CN114214386A - Method for producing astaxanthin by heterotrophic culture of chlorella - Google Patents
Method for producing astaxanthin by heterotrophic culture of chlorella Download PDFInfo
- Publication number
- CN114214386A CN114214386A CN202111521719.6A CN202111521719A CN114214386A CN 114214386 A CN114214386 A CN 114214386A CN 202111521719 A CN202111521719 A CN 202111521719A CN 114214386 A CN114214386 A CN 114214386A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- astaxanthin
- culture
- heterotrophic
- chlorella
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 title claims abstract description 117
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 title claims abstract description 117
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 117
- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 117
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 117
- 241000195649 Chlorella <Chlorellales> Species 0.000 title claims abstract description 42
- 238000004519 manufacturing process Methods 0.000 title abstract description 22
- 239000001963 growth medium Substances 0.000 claims abstract description 64
- 230000006698 induction Effects 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 40
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 36
- 238000000855 fermentation Methods 0.000 claims abstract description 27
- 230000004151 fermentation Effects 0.000 claims abstract description 27
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229930191978 Gibberellin Natural products 0.000 claims abstract description 20
- 239000003448 gibberellin Substances 0.000 claims abstract description 20
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000011780 sodium chloride Substances 0.000 claims abstract description 18
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 238000012136 culture method Methods 0.000 claims abstract description 5
- 230000004663 cell proliferation Effects 0.000 claims abstract description 4
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims description 34
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 23
- 239000008103 glucose Substances 0.000 claims description 23
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims description 18
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 claims description 17
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 claims description 16
- 230000001939 inductive effect Effects 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical group [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 235000019270 ammonium chloride Nutrition 0.000 claims description 5
- 239000012526 feed medium Substances 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 4
- 239000007640 basal medium Substances 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 230000002572 peristaltic effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000005859 coupling reaction Methods 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 230000000052 comparative effect Effects 0.000 description 19
- 241001442241 Chromochloris zofingiensis Species 0.000 description 15
- 240000009108 Chlorella vulgaris Species 0.000 description 9
- 235000007089 Chlorella vulgaris Nutrition 0.000 description 9
- 238000009825 accumulation Methods 0.000 description 7
- 239000002028 Biomass Substances 0.000 description 5
- 241000195493 Cryptophyta Species 0.000 description 5
- GOLXRNDWAUTYKT-UHFFFAOYSA-N 3-(1H-indol-3-yl)propanoic acid Chemical compound C1=CC=C2C(CCC(=O)O)=CNC2=C1 GOLXRNDWAUTYKT-UHFFFAOYSA-N 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 4
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000001651 autotrophic effect Effects 0.000 description 3
- 235000021466 carotenoid Nutrition 0.000 description 3
- 150000001747 carotenoids Chemical class 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- PAJPWUMXBYXFCZ-UHFFFAOYSA-N 1-aminocyclopropanecarboxylic acid Chemical compound OC(=O)C1(N)CC1 PAJPWUMXBYXFCZ-UHFFFAOYSA-N 0.000 description 2
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 2
- 239000005971 1-naphthylacetic acid Substances 0.000 description 2
- 241000168517 Haematococcus lacustris Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 2
- 239000003290 indole 3-propionic acid Substances 0.000 description 2
- 239000003617 indole-3-acetic acid Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000036579 abiotic stress Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000035425 carbon utilization Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 208000012788 shakes Diseases 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Abstract
The invention relates to a method for producing astaxanthin by heterotrophic culture of chlorella, which comprises the following steps: s1, cell proliferation culture: culturing chlorella by heterotrophic culture method to increase dry weight of chlorella cells to a predetermined value; s2, astaxanthin induction culture: under the condition of maintaining heterotrophic culture, three factors are adopted to carry out combined induction on the chlorella to promote the chlorella to accumulate astaxanthin and improve the content of the astaxanthin in cells; the three factors include: adding gibberellin into the heterotrophic culture medium to make the initial concentration of gibberellin in the culture medium be 10mg/L-150 mg/L; adjusting the initial carbon-nitrogen ratio in the heterotrophic culture medium in the fermentation tank to make the carbon-nitrogen ratio C/N equal to 140-; sodium chloride was added to the heterotrophic culture medium to an initial concentration of 0.1M to 0.6M sodium chloride in the culture medium. The invention adopts a three-factor induction method on the basis of heterotrophic culture of chlorella, can obviously improve the content of astaxanthin in cells, and can improve the yield of astaxanthin in both the dry weight of chlorella and the content of astaxanthin in cells.
Description
Technical Field
The invention relates to the technical field of microalgae culture, in particular to a method for producing astaxanthin by heterotrophically culturing chlorella.
Background
Astaxanthin is a kind of rhodochrolone carotenoid, the oxidation resistance of which is 10 times that of other carotenoids, and is widely applied in the fields of aquaculture, food, medicine, cosmetics, health care products and the like in recent years due to the strong oxidation resistance. Chlorella zofingiensis is a unicellular green alga which can perform both photoautotrophy and heterotrophy using organic carbon and can accumulate astaxanthin in cells, and produces natural astaxanthin with the same structure as that produced by Haematococcus pluvialis, so Chlorella zofingiensis has been attracting attention as a production source of natural astaxanthin. Compared with the production of astaxanthin by autotrophic Haematococcus pluvialis, the Chlorella zofingiensis can be cultured at high density under heterotrophic conditions by utilizing the existing fermentation culture equipment, and the accumulation amount of secondary carotenoids such as astaxanthin and the like can be obviously improved under induction conditions to produce natural astaxanthin, which has important significance for the industrialization and commercialization of astaxanthin, so that the technology for producing astaxanthin by heterotrophic culture of Chlorella zofingiensis is concerned by people.
However, although it has been found that the cell dry weight of Chlorella vulgaris zofingiensis cultured under heterotrophic conditions for 11 days can be as high as 160 g.L-1And the astaxanthin content in the fermented cells is lower and only accounts for 0.06 percent of the dry weight of the cells. In view of this limitation, there are great difficulties in producing astaxanthin by heterotrophically cultivating chlorella. In order to solve the technical problem, researchers propose to adopt a heterotrophic-dilution-light-induced culture method under which the maximum biomass of Chlorella zofingiensis can reach 98.4 g.L-1The yield of astaxanthin can reach 73.3 mg.L-1. However, the device is not suitable for use in a kitchenThe method has some disadvantages, such as complex operation, long culture period, high cost and the like. In addition, under autotrophic conditions, some abiotic factors, such as nitrogen deficiency stress, are added to improve the biosynthesis capacity of astaxanthin, but the abiotic stress severely inhibits the growth of microalgae cells, so that the cell density and number cannot be increased, and the astaxanthin production efficiency is still low.
Besides the low content of astaxanthin in chlorella cells in the conventional culture technology, the slow growth speed and low cell density of the chlorella cells under autotrophic conditions are another big difficulty in limiting the production of astaxanthin by the chlorella.
Disclosure of Invention
Technical problem to be solved
In view of the above-mentioned disadvantages and shortcomings of the prior art, the present invention provides a method for heterotrophic culture of chlorella to produce astaxanthin. Under the condition of heterotrophic culture of Chlorella, the content of astaxanthin in Chlorella is greatly improved, the advantages of high cell growth speed, high cell density, great cell stem and the like of the heterotrophic culture can be taken into consideration, the content of astaxanthin in Chlorella zofingiensis can be obviously improved, and the technical support is improved for promoting the industrialization and commercialization of the Chlorella zofingiensis for efficiently producing astaxanthin.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
a method for heterotrophically cultivating chlorella to produce astaxanthin comprises:
s1 cell proliferation culture
Culturing chlorella by heterotrophic culture method to increase dry weight of chlorella cells to a predetermined value;
s2 astaxanthin induction culture
Under the condition of maintaining heterotrophic culture, three factors are adopted to carry out combined induction on the chlorella to promote the chlorella to accumulate astaxanthin and improve the content of the astaxanthin in cells; the three factors include:
adding gibberellin into the heterotrophic culture medium to make the initial concentration of gibberellin in the culture medium be 10mg/L-150 mg/L;
adjusting the initial carbon-nitrogen ratio in the heterotrophic culture medium in the fermentation tank to make the carbon-nitrogen ratio C/N equal to 140-;
sodium chloride was added to the heterotrophic culture medium to an initial concentration of 0.1M to 0.6M sodium chloride in the culture medium.
According to a preferred embodiment of the present invention, in S2, gibberellin is added to the heterotrophic culture medium such that the initial concentration of gibberellin in the culture medium is 10 mg/L; adjusting the initial carbon-nitrogen ratio of the heterotrophic culture medium in the fermentation tank to 180; sodium chloride was added to the heterotrophic culture medium to give an initial concentration of 0.2M sodium chloride in the culture medium.
According to the preferred embodiment of the present invention, in S1-S2, the nitrogen source in the medium during the heterotrophic culture is ammonium chloride and ammonia; the carbon source is glucose.
According to the preferred embodiment of the present invention, in S1, the culture medium is heterotrophic in a fed-batch mode, and the glucose concentration in the fermentation tank is monitored in real time, the feeding speed is adjusted in real time, and the glucose concentration in the fermentation tank is controlled to be 5g/L-20 g/L;
in S2, the culture medium is fed-batch for heterotrophic culture, and the glucose concentration in the fermentor is monitored in real time, and the feeding speed is adjusted in real time to control the glucose concentration in the fermentor to be 4.5-5.5g/L (preferably 5 g/L).
According to a preferred embodiment of the present invention, the heterotrophic culture conditions in S1-S2 are: the culture temperature is 25-28 deg.C (preferably 26 deg.C), dissolved oxygen is set at 15-25% (preferably 20%), stirring speed and dissolved oxygen coupling control; meanwhile, the pH of the culture solution in the fermentation tank is monitored in real time, and the pH of the culture solution in the fermentation tank is controlled to be 6.5 +/-0.2 by feeding ammonia water.
According to a preferred embodiment of the present invention, in S1, the culture medium comprises a basal medium and a feed medium, the basal medium uses ammonium chloride as a unit, wherein the glucose concentration is 20g/L, the initial C/N is 32: 1; when the glucose concentration in the basic culture medium is lower than 5g/L, a peristaltic pump is started to feed the feed culture medium, the components of the feed culture medium are the same as the basic culture medium, but the carbon-nitrogen ratio in the feed culture medium is 1/20 of the carbon-nitrogen ratio in the basic culture medium.
According to the preferred embodiment of the present invention, when the heterotrophic culture of the S1 stage is carried out to make the dry weight of the chlorella cells equal to or greater than 150g/L, the induction culture process of S2 is carried out, and the culture time of the S2 stage is such that the astaxanthin content in the cells is not changed or begins to decrease, the induction culture is completed. The cultivation time at the S1 stage is usually 9-11 days, and the heterotrophic cultivation conditions of the invention S1 allow the dry weight of the chlorella cells to reach about 160g/L, at which time the chlorella has proliferated to a considerably high cell number, followed by the cultivation process for inducing accumulation of astaxanthin, so that the astaxanthin content in the cells is increased, thereby obtaining the highest astaxanthin production.
According to a preferred embodiment of the present invention, in S2, one or both of oxaloacetate and γ -aminobutyric acid are further added to the heterotrophic culture medium of the fermenter.
According to a preferred embodiment of the present invention, in S2, the initial concentration of gamma-aminobutyric acid in the heterotrophic culture medium of the fermentor is 0.25mM to 1mM, but is most preferably 0.5 mM.
According to a preferred embodiment of the invention, the initial concentration of oxaloacetate in the heterotrophic culture medium of the fermentor in S2 is 0.1mM-0.5mM, but is most preferably 0.2 mM.
(III) advantageous effects
The invention divides the process of producing astaxanthin by heterotrophic culture of chlorella into two stages, one is a stage of cell mass proliferation, and the second is an induction stage based on the condition of heterotrophic culture, which promotes the accumulation of astaxanthin in chlorella cells. In the second stage, mainly based on the three-factor induction method, a certain initial concentration of gibberellin and sodium chloride is added into the culture medium of the fermentation tank, and the initial C/N in the culture medium of the fermentation tank is adjusted to be a high carbon-nitrogen ratio (140-; the astaxanthin is obtained by culturing for more than 4 days under induction, stopping culturing under induction until the astaxanthin content in the cells is not changed or begins to decrease continuously, and harvesting the chlorella at the moment. Experiments prove that the method adopts a three-factor induction method, compared with the prior art, the content of astaxanthin in cells can be obviously improved, and meanwhile, the induction culture process hardly has obvious negative influence on the growth speed of the cell number. Based on the high density and high dry weight of chlorella cells obtained in the first stage and the high content of astaxanthin obtained in the second stage, the production efficiency of the chlorella for preparing astaxanthin is improved in two aspects.
On the basis, the invention also optimizes the optimal initial concentration of gibberellin and sodium chloride; furthermore, in the second stage, a certain initial concentration of oxaloacetate and/or gamma-aminobutyric acid is also added into the culture medium of the fermentation tank, and the experimental result proves that compared with the simple three-factor induction, the addition of the certain initial concentration of oxaloacetate and/or gamma-aminobutyric acid is helpful for further inducing the accumulation speed and the accumulation amount of astaxanthin in chlorella cells.
In addition, in the first stage culture process, mainly based on a heterotrophic culture method, the chlorella can be quickly accumulated to the dry cell weight of more than or equal to 150g/L by controlling the glucose concentration, the nitrogen source and the carbon-nitrogen ratio in a basic culture medium, the carbon-nitrogen ratio in a supplemented culture medium, the glucose concentration in a fermentation tank in the whole first stage process within a preset range, a pH control method, a pH control range and other conditions, so that the initial accumulation of the cell number is achieved, and a high base number is provided for the production of the astaxanthin.
Drawings
FIG. 1 is a graph showing the cell growth rate and astaxanthin content of the chlorella vulgaris of example 1 and comparative example 1 during heterotrophic culture; (a) the cell dry weight curve, (b) the astaxanthin content curve, and (c) the astaxanthin production curve.
FIG. 2 is a comparative line graph showing the astaxanthin contents obtained after the completion of the cultivation in examples 1 to 10 and comparative example 1 (the relative contents in other examples or comparative examples were calculated by taking example 1 as 100%).
FIG. 3 is a line graph showing comparison of astaxanthin contents obtained after the completion of the cultivation in comparative examples 1 to 7 and example 1 (relative contents in other comparative examples were calculated by taking example 1 as 100%).
FIG. 4 is a graph showing the cell growth rate and astaxanthin content of the chlorella vulgaris of example 1, example 12 and example 13 during the second stage of induction culture; (a) the cell dry weight curve, (b) the astaxanthin content curve, and (c) the astaxanthin production curve.
FIG. 5 is a bar graph showing comparison of astaxanthin contents obtained after completion of the cultivation in examples 1, 11 and 13 to 15 (relative contents of other examples were calculated by taking example 11 as 100%).
FIG. 6 is a bar graph showing comparison of astaxanthin contents obtained after completion of the cultivation in examples 1, 12 and 16 to 18 (relative contents of other examples were calculated by taking example 12 as 100%).
Detailed Description
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.
Example 1
This example provides a method for heterotrophic culture of chlorella to produce astaxanthin, comprising the steps of:
the method comprises the following steps: cell proliferation culture
And (3) algae strain: chlorella vulgaris zofingiensis
The composition and amount of each medium used in the culture of Chlorella zofingiensis are shown in tables 1-3.
The whole heterotrophic culture process of Chlorella zofingiensis comprises the following steps:
(1) activation of algae seed and culture of primary, secondary and tertiary seeds
Under a sterile environment, single colonies of Chlorella zofingiensis are picked from a well-cultured plate (the Chlorella zofingiensis algae seeds are smeared on the plate until the colonies grow) by using an inoculating loop, streaked on a sterilized plate (the composition of a culture medium is shown in a table 1), and the streaked plate is placed in a dark environment to be cultured at a culture temperature of 25 ℃ for 15-20 days (obvious Chlorella zofingiensis algae single colonies are formed). See table 1 for the composition of the plating medium.
A Chlorella annulata Zofinggiensis (phi 2-3 mm) is picked from an activated fresh plate and is put into a 50mL triangular flask (the liquid loading is 15mL) for shaking culture at the culture temperature of 27 ℃ and the rotation speed of 180rpm for 120-144h (OD750 ═ 8).
Inoculating the cultured primary seed solution into a 250mL secondary shake flask (the liquid loading amount is 100mL) at the inoculation amount of 10% (v/v) for shaking culture, and culturing at the culture temperature of 27 ℃ and the rotation speed of 180rpm for 72-84 h (OD750 ═ 16-20).
The cultured secondary seed solution was inoculated into a 1000mL tertiary flask (300 mL liquid loading) at an inoculum size of 10% (v/v) and subjected to shaking culture at 27 ℃ and 180rpm for 72-84 (OD750 ═ 20-22).
The media components for the first seed culture, the second seed culture and the third seed culture are shown in Table 2.
(2) Heterotrophic culture in a fermentation tank
The culture was carried out on a 7.5L fermenter, the composition of the medium being shown in Table 3. The seed liquid of heterotrophically cultured Chlorella zofingiensis is inoculated into a 7.5L fermentation tank in an inoculation amount of 10% (v/v), ammonia water is used for controlling the pH value to be 6.5 +/-0.2 in the culture process (the ammonia water can be used as an auxiliary nitrogen source to be absorbed and utilized by algae cells and can also be used for regulating and controlling the pH value), the temperature is 26 ℃, the dissolved oxygen is set to be 20%, and the stirring speed and the dissolved oxygen are coupled to control the solution. The fermentation basic culture medium adopts ammonium chloride as a nitrogen source, the initial carbon-nitrogen ratio in the basic culture medium is 32:1, and the glucose concentration in the basic culture medium is 20 g.L-1When the glucose concentration is less than 5 g.L-1When the culture medium is fed by a peristaltic pump with adjustable speed, the feed medium and the basic medium have the same components, but the carbon-nitrogen ratio in the feed medium is 1/20 of the carbon-nitrogen ratio in the basic medium, the glucose concentration is monitored and determined, the feed speed is adjusted timely according to the change of the glucose concentration, and the glucose concentration in the whole culture process is controlled to be 5 g.L-1-20g·L-1Within the range.
Table 1: composition of plate culture medium in seed activation stage
Table 2: the culture medium for the first, second and third stage culture of seed
TABLE 3 composition of basal and feed media for heterotrophic culture in 7.5L fermentors
Step two, astaxanthin induction culture
The dry cell weight of the Chlorella zofingiensis cultured to the 10 th day reaches 160 g.L-1When the culture is performed, the heterotrophic culture and the three-factor induction culture are switched.
Adding gibberellin and sodium chloride into the fermentation tank to make the initial concentration of gibberellin be 10 mg.L-1Adjusting the initial concentration of sodium chloride to 200mM, adjusting the initial carbon-nitrogen ratio in the heterotrophic culture medium in the fermentation tank to 180/1, and controlling the feeding speed according to the change of the glucose concentration in the fermentation tank at proper time to control the glucose concentration in the whole culture process to be 5 g.L-1Left and right.
When the astaxanthin content is measured in several consecutive days by taking a sample at the same time point, the induction process is ended when the astaxanthin content in the sample is not changed or begins to decrease.
Comparative example 1
The first stage of cultivation was carried out under the conditions of the first stage in example 1, and in the second stage, gibberellin and sodium chloride were not added to the fermenter, and the initial carbon-nitrogen ratio in the heterotrophic medium in the fermenter was not adjusted to 180. The remaining conditions were carried out with reference to example 1.
Samples in the fermenter were continuously withdrawn, and the dry cell weight (%, DW), astaxanthin production (g/L) in example 1 and comparative example 1 were measured and calculated and plotted, respectively, and the results are shown in FIG. 1. As can be seen from FIG. 1 (a), the curves of cell dry weight, astaxanthin content and astaxanthin production of example 1 and comparative example 1 almost coincide with each other during the 10-day culture in the first stage. Although the rate of increase in dry cell weight in example 1 was slightly lower than that in comparative example 1 during the second-stage astaxanthin induction culture (due to accumulation of astaxanthin, the increase in biomass of chlorella vulgaris was slightly slower, and it was also in accordance with objective rules), the astaxanthin content and the astaxanthin yield, particularly the astaxanthin yield, were significantly higher than in comparative example 1.
Specifically, the highest biomass of Chlorella vulgaris zofingiensis can reach 235 g.L by adopting the induction scheme of example 1-1The content of astaxanthin can be up to 0.144% of the dry weight of the cells, and the yield of the astaxanthin can reach 0.32 g.L-1。
Examples 2 to 9
Examples 2 to 10 were conducted by changing only the conditions of the three inducing factors of "step two, astaxanthin induction culture" based on example 1, namely, the initial concentrations of gibberellin and sodium chloride and the initial values of the C/N ratio; the remaining conditions and procedure were exactly the same as in example 1. The following table specifically shows:
| group of | C/N | Gibberellins | Sodium chloride |
| Example 2 | 140 | 10mg/L | 0.2M |
| Example 3 | 220 | 10mg/L | 0.2M |
| Example 4 | 280 | 10mg/L | 0.2M |
| Example 5 | 180 | 50mg/L | 0.2M |
| Example 6 | 180 | 100mg/L | 0.2M |
| Example 7 | 180 | 150mg/L | 0.2M |
| Example 8 | 180 | 10mg/L | 0.1M |
| Example 9 | 180 | 10mg/L | 0.4M |
| Example 10 | 180 | 10mg/L | 0.6M |
After the second-stage cultivation was completed (16 days in total), the astaxanthin content in example 1 was taken as a reference value of 100%, and the astaxanthin contents in comparative example 1 and examples 2 to 10 after the completion of the cultivation were counted and the relative contents thereof with respect to the reference value were calculated, respectively. The statistical results are shown in fig. 2.
As can be seen from FIG. 2, the astaxanthin contents of examples 2 to 10 were all lower than that of example 1 but higher than that of comparative example 1, indicating that the astaxanthin contents produced under the conditions of the second stage of the present invention using the three factors induction were higher than those without these induction conditions, but the induction conditions "initial concentration of gibberellin was 10 mg. multidot.L-1The initial concentration of sodium chloride was 200mM, and the induction effect was the best when the initial carbon-nitrogen ratio in the heterotrophic medium was 180/1 ″.
Comparative examples 2 to 7
Comparative examples 2 to 7 were conducted by changing the conditions of three inducing factors of "step two, astaxanthin induction culture" on the basis of example 1 so that one or two of the inducing factors were deleted; the remaining conditions and procedure were exactly the same as in example 1. The following table specifically shows:
after the second-stage cultivation was completed (16 days in total), the astaxanthin contents of comparative examples 2 to 7 were counted with the astaxanthin content of example 1 being 100% as a reference value, and the relative contents thereof with respect to the reference value were calculated. The statistical results are shown in fig. 3.
As can be seen from FIG. 3, the astaxanthin contents of comparative examples 2 to 7 were all much lower than those of example 1. It is shown that in the method of the present invention, three inducing factors in the second culturing stage have a synergistic effect on increasing the astaxanthin content in the cells and the total amount of astaxanthin in the fermentation medium, and one of the three inducing factors is lacking, so that the technical effects of the present invention cannot be achieved.
In addition, under the condition that other conditions are not changed, Gibberellin (GA) in the three inducing factors of the second stage is replaced by indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-butyric acid (IBA), 1-naphthylacetic acid (NAA) and 1-aminocyclopropane-1-carboxylic Acid (ACC), but the content of astaxanthin is not as high as that of gibberellin when the inducing culture is carried out for 4 days under the condition of equal concentration or higher concentration. Therefore, the gibberellin has uniqueness on the function of astaxanthin induced by chlorella under heterotrophic culture conditions, and is not easily replaced by other hormones.
Example 11
In this example, the conditions for inducing astaxanthin induction culture in the second step were changed based on example 1, and specifically, a certain amount of GABA was added to the fermentor medium based on the three inducing factors described in example 1 to make the initial concentration of GABA 0.5 mM. The remaining conditions and procedure were exactly the same as in example 1.
Example 12
In this example, the conditions for inducing astaxanthin induction culture in step two were changed based on example 1, and specifically, oxaloacetate was further added to the fermentor medium in an amount such that the initial concentration of oxaloacetate was 0.2mM based on the three inducing factors described in example 1. The remaining conditions and procedure were exactly the same as in example 1.
In the astaxanthin induction culture stage, samples in the fermenter were continuously taken, and the dry cell weight (g/L), the astaxanthin content (%, DW), and the astaxanthin production (g/L) in examples 11 to 12 were measured and calculated and plotted respectively, and the results are shown in FIG. 4 with reference to the graph of example 1.
As can be seen from FIG. 4 (a), the rate of increase in the dry weight of cells in examples 1 and 11 to 12 was slightly decreased as compared with that in comparative example 1 within 4 days of astaxanthin induction culture (day12-16), but the astaxanthin content and the astaxanthin production, particularly the astaxanthin production, were significantly higher than that in comparative example 1. In particular, the amount of the solvent to be used,
example 12 with 200. mu.M oxaloacetate addition gave a biomass of up to 245g/L when cultured for 16 days. In example 11, the astaxanthin content and the astaxanthin production of Chlorella vulgaris zofingiensis tend to increase and decrease after addition of gamma-aminobutyric acid, and the astaxanthin content accounts for 0.154% of the dry weight of the cells by the 15 th day of culture, and the astaxanthin production is 0.35 g/L. In example 12, the astaxanthin content and the astaxanthin production of Chlorella vulgaris zofingiensis tend to increase after oxaloacetic acid addition, the astaxanthin content can be 0.16% of the dry cell weight by the 16 th day of culture, and the astaxanthin production is 0.39 g/L.
From this, it was found that the fermentation broth obtained in example 12 was the highest in astaxanthin content and astaxanthin yield after the induction culture was completed, and that the fermentation broth obtained in example 11 was the next. Thus, it is proved that if oxaloacetate or gamma-aminobutyric acid is further added for combined induction on the basis of the three-factor induction in example 1, the biomass, the astaxanthin content and the yield of Chlorella zofingiensis are remarkably improved.
Examples 13 to 15
Examples 13 to 15 were conducted by changing only the initial concentration of gamma-aminobutyric acid in "step two, astaxanthin induction culture" based on example 11. The remaining conditions and procedures were exactly the same as in example 11. Specifically, example 13 changed the initial concentration of γ -aminobutyric acid in the fermentor medium to 0.1 mM; example 14 instead, 0.25mM of the initial concentration of gamma-aminobutyric acid was added; example 15 instead, 1mM of the initial concentration of gamma-aminobutyric acid was added.
In the statistical analysis, the astaxanthin content of examples 13 to 15 after the completion of the cultivation was measured with the astaxanthin content of example 11 as a reference value of 100%, and the relative content thereof with respect to the reference value was calculated, while example 1 was used as a control, and the statistical results are shown in FIG. 5.
As can be seen from FIG. 5, under the second stage induction culture conditions of example 1, if γ -aminobutyric acid is further added at a certain initial concentration, it is advantageous to obtain higher astaxanthin yield, and especially, when the initial concentration of γ -aminobutyric acid added is 0.5mM, the inducing effect on Chlorella vulgaris is optimal.
Examples 16 to 18
Examples 16 to 18 were carried out by changing only the initial concentration of oxaloacetate in "step two, astaxanthin induction culture" based on example 12. The remaining conditions and procedures were exactly the same as in example 12. Specifically, example 16 changed the initial concentration of oxaloacetate in the fermentor medium to 0.01 mM; example 14 change to initial concentration of 0.1mM oxaloacetate; example 15 was changed to oxaloacetate at an initial concentration of 0.5 mM.
In the statistical analysis, the astaxanthin content of examples 16 to 18 was measured after the completion of the cultivation with the astaxanthin content of example 12 as a reference value of 100%, and the relative content thereof with respect to the reference value was calculated, while the statistical results of example 1 as a control were shown in FIG. 6.
As can be seen from FIG. 6, under the second stage induction culture conditions of example 1, when oxaloacetate is further added at a certain initial concentration, higher astaxanthin yield can be obtained, and especially, when the initial concentration of oxaloacetate is 0.2mM, the chlorella is induced optimally.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for heterotrophically cultivating chlorella to produce astaxanthin, comprising:
s1 cell proliferation culture
Culturing chlorella by heterotrophic culture method to increase dry weight of chlorella cells to a predetermined value;
s2 astaxanthin induction culture
Under the condition of maintaining heterotrophic culture, three factors are adopted to carry out combined induction on the chlorella to promote the chlorella to accumulate astaxanthin and improve the content of the astaxanthin in cells; the three factors include:
adding gibberellin into the heterotrophic culture medium to make the initial concentration of gibberellin in the culture medium be 10mg/L-150 mg/L;
adjusting the initial carbon-nitrogen ratio in the heterotrophic culture medium in the fermentation tank to make the carbon-nitrogen ratio C/N equal to 140-;
sodium chloride was added to the heterotrophic culture medium to an initial concentration of 0.1M to 0.6M sodium chloride in the culture medium.
2. The method according to claim 1, wherein in S2, gibberellin is added to the heterotrophic culture medium such that the initial concentration of gibberellin in the culture medium is 10 mg/L; adjusting the initial carbon-nitrogen ratio in the heterotrophic culture medium in the fermentation tank to 180; sodium chloride was added to the heterotrophic culture medium to give an initial concentration of 0.2M sodium chloride in the culture medium.
3. The method according to claim 1, wherein in S1-S2, the nitrogen source in the medium during the heterotrophic culture is ammonium chloride and ammonia; the carbon source is glucose.
4. The method of claim 3, wherein in S1, the culture medium is heterotrophic in a fed-batch mode, the glucose concentration in the fermentor is monitored in real time, the feeding speed is adjusted in real time, and the glucose concentration in the fermentor is controlled to be 5g/L-20 g/L;
in S2, the culture medium is heterotrophic in fed-batch mode, and the glucose concentration in the fermentation tank is monitored in real time, the feeding speed is adjusted in real time, and the glucose concentration in the fermentation tank is controlled to be 4.5-5.5 g/L.
5. The method according to claim 4, wherein the heterotrophic culture conditions in S1-S2 are: the culture temperature is 25-28 ℃, the dissolved oxygen is set at 15-25%, and the stirring speed and the dissolved oxygen are controlled in a coupling way; meanwhile, the pH of the culture solution in the fermentation tank is monitored in real time, and the pH of the culture solution in the fermentation tank is controlled to be 6.5 +/-0.2 by feeding ammonia water.
6. The method according to claim 5, wherein in S1, the culture medium comprises a basal medium and a feed medium, the basal medium adopts ammonium chloride as a unit, the glucose concentration is 20g/L, and the initial C/N is 32: 1; when the glucose concentration in the basic culture medium is lower than 5g/L, a peristaltic pump is started to feed the feed culture medium, the components of the feed culture medium are the same as the basic culture medium, but the carbon-nitrogen ratio in the feed culture medium is 1/20 of the carbon-nitrogen ratio in the basic culture medium.
7. The method according to claim 1, wherein the inducing culture process of S2 is performed when the chlorella cells are heterotrophic in S1 stage to have a dry weight of 150g/L or more, and the inducing culture process is terminated when the culturing time of S2 stage is such that the astaxanthin content in the cells is detected to be constant or begins to decrease.
8. The process of any one of claims 1 to 7, wherein in S2, further, one or both of oxaloacetate and gamma-aminobutyric acid are added to the heterotrophic culture medium of the fermentor.
9. The method according to claim 8, wherein in S2, γ -aminobutyric acid is added to the heterotrophic culture medium in the fermentor such that an initial concentration of γ -aminobutyric acid in the heterotrophic culture medium in the fermentor is 0.5 mM.
10. The method according to claim 8, wherein in S2, γ -aminobutyric acid is added to the heterotrophic culture medium in the fermentor such that the initial concentration of oxaloacetate in the heterotrophic culture medium in the fermentor is 0.2 mM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111521719.6A CN114214386A (en) | 2021-12-13 | 2021-12-13 | Method for producing astaxanthin by heterotrophic culture of chlorella |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111521719.6A CN114214386A (en) | 2021-12-13 | 2021-12-13 | Method for producing astaxanthin by heterotrophic culture of chlorella |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114214386A true CN114214386A (en) | 2022-03-22 |
Family
ID=80701519
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111521719.6A Pending CN114214386A (en) | 2021-12-13 | 2021-12-13 | Method for producing astaxanthin by heterotrophic culture of chlorella |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114214386A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113881749A (en) * | 2021-11-11 | 2022-01-04 | 中国科学院海洋研究所 | Method for rapidly promoting accumulation of astaxanthin in haematococcus pluvialis |
| CN115161203A (en) * | 2022-07-20 | 2022-10-11 | 德默特生物科技(珠海)有限公司 | Method for heterotrophic culture of high-yield protein by chlorella |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050214897A1 (en) * | 2004-03-26 | 2005-09-29 | Feng Chen | Methods for production of astaxanthin from the green microalgae Chlorella in dark-heterotrophic cultures |
| CN103045709A (en) * | 2011-10-14 | 2013-04-17 | 中国科学院烟台海岸带研究所 | Method for synthesizing astaxanthin by inducing chlorella vulgaris by using plant hormones and iron ions |
| CN103571906A (en) * | 2012-07-27 | 2014-02-12 | 上海泽元海洋生物技术有限公司 | Novel method for high-efficiently producing astaxanthin by utilizing microalgae |
| CN104662162A (en) * | 2013-09-26 | 2015-05-27 | 华东理工大学 | Method using micro-algae for high-efficiency production of astaxanthin |
| CN109439611A (en) * | 2018-11-20 | 2019-03-08 | 华南理工大学 | Improve the method for inducing and cultivating of color green alga astaxanthin and algae oil accumulation intracellular |
| CN109797104A (en) * | 2019-02-22 | 2019-05-24 | 中国科学院水生生物研究所 | A kind of chlorella Chlorella zofingiensis heterotrophism high-density cultivation method |
| CN110184193A (en) * | 2019-05-21 | 2019-08-30 | 中国科学院水生生物研究所 | It is a kind of efficiently to expand numerous continuous gradient feed process and device applied to haematococcus pluvialis |
-
2021
- 2021-12-13 CN CN202111521719.6A patent/CN114214386A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050214897A1 (en) * | 2004-03-26 | 2005-09-29 | Feng Chen | Methods for production of astaxanthin from the green microalgae Chlorella in dark-heterotrophic cultures |
| CN103045709A (en) * | 2011-10-14 | 2013-04-17 | 中国科学院烟台海岸带研究所 | Method for synthesizing astaxanthin by inducing chlorella vulgaris by using plant hormones and iron ions |
| CN103571906A (en) * | 2012-07-27 | 2014-02-12 | 上海泽元海洋生物技术有限公司 | Novel method for high-efficiently producing astaxanthin by utilizing microalgae |
| CN104662162A (en) * | 2013-09-26 | 2015-05-27 | 华东理工大学 | Method using micro-algae for high-efficiency production of astaxanthin |
| CN109439611A (en) * | 2018-11-20 | 2019-03-08 | 华南理工大学 | Improve the method for inducing and cultivating of color green alga astaxanthin and algae oil accumulation intracellular |
| CN109797104A (en) * | 2019-02-22 | 2019-05-24 | 中国科学院水生生物研究所 | A kind of chlorella Chlorella zofingiensis heterotrophism high-density cultivation method |
| CN110184193A (en) * | 2019-05-21 | 2019-08-30 | 中国科学院水生生物研究所 | It is a kind of efficiently to expand numerous continuous gradient feed process and device applied to haematococcus pluvialis |
Non-Patent Citations (5)
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113881749A (en) * | 2021-11-11 | 2022-01-04 | 中国科学院海洋研究所 | Method for rapidly promoting accumulation of astaxanthin in haematococcus pluvialis |
| CN115161203A (en) * | 2022-07-20 | 2022-10-11 | 德默特生物科技(珠海)有限公司 | Method for heterotrophic culture of high-yield protein by chlorella |
| CN115161203B (en) * | 2022-07-20 | 2023-09-26 | 德默特生物科技(珠海)有限公司 | Method for heterotrophic culture of high-yield protein by chlorella |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114214386A (en) | Method for producing astaxanthin by heterotrophic culture of chlorella | |
| CN102168115A (en) | Industrialized production method of coenzyme Q10 | |
| CN102925503B (en) | Method for preparing ARA (Arachidonic Acid) by culturing mortierella alpina by utilizing solid material culture medium | |
| CN109797104A (en) | A kind of chlorella Chlorella zofingiensis heterotrophism high-density cultivation method | |
| CN111560321A (en) | Efficient fermentation process of rhodotorula benthica | |
| CN111500464A (en) | Method for producing lutein by first mixotrophic-later autotrophic microalgae | |
| CN109234215B (en) | Lactobacillus rhamnosus low-salt culture medium and culture method | |
| CN110982750A (en) | High-density fermentation method for rhodopseudomonas palustris and application of high-density fermentation method | |
| CN117229982B (en) | Application of N- (1, 3-dimethylbutyl) -N' -phenyl p-phenylenediamine-quinone in culture of synechocystis | |
| CN108823123A (en) | It is a kind of regulate and control brown yellow spore streptomycete fermentation production natamycin preparation method | |
| CN116179356B (en) | Method for high-density heterotrophic culture of chlamydomonas reinhardtii and application thereof | |
| CN109628509B (en) | Method for producing pyrroloquinoline quinone by semi-continuous fermentation process | |
| CN115161203B (en) | Method for heterotrophic culture of high-yield protein by chlorella | |
| CN111763636A (en) | Method for producing coenzyme Q10 by industrial fermentation | |
| CN109266578B (en) | Escherichia coli ACThr1032 and application thereof in fermentation production of L-threonine | |
| CN108641961B (en) | Method for high-density culture of guava leaf endophytes | |
| CN113812302B (en) | Method for preparing liquid edible fungus strain | |
| CN112725201B (en) | Liquid submerged fermentation method of pichia pastoris for producing acid protease | |
| CN112852896A (en) | Fermentation production method of L-arginine | |
| CN113528349A (en) | Culture method for improving high-temperature tolerance of dinoflagellate such as Zhanjiang | |
| CN114032176A (en) | Culture system and culture method for promoting growth of microalgae | |
| CN102154129B (en) | Rhodosporidium paludigenum for degrading gossypol and application thereof | |
| CN113136321A (en) | Method and system for heterotrophic-autotrophic co-culture of photosynthetic microorganisms and method for production of biomass and bioenergy | |
| CN110982705A (en) | Fermentation process of schizochytrium limacinum | |
| CN105296407A (en) | Method for culturing avibacterium paragallinarum bacterial solution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |