CN111172039A - Two-stage low-nitrogen low-phosphorus stress microalgae culture method - Google Patents
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 45
- 229910052698 phosphorus Inorganic materials 0.000 title claims abstract description 45
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- 238000005286 illumination Methods 0.000 claims abstract description 13
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- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 8
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- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
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- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-N disodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound [Na+].[Na+].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O ZGTMUACCHSMWAC-UHFFFAOYSA-N 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
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- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
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- 239000011684 sodium molybdate Substances 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- FDEIWTXVNPKYDL-UHFFFAOYSA-N sodium molybdate dihydrate Chemical compound O.O.[Na+].[Na+].[O-][Mo]([O-])(=O)=O FDEIWTXVNPKYDL-UHFFFAOYSA-N 0.000 description 1
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Abstract
A two-stage low-nitrogen low-phosphorus stress microalgae culture method is characterized by comprising the following steps: the method comprises the following steps: (1) culturing under the optimal culture condition to obtain a logarithmic phase microalgae cell pre-culture solution; (2) centrifuging the logarithmic phase microalgae cell pre-culture solution, and collecting to obtain osmunda cells; (3) culturing collected Osmunda japonica cells in a low-nitrogen and low-phosphorus stress culture medium in an amplification manner again; (4) culturing at 25 deg.C under illumination and aeration for 10-14 days. The microalgae cells with the significantly increased accumulation of polyunsaturated fatty acids (PUFAs) can be obtained.
Description
Technical Field
The invention belongs to the technical field of microalgae culture, and particularly relates to a two-section type low-nitrogen low-phosphorus stress microalgae cell culture method.
Background
Microalgae is a generic name of small and medium-sized algae, is an autotroph which can synthesize various organic matters in cells of the microalgae by using light energy, carbon dioxide and water as pioneer species on the earth, and has extremely strong adaptability to the environment. The strong adaptability is closely related to the efficient metabolic path formed by the algae in the long-term evolution process and various damage repair active substances which are generated and accumulated in large quantity. Numerous studies have reported that microalgae produce and accumulate various active substances for repairing damage in cells under the stress of nutrient deficiency, and the important one is polyunsaturated fatty acids (PUFAs). PUFAs are fatty acids with more than or equal to two double bonds, and play an important role in regulating various physiological functions of a body. Simultaneously, PUFAs also have important physiological functions on human bodies, can regulate lipid metabolism of the human bodies, promote growth and development, and treat and prevent cardiovascular and cerebrovascular diseases. In addition, it has important physiological functions in the aspects of immunoregulation, cancer resistance, weight reduction, senility delay, beauty treatment and the like. Development of many food resources rich in PUFAs is becoming a hot field of functional food research, and extracted PUFAs have been applied in health food and medical research.
Various inorganic nutrient elements are required for normal growth and metabolism of microalgae cells, N, P is particularly important in the nutrient elements, wherein N element is a constituent element of various biomacromolecules such as protein, nucleic acid, amino acid and the like, and P element is a constituent element of nucleic acid, ATP, NADPH and a biological membrane system. Considerable research reports indicate that the content of N and P elements in a microalgae cell culture medium has great influence on the growth rate and the oil content of microalgae, and the more sufficient the N and P elements provide in a certain concentration range, the faster the growth rate of the microalgae is; and when the deficiency of the N and P elements occurs, the generation and accumulation of the oil in the microalgae cells, particularly the oil consisting of PUFAs, are obviously increased. This is probably because the protein synthesis pathway is hindered, and more NADPH obtained by photosynthesis flows to the lipid synthesis pathway, thereby increasing the lipid content composed of PUFAs in the microalgal cells.
Different algae are cultured in environments with different N, P nutrient element contents, and the generation and accumulation of polyunsaturated fatty acids in cells are obviously different, so that the supply of N, P nutrient elements is explored and controlled by optimizing the nutrient components of the microalgae cell culture solution, and the method is a wide market prospect for producing the polyunsaturated fatty acids and other active substances by utilizing microalgae. At present, no method for carrying out stress culture on microalgae by using low N, P nutrient elements so as to massively produce and accumulate damage repair active substances is disclosed.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: a two-stage culture method of microalgae cells with low nitrogen and phosphorus stress can realize rapid proliferation growth of the microalgae cells under the optimal culture condition through two-stage culture, and polyunsaturated fatty acids (PUFAs) with various physiological activities and important market application values are massively produced and accumulated under the culture condition with low nitrogen and phosphorus stress. Is a good method for efficiently producing injury repair active substances such as polyunsaturated fatty acids (PUFAs).
In order to solve the technical problems, the invention provides the following technical scheme: a two-stage low-nitrogen low-phosphorus stress microalgae cell culture method.
The method is characterized by comprising the following steps: (1) culturing under the optimal culture condition to obtain a logarithmic phase microalgae cell pre-culture solution; (2) centrifuging the logarithmic phase microalgae cell pre-culture solution, and collecting to obtain osmunda cells; (3) culturing collected Osmunda japonica cells in a low-nitrogen and low-phosphorus stress culture medium in an amplification manner again; (4) culturing at 25 deg.C under illumination and aeration for 10-14 days. The microalgae cells with the significantly increased accumulation of polyunsaturated fatty acids (PUFAs) can be obtained.
Preferably, the optimum culture conditions in step (1) are illumination intensity of 100. mu. mol/(m)2S) at 25 deg.C, pre-culturing to obtain logarithmic phase microalgae cell culture solution by using Chlamydomonas, Chlorella, and Phaeodactylum tricornutum as algae seed, inoculating, and culturing until the cell concentration is 1.0x106Per L-1.2x 106Microalgae cell culture solution in the range of per liter.
Preferably, the rotation speed of the centrifuge during centrifugal collection in the step (2) is 2000-5000rpm, and the centrifugation time is 3-5 min.
Preferably, the low-nitrogen low-phosphorus stress culture for the amplification culture in the step (3) is optimized on the basis of the formulas of TAP and BG11 culture mediums, the total nitrogen (calculated by N) concentration in the optimized 1X culture solution is less than 0.200mg/L, the total phosphorus concentration is less than 0.010mg/L, and other components are unchanged.
Preferably, when chlamydomonas, chlorella, or phaeodactylum tricornutum is used as the algal species for the scale-up culture, the culture media used are respectively optimized low-nitrogen and low-phosphorus TAP culture medium, low-nitrogen and low-phosphorus BG11, and low-nitrogen and low-phosphorus BG 11.
Preferably, the light intensity of the 25 ℃ light aeration culture in the step (4) is 100. mu. mol/(m)2S) the gas introduced is sterile air. The culture time varies according to the species of microalgae. When Chlamydomonas, Chlorella and Phaeodactylum tricornutum are used as the algae seeds, the culture time is 10 days, 12 days and 14 days respectively.
Advantageous effects
1. The two-stage culture adopted by the method is as follows: optimum conditions (25 ℃, 100 mu mol/(m) are adopted in the pre-culture stage2S)) to culture Osmunda cells from initial inoculum size to log phase (microalgae cell concentration in culture medium ranging from 1.0x106a/L of-1.2 x106Per L), and then culturing the centrifugally collected osmunda cells in a low-nitrogen and low-phosphorus stress culture medium for producing and accumulating polyunsaturated fats (PUFAs) in an amplification mode. The two-stage culture operation can greatly shorten the culture period, reduce the cell death rate and improve the production efficiency of the injury repair active substances.
2. The formula of the low-nitrogen and low-phosphorus stress culture medium used in the invention is obtained by repeated experiments and adjustments of the inventor, has the effects of inducing microalgae cells to efficiently generate and accumulate a large amount of damage repair active substances such as polyunsaturated fatty acids (PUFAs) and the like, and has small influence on other physiological activities of the microalgae cells and low death rate.
In summary, the present invention: a two-stage culture method of microalgae cells with low nitrogen and phosphorus stress can realize rapid proliferation growth of microalgae cells under an optimal culture condition through two-stage culture, and can produce and accumulate a large amount of polyunsaturated fatty acids (PUFAs) with various physiological activities and important market application values under the culture condition with low nitrogen and phosphorus stress. Is a good method for efficiently producing injury repair active substances such as polyunsaturated fatty acids (PUFAs).
Drawings
FIG. 1 schematic view of a culture apparatus (triangular flask)
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown to illustrate how the invention may be practiced, and which will make apparent and understood by those skilled in the art. The present invention may be embodied in many different forms of embodiments, and the scope of protection is not limited to the embodiments described herein, which are illustrative rather than restrictive in nature.
The experimental procedures in the following examples are conventional unless otherwise specified.
The raw materials, reagents and the like used in the following examples are commercially available or disclosed unless otherwise specified.
Example 1:
a two-stage low-nitrogen low-phosphorus stress microalgae cell culture method is carried out by taking chlamydomonas (sp.cw15, purchased from Duke university algae seed bank in America) cells as algae seeds, and comprises the following specific steps:
1. culturing to obtain a logarithmic phase microalgae cell pre-culture solution. Preparing TAP culture medium 6L, the specific formula is shown in Table 1 below, inoculating Chlamydomonas cells after sterilization, placing the inoculated culture solution at 25 deg.C with fluorescent lamp illumination intensity of 100 μmol/(m)2S) and culturing for 7 days in a shaking table illumination incubator with the rotation speed of 20rpm to obtain the chlamydomonas cell pre-culture solution in the logarithmic phase.
TABLE 1 TAP Medium formulation
2. Detecting log-phase predictionsThe cell concentration of Chlamydomonas in the culture solution. The chlamydomonas cell pre-culture solution was shaken up and then counted under a microscope using a blood count plate. Calculating the chlamydomonas cell concentration of 1.14x10 in the pre-culture solution in logarithmic phase6And (2) per liter.
3. And (4) carrying out even centrifugation. Shaking up 6L of pre-cultured Chlamydomonas cell sap, respectively adding into 1L centrifuge tube, setting the centrifugation speed at 2000rpm, and centrifuging for 5 minutes. And discarding the supernatant, and collecting the chlamydomonas cells for later use.
4. And (5) performing grouped amplification culture. TAP culture solution (formula shown in Table 1) and low-nitrogen low-phosphorus stress TAP culture solution 3L are prepared respectively, and the specific formula of the low-nitrogen low-phosphorus stress TAP culture medium is shown in Table 2 below. And (3) respectively using the prepared sterilized culture solution, correspondingly re-suspending the microalgae cells centrifugally collected in the step (3), and performing expanded culture in two groups of 6 sterilized triangular flasks (three flasks in each group, wherein the flasks are connected as shown in the attached drawing 1) with the numbers of A and B, wherein the group A is the normal TAP culture solution, and the group B is the low-nitrogen low-phosphorus stress TAP culture solution. Introducing sterile air into the triangular bottles of group A and group B through 1 port, sealing, placing the bottles of group A and group B at 25 deg.C with illumination intensity of 100 μmol/(m)2S) for 10 days in a closed incubator.
TABLE 2 Low-nitrogen and phosphorus stress TAP culture recipe
| Compound (I) | Final concentration |
| CaCl2·2H2O | 84.6mg/l |
| MgSO4·7H2O | 204.2mg/l |
| KCL | 324.1mg/l |
| Glacial acetic acid | 1ml |
| NaEDTA·2H2O | 45.05mg/l |
| ZnSO4·7H2O | 39.03mg/l |
| H3BO3 | 11.41mg/l |
| MnCl2·4H2O | 7916.40mg/l |
| FeSO4·7H2O | 9146.20mg/l |
| CoCl2·6H2O | 2926.54mg/l |
| CuSO4·6H2O | 1720mg/l |
| Na7Mo7O24 | 1129mg/l |
5. And detecting the content of grease and polyunsaturated fatty acids (PUFAs). And after 10 days, respectively centrifugally collecting chlamydomonas cells in triangular flasks of the group A and the group B, and measuring and collecting the contents of cell grease and polyunsaturated fatty acid. And measuring the oil content by adopting a Nile red-fluorescence spectrometry method, resuspending part of centrifuged algae cells by using a dimethyl sulfoxide (DMSO) aqueous solution with the volume fraction of 4% until OD750 is 0.3 and the mass concentration of Nile red is 0.8 mu g/mL, keeping the temperature of a water bath at 40 ℃ away from light for 10min, measuring the fluorescence value at the excitation wavelength of 520nm, and then comparing with a standard curve to calculate the oil content. The method for measuring the content of the polyunsaturated fatty acid refers to the determination of the polyunsaturated fatty acid with cis, cis-1, 4-diene structure of animal and vegetable fat (see the national standard GB/T21495-2008/ISO 7847: 1987).
6. And (5) experimental results. The results of measuring the content of the chlamydomonas cell damage repairing active substance are shown in Table 3. The experimental results show that the content of polyunsaturated fatty acids (PUFAs) in the Chlamydomonas B cells is remarkably higher than that in the Chlamydomonas A.
TABLE 3 measurement results of contents of fats and oils and polyunsaturated fatty acids (PUFAs)
Example 2:
chlorella (sp.dh8248, stored in laboratory and collected in east lake of Wuhan) cells are adopted as a two-stage low-nitrogen low-phosphorus stress microalgae cell culture method, which comprises the following specific steps:
1. culturing to obtain a logarithmic phase microalgae cell pre-culture solution. Preparing TAP culture medium 6L, the specific formula is shown in Table 1 below, inoculating small spherical cells after sterilization, placing the inoculated culture solution at 25 deg.C with fluorescent lamp illumination intensity of 100 μmol/(m)2S) in a shaking table illumination incubator with the rotation speed of 20rpm for 7 days, and then the spherule cell preculture solution in the logarithmic phase can be obtained.
TABLE 4BG11 Medium recipe
| Compound (I) | Final concentration |
| NaNO3 | 1.5g/L |
| K2HPO4 | 0.04g/L |
| MgSO4·7H2O | 0.075g/L |
| CaCl2·2H2O | 0.036g/L |
| Na2CO3 | 0.02g/L |
| Citric acid | 0.006g/L |
| Ferric ammonium citrate | 0.006g/L |
| EDTANa2 | 0.001g/L |
| H3BO4 | 2.86mg/L |
| MnCl2·4H2O | 1.81mg/L |
| ZnSO4·4H2O | 0.222mg/L |
| Na2MoO4·2H2O | 0.39mg/L |
| CuSO4·5H2O | 0.079mg/L |
| Co(NO3)2·6H2O | 0.049mg/L |
2. And detecting the concentration of chlorella cells in the logarithmic phase pre-culture solution. The chlorella cell pre-culture solution is shaken up and then counted under a microscope by using a blood counting plate. Calculating the concentration of chlorella cells in the logarithmic phase pre-culture solution to be 1.01x106And (2) per liter.
3. And (4) carrying out even centrifugation. Shaking up 6L of pre-cultured chlorella cell sap, respectively adding into 1L centrifuge tubes, setting the centrifugation speed at 2000rpm, and centrifuging for 5 minutes. And discarding the supernatant, and collecting chlorella cells for later use.
4. And (5) performing grouped amplification culture. BG11 culture solution (formula shown in Table 4) and BG11 culture solution 3L with low nitrogen and phosphorus stress were prepared respectively, and the specific formula of BG11 culture medium with low nitrogen and phosphorus stress is shown in Table 5 below. And (3) respectively using the prepared sterilized culture solution, correspondingly re-suspending the microalgae cells centrifugally collected in the step (3), and performing expanded culture in two groups of 6 sterilized triangular flasks (three flasks in each group, wherein the flasks are connected as shown in the attached drawing 1) with the numbers of A and B, wherein the group A is the normal BG11 culture solution, and the group B is the low-nitrogen and low-phosphorus stress BG11 culture solution. Introducing sterile air into the triangular bottles of group A and group B through 1 port, sealing, placing the bottles of group A and group B at 25 deg.C with illumination intensity of 100 μmol/(m)2S) for 12 days in a light incubator.
TABLE 5 Low Nitrogen and phosphorus stress optimized BG11 culture formula
| Compound (I) | Final concentration |
| KCl | 0.034g/l |
| MgSO4.7H2O | 0.075g/l |
| CaCl2.2H2O | 0.036g/l |
| Na2CO3 | 0.02g/l |
| Citric acid | 0.006g/l |
| Ferric ammonium citrate | 0.006g/l |
| EDTA Na2 | 0.001g/l |
| H3BO4 | 2.86mg/l |
| MnCl2.4H2O | 1.81mg/l |
| ZnSO4.4H2O | 0.222mg/l |
| Na2MoO4.2H2O | 0.39mg/l |
| CuSO4.5H2O | 0.079mg/l |
| CoCl2 | 0.022mg/l |
5. And detecting the content of grease and polyunsaturated fatty acids (PUFAs). Centrifuging and collecting chlorella cells in the triangular flasks of group A and group B after 12 days, and measuring and collecting cell oil and polyunsaturated fatty acid content. And measuring the oil content by adopting a Nile red-fluorescence spectrometry method, resuspending part of centrifuged algae cells by using a dimethyl sulfoxide (DMSO) aqueous solution with the volume fraction of 4% until OD750 is 0.3 and the mass concentration of Nile red is 0.8 mu g/mL, keeping the temperature of a water bath at 40 ℃ away from light for 10min, measuring the fluorescence value at the excitation wavelength of 520nm, and then comparing with a standard curve to calculate the oil content. The method for measuring the content of polyunsaturated fatty acids refers to the determination of polyunsaturated fatty acids with cis, cis-1, 4-diene structure in animal and vegetable fats (see national standard GB/T21495-2008/ISO 7847: 1987).
6. And (5) experimental results. The detection results of chlorella cell oil and polyunsaturated fatty acids (PUFAs) are shown in Table 6, and the experimental results show that the content of polyunsaturated fatty acids (PUFAs) in chlorella cells in group B is significantly higher than that in group A.
TABLE 6 measurement results of contents of fats and oils and polyunsaturated fatty acids (PUFAs)
Claims (5)
1. A two-stage low-nitrogen low-phosphorus stress microalgae cell culture method is characterized by comprising the following steps:
(1) culturing under the optimal culture condition to obtain a logarithmic phase microalgae cell pre-culture solution;
(2) centrifuging the logarithmic phase microalgae cell pre-culture solution, and collecting to obtain osmunda cells;
(3) culturing collected Osmunda japonica cells in a low-nitrogen and low-phosphorus stress culture medium in an amplification manner again;
(4) culturing at 25 deg.C under illumination and aeration for 10-14 days.
2. The two-stage cultivation method of microalgae with low nitrogen and phosphorus stress according to claim 1, comprising: the optimal culture condition of the step (1) is that the illumination intensity is 100 mu mol/(m)2S) and a temperature of 25 ℃. The logarithmic phase microalgae cell culture solution obtained by pre-culture is obtained by using Chlamydomonas, Chlorella and Phaeodactylum tricornutum as algae species for initial inoculation, and culturing until the concentration of algae cells is 1.0x106a/L-1.2 x106Microalgae cell culture solution in the range of per liter.
3. The two-stage cultivation method of microalgae with low nitrogen and phosphorus stress according to claim 1, comprising: and (3) when the centrifugal collection in the step (2) is carried out, the rotating speed of a centrifugal machine is 2000-5000rpm, and the centrifugal time is 3-5 min.
4. The two-stage microalgae cultivation method according to claim 1, characterized in that: the low-nitrogen and low-phosphorus stress culture medium for the amplified culture in the step (3) is optimized on the basis of the formulas of TAP and BG11 culture mediums. The concentration of total nitrogen (calculated by N) in the optimized 1X culture solution is less than 0.200mg/L, the concentration of total phosphorus is less than 0.010mg/L, and other components are unchanged. When chlamydomonas, chlorella and phaeodactylum tricornutum are used as algal species for carrying out amplification culture, the adopted culture media are respectively an optimized low-nitrogen low-phosphorus stress TAP culture medium, a low-nitrogen low-phosphorus stress BG11 and a low-nitrogen low-phosphorus stress BG 11.
5. The two-stage low-nitrogen low-phosphorus stress microalgae cell culture method according to claim 1, characterized in that: the illumination intensity of the 25 ℃ illumination aeration culture in the step (4) is 100 mu mol/(m)2S) the gas introduced is sterile air. The culture time is not dependent on the species of microalgaeThe same is true. When Chlamydomonas, Chlorella and Phaeodactylum tricornutum are used as the algae seeds, the culture time is 10 days, 12 days and 14 days respectively.
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