CN115011511A - Mixed nutrient culture method suitable for growth of nostoc sphaeroides - Google Patents
Mixed nutrient culture method suitable for growth of nostoc sphaeroides Download PDFInfo
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- 241000452732 Nostoc sphaeroides Species 0.000 title claims abstract description 46
- 238000012136 culture method Methods 0.000 title claims abstract description 24
- 235000015097 nutrients Nutrition 0.000 title claims abstract description 21
- 230000012010 growth Effects 0.000 title abstract description 13
- 239000001963 growth medium Substances 0.000 claims abstract description 46
- 241000195493 Cryptophyta Species 0.000 claims abstract description 24
- 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 abstract description 14
- 239000008103 glucose Substances 0.000 claims abstract description 14
- 238000005286 illumination Methods 0.000 claims abstract description 5
- 238000011081 inoculation Methods 0.000 claims description 9
- 230000009105 vegetative growth Effects 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical compound OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
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- 235000013817 Nostoc commune Nutrition 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 6
- 239000002028 Biomass Substances 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 238000009825 accumulation Methods 0.000 abstract description 2
- 230000037208 balanced nutrition Effects 0.000 abstract description 2
- 235000019046 balanced nutrition Nutrition 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000011521 glass Substances 0.000 description 11
- 241000192656 Nostoc Species 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 5
- 241000192700 Cyanobacteria Species 0.000 description 4
- 235000021529 Nostoc flagelliforme Nutrition 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000692932 Nostoc flagelliforme Species 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000579895 Chlorostilbon Species 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000001140 Night Blindness Diseases 0.000 description 1
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- 229930003268 Vitamin C Natural products 0.000 description 1
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(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)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 description 1
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- 235000013734 beta-carotene Nutrition 0.000 description 1
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- 229960002747 betacarotene Drugs 0.000 description 1
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- 229930002875 chlorophyll Natural products 0.000 description 1
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
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- 229910052876 emerald Inorganic materials 0.000 description 1
- 239000010976 emerald Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
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- 239000004009 herbicide Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
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- 238000011084 recovery Methods 0.000 description 1
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- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
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- 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
- C12N1/20—Bacteria; Culture media therefor
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- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses a mixed nutrient culture method suitable for nostoc sphaeroides growth, which comprises the steps of adjusting the pH value of a BG11 culture medium, adding a certain amount of glucose to enable the glucose concentration to be 0.09-0.11mol/L to obtain an improved BG11 culture medium for later use; selecting nostoc sphaeroides with the grain diameter of 3 +/-1 mm, elasticity, compact structure and dark green color as an algae seed, and inoculating the algae seed on an improved BG11 culture medium; then, the modified BG11 culture medium inoculated with the algae is cultured under the following conditions: temperature: at the temperature of 21-23 ℃, illumination: 50 μmol · m-2 · s-1, light dark period L: d =10 h: and 14 h. The method of the invention is simple to operate; the obtained improved culture medium has balanced nutrition, and under the physical and chemical conditions, the growth rate and biomass accumulation of nostoc sphaeroides can be improved, so that the method is particularly beneficial to improving the factory production efficiency and improving the factory microalgae culture efficiency under the existing condition.
Description
Technical Field
The invention relates to a mixed nutrient culture method suitable for microalgae growth, in particular to a mixed nutrient culture method suitable for nostoc sphaeroides growth.
Background
Cyanobacteria, also known as cyanobacteria, is a large unicellular prokaryotic organism with a long evolutionary history, gram-negative staining, no flagella, chlorophyll-containing a, but no chloroplast (distinct from eukaryotic algae), capable of aerobic photosynthesis. Nostoc sphaeroids KutzNostoc sphaeroides) The Nostoc globiformis is named as Nostoc globiformis, belongs to Nostoc of Nostocales of Cyanophyta, and is a precious and wild dual-purpose cyanobacteria for food and medicine, which is exported traditionally in China. Nostoc sphaeroids Kutz has the efficacy of treating nyctalopia, rectocele and scald and beautifying according to records of Ben Cao gang mu and so on. According to the research, the nostoc sphaeroides is high in protein content, good in protein quality, high in nutritional value and high in quality, the amino acid varieties in the protein are complete, the structure is reasonable, and the nostoc sphaeroides basically accords with the essential amino acid mode of a human body promoted by FAO/WHO expert committee in 1973, and the nostoc sphaeroides is also rich in polysaccharide, vitamin C, B complex with special health care function, beta-carotene, trace elements and other nutritional ingredients. Meanwhile, the method has double ecological values of carbon fixation and nitrogen fixation.
The nostoc sphaeroides is mainly inhabited in the Hefeng county in Hubei province of China, but in recent years, the wild resource quantity is rapidly and sharply reduced, farmers do not apply farmyard manure but apply chemical fertilizers, the soil structure is changed, and fertilizers such as ammonium bicarbonate and the like change the acidity and alkalinity of soil; the use of a large amount of herbicide changes the growth environment in which nostoc sphaeroides lives, and the number of wild nostoc sphaeroides is sharply reduced. The research on the recovery method of nostoc resources and the analysis of sharp reduction reasons are widely concerned by relevant scholars. An alga, Nostoc flagelliforme (C.flagelliforme, C.Nostoc flagelliforme) Commonly known as Nostoc flagelliforme, is also classified as a national first-level important protection wild plant due to the sharp decrease of wild resource amount. Therefore, the increase of the biomass of nostoc is one of the problems which need to be solved urgently at present.
The nostoc sphaeroides is generally cultured by an autotrophic culture method and using BG11 culture medium, the method is used for many years in laboratories and industrial culture, and has good effect on the culture of nostoc sphaeroides. However, the cultivation method has disadvantages, so that the nostoc sphaeroides is not high in yield and cannot meet the market demand.
Disclosure of Invention
The invention aims to solve the technical problem of providing a mixed nutrient culture method suitable for the vegetative growth of nostoc sphaeroides aiming at the defects in the prior art, and the traditional nostoc sphaeroides culture method is improved, so that the biomass of nostoc sphaeroides can be effectively improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a mixed nutrient culture method suitable for the vegetative growth of nostoc sphaeroides comprises the following steps:
(1) preparing a nostoc sphaeroides culture medium: after the pH of the BG11 culture medium is adjusted, adding a certain amount of glucose to ensure that the glucose concentration is 0.09-0.11mol/L to obtain an improved BG11 culture medium for later use;
(2) inoculating algae seeds: selecting nostoc sphaeroides with the grain diameter of 3 +/-1 mm, elasticity, compact structure and dark green color as an algae seed, and inoculating the algae seed on the improved BG11 culture medium in the step (1);
(3) mixed nutrient culture: culturing the modified BG11 culture medium inoculated with the algae seeds in the step (2) under the following conditions: temperature: at the temperature of 21-23 ℃, illumination: 50 μmol. m -2 ·s -1 Light-dark period L: d =10 h: and 14 h.
In the technical scheme, in the step (1), the adjusting of the pH of the BG11 culture medium refers to adjusting the pH to 7.0-7.2; then carrying out high-temperature 100kpa high-pressure sterilization on the culture medium for 20 minutes at 120 ℃ to obtain an incomplete culture medium, cooling the incomplete culture medium, and adding glucose to enable the glucose concentration to be 0.09-0.11mol/L to obtain an improved BG11 culture medium.
In the above technical solution, the glucose concentration is preferably 0.1 mol/L.
In the above technical scheme, in the step (2), the inoculation density is as follows: 6.64-6.70g algae/L medium, preferably 6.67g algae/L medium.
In the above technical solution, in the step (3), the conditions of the mixed nutrient culture are preferably: temperature: 22 ℃ and illumination: 50 μmol. m -2 ·s -1 Light-dark period L: d =10 h: and 14 h.
In the technical scheme, the mixed culture method for vegetative growth of nostoc sphaeroides is used for large-scale culture of nostoc sphaeroides.
In the technical scheme, the hybrid culture method for vegetative growth of nostoc sphaeroides is used for large-scale culture of common nostoc sphaeroides or emerald nostoc sphaeroides.
The mixed nutrient culture method for the growth of nostoc sphaeroides is simple to operate; the obtained improved culture medium has balanced nutrition, and under the physical and chemical conditions, the growth rate and biomass accumulation of nostoc sphaeroides can be improved, so that the method is particularly beneficial to improving the factory production efficiency and improving the factory microalgae culture efficiency under the existing condition.
Detailed Description
The mixed culture method for vegetative growth of nostoc sphaeroides according to the present invention will be described in detail with reference to the following examples:
example 1:
a mixed nutrient culture method suitable for growth of nostoc sphaeroides comprises the following steps:
(1) BG11 medium and modified BG11 medium containing glucose at a concentration of 0.1mol/L were prepared separately.
(2) Preparing: 6 autoclaved transparent glass conical flasks with 450mL are arranged, wherein 3 500mL transparent glass conical flasks of an experimental group contain the modified BG11 culture medium, 3 500mL transparent glass conical flasks of a control group contain a BG11 culture medium, the pH value is adjusted to 7.1 by using a pH meter, the culture media are prepared in situ and each flask contains 450mL culture medium;
(3) inoculation: on the 1 st day, the experimental group and the control group are respectively inoculated with algae seeds in the index growth period of nostoc sphaeroides, the diameter of the algae seeds is 3 +/-1 mm, the algae seeds are elastic, the structure is compact, the color is dark green, and the cell inoculation density in each conical flask is 450mL of culture medium/3.0 g of nostoc sphaeroides;
(4) culturing: the experimental group and the control group were cultured in the same light incubator, and the light-dark period: l: d =10 h: 14h, the temperature is 22 ℃, and the light intensity is 50 mu mol · m < -2 >. s < -1 >; ventilating both the experimental group and the control group, and replacing the culture medium every 3 days;
(5) and (4) comparing the results: after 12 days of culture, the yield of nostoc sphaeroides in the experimental group is improved by 57.14 percent compared with the control group.
Application example 1: the invention cultivates common nostoc:
(1) preparing: 6 autoclaved transparent glass conical flasks with 450mL are arranged in 3 500mL transparent glass conical flasks of an experimental group, the improved BG11 culture medium is contained in the 3 500mL transparent glass conical flasks of a control group, the BG11 culture medium is contained in the 3 mL transparent glass conical flasks of the control group, the pH is adjusted to 7.1 by using a pH meter, the culture medium is prepared in situ, and each flask contains 450mL culture medium;
(2) inoculation: on day 1, the experimental group and the control group are respectively inoculated with algae seeds in the index growth period green nutrition stage of nostoc sphaeroides, the grain diameter is 3 +/-1 mm, the algae seeds are elastic, the structure is compact, the color is dark green, and the cell inoculation density in each conical flask is 450mL of culture medium/3.0 g of nostoc sphaeroides;
(3) culturing: the experimental group and the control group were cultured in the same light incubator, and the light-dark period: l: d =10 h: 14h, the temperature is 22 ℃, and the light intensity is 50 mu mol · m < -2 >. s < -1 >; ventilating both the experimental group and the control group, and replacing the culture medium every 3 days;
(4) and (4) comparing the results: after 12 days of culture, the yield of nostoc sphaeroides in the experimental group is improved by 57.14 percent compared with the control group.
Application example 2: the invention cultivates emerald green:
(1) preparing: 6 autoclaved transparent glass conical flasks with 450mL are arranged in 3 500mL transparent glass conical flasks of an experimental group, the improved BG11 culture medium is contained in the 3 500mL transparent glass conical flasks of a control group, the BG11 culture medium is contained in the 3 mL transparent glass conical flasks of the control group, the pH is adjusted to 7.1 by using a pH meter, the culture medium is prepared in situ, and each flask contains 450mL culture medium;
(2) inoculation: on the 1 st day, the experimental group and the control group are respectively inoculated with algae seeds in the index growth period of nostoc sphaeroides, the diameter of the algae seeds is 3 +/-1 mm, the algae seeds are elastic, the structure is compact, the color is dark green, and the cell inoculation density in each conical flask is 450mL of culture medium/3.0 g of nostoc sphaeroides;
(3) culturing: the experimental group and the control group were cultured in the same light incubator, and the light-dark period: l: d =10 h: 14h, the temperature is 22 ℃, and the light intensity is 50 mu mol · m < -2 >. s < -1 >; ventilating both the experimental group and the control group, and replacing the culture medium every 3 days;
(4) and (4) comparing the results: after 12 days of culture, the yield of nostoc sphaeroides in the experimental group is improved by 55.28 percent compared with the control group.
From the above results, it is understood that the increase of the culture medium and the physicochemical conditions of glucose BG11 in the present invention can increase the cell concentration by 57.14%, which is beneficial to the vegetative growth of nostoc cells.
The above examples are only for illustrating the technical concept and features of the present invention, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (8)
1. A mixed nutrient culture method suitable for the vegetative growth of nostoc sphaeroides is characterized by comprising the following steps:
(1) preparing a nostoc sphaeroides culture medium: after the pH of the BG11 culture medium is adjusted, adding a certain amount of glucose to ensure that the glucose concentration is 0.09-0.11mol/L to obtain an improved BG11 culture medium for later use;
(2) inoculating algae seeds: selecting nostoc sphaeroides with the grain diameter of 3 +/-1 mm, elasticity, compact structure and dark green color as an algae seed, and inoculating the algae seed on the improved BG11 culture medium in the step (1);
(3) mixed nutrient culture: culturing the modified BG11 culture medium inoculated with the algae seeds in the step (2) under the following conditions: temperature: at the temperature of 21-23 ℃, illumination: 50 μmol. m -2 ·s -1 Light-dark period L: d =10 h: and 14 h.
2. The mixed nutrient culture method of claim 1, wherein in step (1), the BG11 medium is adjusted to pH 7.0-7.2; then carrying out high-temperature 100kpa high-pressure sterilization on the culture medium for 20 minutes at 120 ℃ to obtain an incomplete culture medium, cooling the incomplete culture medium, and adding glucose to enable the glucose concentration to be 0.09-0.11mol/L to obtain an improved BG11 culture medium.
3. The mixed nutrient culture method of claim 1, wherein the glucose concentration is 0.1 mol/L.
4. The mixed nutrient culture method of claim 1, wherein in step (2), the inoculation is carried out at a density of: 6.64-6.70g algae/L culture medium.
5. The mixed nutrient culture method of claim 4, wherein the inoculation is performed at a density of: 6.67g algae/L medium.
6. The mixed nutrient culture method according to claim 1, wherein in step (3), the conditions of the mixed nutrient culture are preferably: temperature: 22 ℃ and illumination: 50 μmol. m -2 ·s -1 Light-dark period L: d =10 h: and 14 h.
7. The mixed nutrient culture method as claimed in claim 1, wherein the mixed culture method for vegetative growth of nostoc sphaeroides is used for scale culture of nostoc sphaeroides.
8. The mixed nutrient culture method as claimed in claim 1, wherein the mixed culture method for vegetative growth of nostoc sphaeroides is used for large-scale culture of nostoc commune or emerald green nostoc sphaeroides.
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| CN112831449A (en) * | 2021-04-13 | 2021-05-25 | 中国海洋大学 | Seed expansion method for carrying out Nostoc sphaeroides cultivation section generation by utilizing temperature rise |
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- 2022-05-24 CN CN202210568810.1A patent/CN115011511A/en active Pending
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|---|---|---|---|---|
| WO2010123848A2 (en) * | 2009-04-20 | 2010-10-28 | University Of Georgia Research Foundation, Inc. | Mixotrophic algae for the production of algae biofuel feedstock on wastewater |
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