |
| C18:0 | 1.44 |
| C18:1 | 0.05 |
| C18:2 | 0.05 |
| C18:3 | 0.07 |
| C20:3 | 0.36 |
| Squalene | 0.45 |
| C20:5 | 0.39 |
| C22:0 | 0.27 |
| C22:5 | 12.97 |
| C22:6 | 51.12 |
Embodiment 2
Embodiment 2 compared with embodiment 1 except the method for cultivating in 3000L fermentation cylinder for fermentation is different, all the other are all identical, be specially: the seed liquor 150L in embodiment 1 first class seed pot is all seeded in the 3000L fermentor tank that 1500L fermentation tank culture medium is housed and carries out fermentation culture, culture temperature is 28 DEG C, air flow is 0.5vvm, and whole fermenting process utilizes 30% ammoniacal liquor to control the pH of fermented liquid between 5.5 ~ 6.0.Ferment to starting stream during 20h and add the glucose solution of 750g/L and the mixed nitrogen solution (this solution comprises 30g/L yeast extract, 100g/L Sodium Glutamate, 100g/L Dried Corn Steep Liquor Powder) with 230g/L, glucose and total nitrogen content in fermented liquid is detected, by stream rate of acceleration control fermented liquid, glucose concn maintains 30 ~ 50g/L all the time, total nitrogen content maintains 15 ~ 25g/L every 3h sampling.Fermentation culture carries out low temperature stress cultivation to during 77h, in 30min, culture temperature is reduced to 20 DEG C, continues to cultivate 37h, terminates fermentation.After testing, when putting tank, in fermented liquid, dry cell weight reaches 138.5g/L, and compound lard content accounts for 63.5% of dry cell weight, and DHA content accounts for 56.85% of compound lard dry weight, and in fermented liquid, the content of DHA can reach 49.99g/L.After this Batch fermentation, the component analysis of gained compound lard is in table 2:
Table 2
| Fatty acid species | Percent mass composition (%) |
| C12:0 | 0.02 |
| C14:0 | 0.58 |
| C16:0 | 26.27 |
| C16:1 | 0.06 |
| C18:0 | 1.26 |
| C18:1 | 0.05 |
| C18:2 | 0.08 |
| C18:3 | 0.05 |
| C20:3 | 0.27 |
| Squalene | 0.65 |
| C20:5 | 0.32 |
| C22:0 | 0.30 |
| C22:5 | 13.24 |
| C22:6 | 56.85 |
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (6)
1. cultivate a method of schizochytrium limacinum high yield DHA, it is characterized in that comprising the steps:
(1) by schizochytrium limacinum strain inoculation in the shaking flask that substratum is housed, in 25 ~ 35 DEG C of shaking table, carry out the permanent rotating speed of constant temperature with 150 ~ 250rpm rotating speed cultivate 20 ~ 35h;
(2) seed liquor that step (1) obtains is transferred to is equipped with in the shaking flask of substratum, inoculation volume is 5 ~ 10% of substratum liquid amount, in 25 ~ 30 DEG C, carry out the permanent rotating speed of constant temperature under 150 ~ 250rpm condition and cultivate 16 ~ 20h, measure the dry cell weight of gained seed liquor, if dry cell weight reaches 18 ~ 22g/L in the seed liquor obtained, then directly carry out step (4);
(3) if dry cell weight does not reach 18 ~ 22g/L in the seed liquor that obtains of step (2), then it is transferred to further and is equipped with in the shaking flask of substratum, cultivate according to the method in step (2); Repeat this step one or many extremely to cultivate dry cell weight in the seed liquor obtained and reach 18 ~ 22g/L;
(4) above-mentioned dry cell weight is reached the seed liquor of 18 ~ 22g/L with 5 ~ 10%(V/V) inoculum size be inoculated in the seeding tank that substratum is housed and carry out fermentation culture; Controlling fermentation pH by ammoniacal liquor or liquid caustic soda maintains between 5.5 ~ 6.5, and culture temperature controls at 25 ~ 35 DEG C, and air flow controls at 0.3 ~ 0.5vvm, is cultured to glucose in fermented liquid and exhausts, and stops cultivating;
(5) by the fermented liquid in step (4) seeding tank with 5 ~ 10% inoculum size be inoculated in the fermentor tank that substratum is housed and carry out fermentation culture; Culture temperature controls at 22 ~ 33 DEG C, air flow controls at 0.3 ~ 0.5vvm, add glucose concn in glucose solution control fermented liquid by stream and maintain 30 ~ 50g/L, add total nitrogen concentration in mixed nitrogen solution control fermenting process by stream and maintain 15 ~ 25g/L, in this mixed nitrogen, comprise one or more in yeast extract, Dried Corn Steep Liquor Powder, Sodium Glutamate, peptone; Control fermenting process pH by Feeding ammonia water or liquid caustic soda and maintain 5.5 ~ 6.5; Carry out low temperature stress cultivation after fermentation culture to 70 ~ 100h, temperature is reduced to 18 ~ 22 DEG C, all the other conditions are constant, continue cultivation 30 ~ 50h;
Above-mentioned steps (1), (2) formula of the substratum and in (3) is: glucose 40 ~ 50g/L, yeast extract 10 ~ 25g/L, sodium sulfate 5.0 ~ 10.0g/L, Repone K 0.6 ~ 0.8g/L, magnesium sulfate 1. 8 ~ 2.2g/L, potassium primary phosphate 1.9 ~ 2.lg/L, calcium chloride 0.14 ~ 0.16g/L, ammonium sulfate 0.8 ~ 1.2g/L, tetrahydrate manganese chloride 1.68 ~ 1.72mg/L, Zinc Sulphate Heptahydrate 2.8 ~ 3.0mg/L, cupric sulfate pentahydrate 1.4 ~ 1.6mg/L, Sodium Molybdate Dihydrate 0 ~ 0.05mg/L, CoCL2 6H2O 0 ~ 0.05mg/L,
The formula of the substratum in above-mentioned steps (4) and (5) is: glucose 75.0 ~ 85.0g/L, Dried Corn Steep Liquor Powder 15 ~ 20g/L, yeast extract 2.9 ~ 3.lg/L, Sodium Glutamate 5.0 ~ 15.0g/L, sodium sulfate 5.0 ~ 10.0g/L, Repone K 0.6 ~ 0.8g/L, magnesium sulfate 1.8 ~ 2.2g/L, potassium primary phosphate 1.9 ~ 2.lg/L, calcium chloride 0.14 ~ 0.16g/L, ammonium sulfate 0.8 ~ 1.2g/L, tetrahydrate manganese chloride 1.68 ~ 1.72mg/L, Zinc Sulphate Heptahydrate 2.8 ~ 3.0mg/L, cupric sulfate pentahydrate 1.4 ~ 1.6mg/L, Sodium Molybdate Dihydrate 0 ~ 0.05mg/L, CoCL2 6H2O 0 ~ 0.05mg/L.
2. the method for producing DHA by Schizochytrium in high-density culture through fermentation according to claim 1, is characterized in that: the schizochytrium limacinum bacterial classification described in step (1) is schizochytrium limacinum ATCC20888.
3. the method for producing DHA by Schizochytrium in high-density culture through fermentation according to claim 1, it is characterized in that: step (1) is: by the schizochytrium limacinum ATCC20888 strain inoculation of preserving with glycerine in the 250m shaking flask that 50mL substratum is housed, in 25 ~ 30 DEG C of shaking tables, carry out the permanent rotating speed of constant temperature with 150 ~ 250rpm rotating speed cultivate 25 ~ 35h.
4. the method for producing DHA by Schizochytrium in high-density culture through fermentation according to claim 1, is characterized in that: the concentration of step (4) and the ammoniacal liquor described in (5) is 28 ~ 30%(V/V), described liquid paper mill wastewater is 30 ~ 40%(W/V).
5. the method for producing DHA by Schizochytrium in high-density culture through fermentation according to claim 1, is characterized in that: the concentration of the glucose solution described in step (5) is 700 ~ 750g/L, and the concentration of described mixed nitrogen solution is 200 ~ 500g/L.
6. the method for producing DHA by Schizochytrium in high-density culture through fermentation according to claim 1, is characterized in that:
The volume of the seeding tank described in step (4) is 200L, and tank body liquid amount is 150L;
The volume of the fermentor tank described in step (5) is 3000L, and tank body liquid amount is 1500L.
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