CN112694977A - Chlorella culture method - Google Patents
Chlorella culture method Download PDFInfo
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- CN112694977A CN112694977A CN202110038606.4A CN202110038606A CN112694977A CN 112694977 A CN112694977 A CN 112694977A CN 202110038606 A CN202110038606 A CN 202110038606A CN 112694977 A CN112694977 A CN 112694977A
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- 241000195649 Chlorella <Chlorellales> Species 0.000 title claims abstract description 104
- 238000012136 culture method Methods 0.000 title claims abstract description 11
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 66
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 59
- 238000000034 method Methods 0.000 claims abstract description 41
- 241000195493 Cryptophyta Species 0.000 claims abstract description 38
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 34
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 33
- 238000012258 culturing Methods 0.000 claims abstract description 28
- 230000001954 sterilising effect Effects 0.000 claims abstract description 21
- 230000012010 growth Effects 0.000 claims abstract description 18
- 238000003306 harvesting Methods 0.000 claims abstract description 6
- 235000015097 nutrients Nutrition 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 83
- 238000004659 sterilization and disinfection Methods 0.000 claims description 27
- 239000007864 aqueous solution Substances 0.000 claims description 25
- 238000005286 illumination Methods 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 21
- 239000000843 powder Substances 0.000 claims description 19
- ODINCKMPIJJUCX-UHFFFAOYSA-N Calcium oxide Chemical compound [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 claims description 18
- ZKQDCIXGCQPQNV-UHFFFAOYSA-N Calcium hypochlorite Chemical compound [Ca+2].Cl[O-].Cl[O-] ZKQDCIXGCQPQNV-UHFFFAOYSA-N 0.000 claims description 14
- 239000007844 bleaching agent Substances 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 13
- 239000013505 freshwater Substances 0.000 claims description 11
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 10
- 238000009835 boiling Methods 0.000 claims description 10
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 10
- 239000000292 calcium oxide Substances 0.000 claims description 9
- 235000012255 calcium oxide Nutrition 0.000 claims description 9
- 239000012286 potassium permanganate Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 229930003451 Vitamin B1 Natural products 0.000 claims description 7
- 229930003779 Vitamin B12 Natural products 0.000 claims description 7
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- 229960003495 thiamine Drugs 0.000 claims description 7
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 7
- 235000010374 vitamin B1 Nutrition 0.000 claims description 7
- 239000011691 vitamin B1 Substances 0.000 claims description 7
- 235000019163 vitamin B12 Nutrition 0.000 claims description 7
- 239000011715 vitamin B12 Substances 0.000 claims description 7
- 238000001556 precipitation Methods 0.000 claims description 6
- 239000013535 sea water Substances 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 claims description 5
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 5
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 5
- 229940037003 alum Drugs 0.000 claims description 5
- 239000011609 ammonium molybdate Substances 0.000 claims description 5
- 235000018660 ammonium molybdate Nutrition 0.000 claims description 5
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 claims description 5
- 229940010552 ammonium molybdate Drugs 0.000 claims description 5
- 229910052810 boron oxide Inorganic materials 0.000 claims description 5
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 5
- JKWMSGQKBLHBQQ-UHFFFAOYSA-N diboron trioxide Chemical compound O=BOB=O JKWMSGQKBLHBQQ-UHFFFAOYSA-N 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 5
- 239000011565 manganese chloride Substances 0.000 claims description 5
- 235000002867 manganese chloride Nutrition 0.000 claims description 5
- 229940099607 manganese chloride Drugs 0.000 claims description 5
- 238000000643 oven drying Methods 0.000 claims description 5
- 239000011592 zinc chloride Substances 0.000 claims description 5
- 235000005074 zinc chloride Nutrition 0.000 claims description 5
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 4
- 239000000460 chlorine Substances 0.000 claims description 4
- 229910052801 chlorine Inorganic materials 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000010298 pulverizing process Methods 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 239000004568 cement Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000002604 ultrasonography Methods 0.000 claims description 2
- 239000013589 supplement Substances 0.000 abstract description 3
- 230000009467 reduction Effects 0.000 description 11
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 4
- 230000001651 autotrophic effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 241000196319 Chlorophyceae Species 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 241000700141 Rotifera Species 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000195627 Chlamydomonadales Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000196127 Osmunda Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 230000035425 carbon utilization Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- 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
- 238000012364 cultivation method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000009569 heterotrophic growth Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000021049 nutrient content Nutrition 0.000 description 1
- 238000009372 pisciculture Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- CEIZFXOZIQNICU-UHFFFAOYSA-N tenuazonic acid Chemical compound CCC(C)C1NC(=O)C(C(C)=O)=C1O CEIZFXOZIQNICU-UHFFFAOYSA-N 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- 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
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- 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
- C12N1/38—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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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of microalgae biology, and particularly discloses a culture method of chlorella. The method comprises the steps of sterilizing a culture container, preparing nutrient solution, inoculating, culturing, harvesting and the like. The method comprises the steps of firstly culturing the chlorella under the most appropriate growth conditions (the light intensity is 3000-4500 Lx, the water temperature is 25-30 ℃, and the carbon dioxide supplement amount is 2-3 mg/L) for 2-4 days, enabling the chlorella to propagate and grow in a large amount, improving the initial concentration of chlorella cells in a culture solution, then culturing at the low temperature (0-5 ℃) for 5-10 days, enabling the chlorella to adapt to the low-temperature growth environment gradually, finally raising the temperature to 5-10 ℃, and continuing to culture for 1-2 days, so that the chlorella can continue to grow, and can be directly used as an algae source cultured in an outdoor pond (the water temperature is 6-9 ℃) in winter. The method can make chlorella grow at low temperature, and has simple culture method, reduced death rate of chlorella, and no other miscellaneous algae.
Description
Technical Field
The invention relates to the field of microalgae biology, in particular to a culture method of chlorella.
Background
Chlorella is a unicellular plant, and has many kinds, most of which live in fresh water, and few of which live in sea. According to the classification of botanicals, the chlorella belongs to the organisms of the genus chlorella of the family ascomycetaceae of the order Chlorococcales of the class Chlorophyceae of the phylum Chlorophyceae, and has a small size, generally 3-8 μm in diameter, and can be seen only by magnifying by more than 400-600 times under a microscope. The chlorella contains high nutrient content, protein content of 50-60% (5 times of egg), fat content of 10-30%, and multiple vitamins, minerals, nucleic acid and chlorophyll. The chlorella biological active substances, such as glycoprotein and polysaccharide, as well as nucleic acid with the concentration of up to 13 percent, have the functions of enhancing the immunity of human bodies, resisting cancers, reducing blood pressure, inhibiting the rise of blood sugar, eliminating vivotoxin, quickly recovering the injury of the organisms and the like, also have great use value in the production application of the fishery industry and create extremely high economic benefit. Therefore, the culture of the chlorella has wide application prospect.
The chlorella can be cultured by both autotrophy and heterotrophy. The traditional culture method mostly adopts autotrophic culture, the autotrophic culture of the chlorella can utilize natural illumination or artificial illumination, and a culture medium mainly comprises inorganic compounds. The culture mode of photoautotrophy currently comprises open pond culture and closed culture, wherein the former has the advantages of simple equipment, low technical content and low investment, but the yield is low and the culture mode is easily influenced by natural conditions. Since Lewis and the like firstly discover that some algae can utilize organic matters as a unique carbon source and energy source for heterotrophic growth, people have great interest in heterotrophic culture of microalgae, the heterotrophic culture of microalgae can overcome the defects of the autotrophic culture, the culture can be carried out in pure species, the conditions are easier to control, high-density culture is realized, and faster growth rate and higher yield can be obtained.
At present, the general microalgae are cultured at the temperature of 25-35 ℃, and CO is generated2The solubility is not very different, when the culture temperature is reduced to 5-15 ℃, CO2The solubility in the culture solution is improved by 3-4 times, which provides a preferential condition for the culture of the microalgae. However, the temperature decrease may seriously affect the growth rate of microalgae, even making most microalgae unable to grow.
To this end, the chinese patent (application No. 201010220906.6) discloses a method for culturing chlorella at low temperature, which is to allow normal growth of cultured chlorella at 0-15 ℃ by stepwise cooling, by means of proper temperature control and synergistic effect of proper culture medium and culture conditions. Meanwhile, chinese patent (application No. 202010093875.6) discloses a high and low temperature continuous stress microalgae cultivation method, which adopts two-stage cultivation: in the pre-culture stage, the optimum conditions are set at [25 ℃, 100 [ mu ] mol/(m)2·s)]Culturing Osmunda cells to 1.0 × 106Per L-1.2X 106In the concentration range of one/L, the microalgae pre-culture solution is used for continuous high-low temperature stress with the maximum temperature of 35-37 ℃,the lowest temperature is 2-4 ℃, and the method can greatly shorten the culture period of the microalgae. However, the high temperature easily causes the death of part of the chlorella, the dead chlorella can acidify water, the death of the chlorella is further accelerated, and finally the appearance that the chlorella is suddenly dead is caused; meanwhile, the method takes 24 hours as a period, high and low temperature adjustment is needed every 8 hours, cultivation is needed for 10-14 periods, the cultivation mode is complicated, and the temperature adjustment time is too frequent, so that the growth and development of chlorella are easy to be disordered.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a method for culturing chlorella, which enables normal growth of chlorella at low temperature by high-low temperature shift culture, and which is simpler in culture method than the prior art and can reduce the death rate of chlorella.
In order to achieve the above objects and other related objects, the present invention provides a method for culturing chlorella, comprising the steps of:
(1) sterilizing the culture container by a high-temperature sterilization method or a chemical sterilization method, and then cleaning the culture container by water;
(2) preparing a culture solution;
(3) inoculation: inoculating according to the ratio of 1: 3-4 of algae liquid and culture solution;
(4) culturing:
the first stage is as follows: the illumination intensity is 3000-4500 Lx, the water temperature is 25-30 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 2-3 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 2-4 days;
and a second stage: the illumination intensity is 4000-5500 Lx, the water temperature is 0-5 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 4-6 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 5-10 days;
and a second stage: the illumination intensity is 2000-4500 Lx, the water temperature is 5-10 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 3-5 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 1-2 days;
(5) harvesting:
when the content of chlorella in the culture solution is more than or equal to 200/liter, the culture solution can be harvested.
Further, in the step (1), the high-temperature sterilization method is selected from one of boiling sterilization and oven drying sterilization. The boiling sterilization is to put the culture container into a pot, add water to boil for sterilization, the boiling time is 10-20 minutes, or add fresh water to the culture container, seal, put on a heating furnace to heat and boil for 5-10 minutes, so as to sterilize the inner wall of the whole container. After the sterilization, the bottle mouth is bound with sterilized paper or sterilized gauze. The method is suitable for sterilizing small container tools. The drying and disinfection of the oven comprises the steps of washing a culture container with clear water, putting the culture container into the oven, closing the oven door, opening the vent hole, switching on a power supply for heating, closing the vent hole when the temperature rises to 120 ℃, and stopping heating; if pure culture is carried out, the culture container must be sterilized, when the temperature rises to 105 ℃, the vent hole is closed, the temperature is continuously heated to 160 ℃, the temperature is kept, the constant temperature is kept for 2 hours or more, and then the heating is stopped; and opening the oven door when the temperature is reduced to below 60 ℃. The culture container with cotton plug and paper wrapping must not exceed 180 deg.C to avoid scorching.
Further, in the step (1), the chemical disinfection method is to soak the culture container with bleaching powder aqueous solution, potassium permanganate aqueous solution or quicklime aqueous solution for disinfection.
Optionally, the bleaching powder contains 25-35% of available chlorine, and the concentration of the bleaching powder aqueous solution is 200-300 mg/L; the concentration of the potassium permanganate aqueous solution is 5-6%, and the concentration of the quicklime aqueous solution is 2-3%.
Optionally, the soaking time of the bleaching powder aqueous solution or the quicklime aqueous solution in the culture container is not less than 0.5 hour, and the soaking time of the potassium permanganate aqueous solution in the culture container is not less than 5 minutes.
Further, in the step (1), when the culture is carried out on a small scale, the culture container is a barrel or a jar, and when the culture is carried out on a large scale, the culture container is a cement pond or a transparent tank.
Further, in the step (2), the preparation method of the culture solution comprises the following steps:
the component A comprises: 45-50 mg of ferric trichloride, 1.8-2.0 mg of manganese chloride, 210 mg of 45% boron oxide 200-;
and B component: 0.5-0.6 g of zinc chloride, 0.5-0.6 g of cobalt chloride, 0.2-0.25 g of ammonium molybdate and 0.5-0.6 g of copper sulfate;
the component A and the component B are put into 1 ton of water to be fully dissolved, then 2.0-2.5 g of vitamin B1 and B12 are added respectively, and the mixture is stirred uniformly to prepare a culture solution.
Further, the culture solution also comprises a component C: 2-5 g of chlorella powder, putting the component A, the component B and the component C into 1 ton of water, fully and uniformly mixing, then adding 2.0-2.5 g of vitamin B1 and B12 respectively, uniformly stirring, performing ultrasonic treatment, and filtering to obtain the culture solution.
Optionally, the preparation method of the chlorella powder comprises the following steps: drying chlorella at 50-60 deg.C, wherein the water content of the dried chlorella is lower than 5%, and pulverizing into 200-mesh 300-mesh powder;
optionally, the ultrasound conditions are: 50-60 ℃ for 6-8 hours.
Further, in the step (2), the water used for preparing the culture solution is seawater or fresh water, and the seawater or fresh water is precipitated and filtered.
Further, in the step (2), the culture solution is heated and sterilized before use, wherein the heating temperature is 85-100 ℃, and the heating time is 5-6 minutes or is heated to boiling. Since some organic substances in the seawater which promote the growth of algae are easily destroyed at high temperature, the heating time is not too long.
Further, in the step (3), the algae species with strong vitality and vigorous growth are selected, and inoculated in the morning of sunny weather, preferably 8-10 am, and the time is the time when the metabolism of algae cells is most vigorous.
Further, in the step (4), the temperature is reduced from 25-30 ℃ to 0-5 ℃ in a stepped temperature reduction mode from the first stage to the second stage, and the temperature reduction rate is 1-2 ℃/h, so that the phenomenon that the temperature is suddenly reduced to cause death of parts of the chlorella is avoided.
Further, in the step (4), in the culture process, the culture solution is stirred, the position of the algae is continuously changed, the illumination, the nutrients and the water temperature are balanced, and the algae solution is observed and pollution control is paid attention.
Further, in the step (4), in the process of culturing the chlorella, whether the color of the chlorella liquid is normal, whether the precipitation and the wall attachment phenomenon exist, and whether the liquid surface has a mycoderm are often observed; if protozoa live under the microscope, they can be killed with 1mg/L formalin.
Further, in the step (5), when the chlorella is collected, 0.3 to 0.5 percent of alum powder is dissolved in the culture solution, and after the chlorella is settled at the bottom of the water, the water on the upper layer is removed, so that the concentrated solution of the chlorella can be obtained.
As described above, the method for culturing chlorella of the present invention has the following advantageous effects:
the method firstly cultures the chlorella for 2-4 days under the most appropriate growth conditions (the light intensity is 3000-4500 Lx, the water temperature is 25-30 ℃, and the supplement amount of carbon dioxide is 2-3 mg/L), so that the chlorella is propagated and grown in large quantity, the initial concentration of the chlorella cells in the culture solution is improved, then reducing the temperature from 25-30 ℃ to 0-5 ℃ by adopting a step-type cooling mode, culturing the chlorella at a low temperature (0-5 ℃) for 5-10 days to ensure that the chlorella gradually adapts to a low-temperature growth environment, and properly improving the illumination intensity and the carbon dioxide supplement amount to avoid slow growth and reproduction of the chlorella at low temperature, finally raising the temperature to 5-10 ℃, continuously culturing for 1-2 days to ensure that the chlorella continuously grows, and directly using the chlorella as an algae source for culturing in an outdoor pond (with the water temperature of 6-9 ℃) in winter. Meanwhile, the low-temperature culture can also avoid the generation of other miscellaneous algae.
The culture solution disclosed by the invention comprises the C component, namely the chlorella powder, besides the conventional raw materials (the A component and the B component), and the chlorella powder is used for preparing the culture solution, so that the culture solution is more suitable for the growth of the chlorella, the adaptability of the chlorella is improved, the death rate of the chlorella is reduced, and meanwhile, the culture solution can also play a role in promoting the low-temperature normal growth of the chlorella.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
In the following examples, chlorella as a culture raw material was collected from living chlorella seed liquid or relatively clean fish-farming ponds or reservoirs, and the purity was examined under a microscope and then reused, or was purchased from research institutes or research institutions.
In the present invention, the sterilization method of the culture vessel includes a high-temperature sterilization method and a chemical sterilization method.
Specifically, the high-temperature disinfection method comprises boiling disinfection and oven drying disinfection.
Boiling and sterilizing, namely putting the culture container into a pot, adding water, boiling and sterilizing for 10-20 minutes, or adding fresh water into the culture container, sealing, and placing on a heating furnace for boiling for 5-10 minutes to sterilize the inner wall of the whole container. After the sterilization, the bottle mouth is bound with sterilized paper or sterilized gauze. The method is suitable for sterilizing small container tools.
Drying and disinfecting the oven, namely, after the culture container is cleaned by clear water, putting the culture container into the oven, closing the oven door, opening the vent hole, switching on a power supply for heating, and when the temperature rises to 120 ℃, closing the vent hole and stopping heating; if pure culture is carried out, the culture container must be sterilized, when the temperature rises to 105 ℃, the vent hole is closed, the temperature is continuously heated to 160 ℃, the temperature is kept, the constant temperature is kept for 2 hours or more, and then the heating is stopped; and opening the oven door when the temperature is reduced to below 60 ℃. The culture container with cotton plug and paper wrapping must not exceed 180 deg.C to avoid scorching.
The chemical disinfection method is to soak the culture container with bleaching powder water solution, potassium permanganate water solution or quicklime water solution for disinfection.
Specifically, the bleaching powder contains 25-35% of available chlorine, and the concentration of the bleaching powder aqueous solution is 200-300 mg/L; the concentration of the potassium permanganate aqueous solution is 5-6%, and the concentration of the quicklime aqueous solution is 2-3%.
Specifically, the soaking time of the bleaching powder aqueous solution or the quicklime aqueous solution in the culture container is not less than 0.5 hour, and the soaking time of the potassium permanganate aqueous solution in the culture container is not less than 5 minutes.
When the rotifer is cultured on a small scale, the culture container adopts a barrel or a jar, and when the rotifer is cultured on a large scale, the culture container adopts a cement pond or a transparent tank body.
In the following examples, chlorella is cultured in a 5L glass container, bleaching powder with 30% chlorine content is prepared into 200mg/L aqueous solution of bleaching powder with water, the glass container and other tools used for culturing chlorella are soaked for half an hour, and then washed with clear water for 3-4 times.
Example 1
A culture method of chlorella comprises the following steps:
(1) the culture vessel is sterilized and then washed clean with water.
(2) Preparing a culture solution, wherein the preparation method of the culture solution comprises the following steps:
the component A comprises: 50 mg of ferric trichloride, 2.0 mg of manganese chloride, 200mg of 45% boron oxide, 1 g of monopotassium phosphate, 200mg of tetrasodium ethylene diamine tetraacetate, and 100 mg of sodium carbonate or EDTA sodium.
And B component: 0.5 g of zinc chloride, 0.5 g of cobalt chloride, 0.2 g of ammonium molybdate and 0.5 g of copper sulfate.
The component A and the component B are put into 1 ton of fresh water to be fully dissolved, then 2.0 g of each of vitamin B1 and B12 is added to be uniformly stirred to prepare a culture solution. The culture broth was heated at 90 ℃ for 5 minutes and reused.
(3) Inoculation:
selecting algae species with strong vitality and vigorous growth, and inoculating in the morning of clear weather, specifically 8 am. Inoculating according to the ratio of 1: 3 of algae liquid and culture solution.
(4) Culturing:
the first stage is as follows: the illumination intensity is 4000Lx, the water temperature is 28-30 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 2 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 3 days;
and a second stage: the illumination intensity is 5000Lx, the water temperature is 0-2 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 5 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 8 days;
and a second stage: the light intensity is 4500Lx, the water temperature is 6-8 deg.C, carbon dioxide is supplemented between 9 am and 2 pm, and each liter of culture solution is supplemented with 4 mg of carbon dioxide for 1 day.
Wherein, the temperature is reduced from 28-30 ℃ to 0-2 ℃ in a step-type temperature reduction manner from the first stage to the second stage, and the temperature reduction rate is 1-2 ℃/h, so as to avoid sudden temperature reduction and partial death of chlorella.
In the culture process, the culture solution is stirred for 2-3 times every day, so that the position of the algae is changed continuously, and the illumination, the nutrients and the water temperature are balanced; observing the algae liquid and preventing pollution, and often observing whether the color of the algae liquid is normal, whether the algae liquid has precipitation and wall attachment phenomena, and whether the liquid surface has a mycoderm; if protozoa live under the microscope, they can be killed with 1mg/L formalin.
(5) Harvesting:
when the content of chlorella in the culture solution is more than or equal to 200/liter, the culture solution can be harvested.
During the collection, 0.3% alum powder is dissolved in the culture solution, and after the chlorella is settled in the water bottom, the water on the upper layer is removed, so that the concentrated solution of the chlorella can be obtained.
The concentration of the algae cells in the obtained chlorella concentrate is 0.7588 × 108One/ml. Example 2
A culture method of chlorella comprises the following steps:
(1) the culture vessel is sterilized and then washed clean with water.
(2) Preparing a culture solution, wherein the preparation method of the culture solution comprises the following steps:
the component A comprises: 45 mg of ferric trichloride, 1.8 mg of manganese chloride, 210 mg of 45% boron oxide 200-.
And B component: 0.6 g of zinc chloride, 0.6 g of cobalt chloride, 0.25 g of ammonium molybdate and 0.6 g of copper sulfate.
And C, component C: 2 g of chlorella powder. The preparation method of the chlorella powder comprises the following steps: oven drying Chlorella at 60 deg.C until water content is lower than 5%, and pulverizing into 200 mesh powder. The component A, the component B and the component C are put into 1 ton of fresh water and fully dissolved, then 2.2 grams of vitamin B1 and B12 are added respectively, the mixture is evenly stirred and is subjected to ultrasonic treatment at 60 ℃ for 6 hours, and the culture solution is prepared after filtration. The culture solution is heated to boiling and then used.
(3) Inoculation:
selecting algae species with strong vitality and vigorous growth, and inoculating in the morning of sunny weather, specifically 9 am, which is the time when the metabolism of algae cells is most vigorous. Inoculating according to the ratio of 1: 4 of algae liquid and culture solution.
(4) Culturing:
the first stage is as follows: supplementing carbon dioxide at the illumination intensity of 3000Lx and the water temperature of 25-27 ℃ between 9 am and 2 pm, supplementing 2 mg of carbon dioxide per liter of culture solution, and culturing for 4 days;
and a second stage: the illumination intensity is 4000Lx, the water temperature is 3-5 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 4 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 10 days;
and a second stage: the illumination intensity is 2000Lx, the water temperature is 5-8 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 3 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 2 days.
Wherein, the temperature is reduced from 25-27 ℃ to 3-5 ℃ in a step-type temperature reduction manner from the first stage to the second stage, and the temperature reduction rate is 1-2 ℃/h, so as to avoid sudden temperature reduction and partial death of chlorella.
In the culture process, the culture solution is stirred for 2-3 times every day, so that the position of the algae is changed continuously, and the illumination, the nutrients and the water temperature are balanced; observing the algae liquid and preventing pollution, and often observing whether the color of the algae liquid is normal, whether the algae liquid has precipitation and wall attachment phenomena, and whether the liquid surface has a mycoderm; if protozoa live under the microscope, they can be killed with 1mg/L formalin.
(5) Harvesting:
when the content of chlorella in the culture solution is more than or equal to 200/liter, the culture solution can be harvested.
During the collection, 0.5% alum powder is dissolved in the culture solution, and after the chlorella is settled in the water bottom, the water on the upper layer is removed, so that the concentrated solution of the chlorella can be obtained.
The concentration of the algae cells in the obtained chlorella concentrate is 0.8546 × 108One/ml.
Example 3
A culture method of chlorella comprises the following steps:
(1) the culture vessel is sterilized and then washed clean with water.
(2) Preparing a culture solution, wherein the preparation method of the culture solution comprises the following steps:
the component A comprises: 48 mg of ferric trichloride, 2.0 mg of manganese chloride, 200mg of 45% boron oxide, 1 g of monopotassium phosphate, 200mg of tetrasodium ethylenediamine tetraacetate, 300mg of sodium carbonate or 100 mg of sodium EDTA;
and B component: 0.5 g of zinc chloride, 0.5 g of cobalt chloride, 0.2 g of ammonium molybdate and 0.5 g of copper sulfate;
and C, component C: and 5 g of chlorella powder. The preparation method of the chlorella powder comprises the following steps: oven drying Chlorella at 50 deg.C, the water content of the oven dried Chlorella is less than 5%, and pulverizing into 300 mesh powder.
The component A, the component B and the component C are put into 1 ton of fresh water and fully dissolved, then 2.5 g of vitamin B1 and B12 are added respectively, the mixture is evenly stirred and is subjected to ultrasonic treatment at 50 ℃ for 8 hours, and the culture solution is prepared after filtration. The culture broth was heated at 85 ℃ for 5 minutes and reused.
(3) Inoculation:
and selecting algae species with strong vitality and vigorous growth, and inoculating in the morning of sunny weather, preferably 8-10 am, wherein the time is the time when the metabolism of algae cells is most vigorous. Inoculating the algae solution and the culture solution according to the ratio of 1: 3-4.
(4) Culturing:
the first stage is as follows: the illumination intensity is 4500Lx, the water temperature is 26-28 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 3 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 2 days;
and a second stage: the illumination intensity is 5500Lx, the water temperature is 2-4 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 6 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 5 days;
and a second stage: the illumination intensity is 4000Lx, the water temperature is 6-8 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 5 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 2 days.
Wherein, the temperature is reduced from 26-28 ℃ to 2-4 ℃ in a step-type temperature reduction manner from the first stage to the second stage, and the temperature reduction rate is 1-2 ℃/h, so as to avoid sudden temperature reduction and partial death of chlorella.
In the culture process, the culture solution is stirred for 2-3 times every day, so that the position of the algae is changed continuously, and the illumination, the nutrients and the water temperature are balanced; observing the algae liquid and preventing pollution, and often observing whether the color of the algae liquid is normal, whether the algae liquid has precipitation and wall attachment phenomena, and whether the liquid surface has a mycoderm; if protozoa live under the microscope, they can be killed with 1mg/L formalin.
(5) Harvesting:
when the content of chlorella in the culture solution is more than or equal to 200/liter, the culture solution can be harvested.
During the collection, 0.5% alum powder is dissolved in the culture solution, and after the chlorella is settled in the water bottom, the water on the upper layer is removed, so that the concentrated solution of the chlorella can be obtained.
The concentration of the algae cells in the obtained chlorella concentrate is 0.8825 × 108One/ml.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A culture method of chlorella is characterized by comprising the following steps:
(1) sterilizing the culture container by a high-temperature sterilization method or a chemical sterilization method, and then cleaning the culture container by water;
(2) preparing a culture solution;
(3) inoculation: inoculating according to the ratio of 1: 3-4 of algae liquid and culture solution;
(4) culturing:
the first stage is as follows: the illumination intensity is 3000-4500 Lx, the water temperature is 25-30 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 2-3 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 2-4 days;
and a second stage: the illumination intensity is 4000-5500 Lx, the water temperature is 0-5 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 4-6 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 5-10 days;
and a second stage: the illumination intensity is 2000-4500 Lx, the water temperature is 5-10 ℃, carbon dioxide is supplemented between 9 am and 2 pm, 3-5 mg of carbon dioxide is supplemented to each liter of culture solution, and the culture is carried out for 1-2 days;
(5) harvesting: when the content of chlorella in the culture solution is more than or equal to 200/liter, the culture solution can be harvested.
2. The method for culturing chlorella according to claim 1, wherein: in the step (1), the high-temperature disinfection method is selected from one of boiling disinfection and oven drying disinfection;
and/or in the step (1), the chemical disinfection method is to soak the culture container with bleaching powder aqueous solution, potassium permanganate aqueous solution or quicklime aqueous solution for disinfection;
and/or, in the step (1), in small-scale culture, a barrel or a jar is adopted as a culture container, and in large-scale culture, a cement pond or a transparent tank is adopted as the culture container.
3. The method for culturing chlorella according to claim 2, wherein: the bleaching powder contains 25-35% of available chlorine, and the concentration of the bleaching powder aqueous solution is 200-300 mg/L; the concentration of the potassium permanganate aqueous solution is 5-6%, and the concentration of the quicklime aqueous solution is 2-3%;
and/or the soaking time of the bleaching powder aqueous solution or the quicklime aqueous solution in the culture container is not less than 0.5 hour, and the soaking time of the potassium permanganate aqueous solution in the culture container is not less than 5 minutes.
4. The method for culturing chlorella according to claim 1, wherein: in the step (2), the preparation method of the culture solution comprises the following steps:
the component A comprises: 45-50 mg of ferric trichloride, 1.8-2.0 mg of manganese chloride, 210 mg of 45% boron oxide 200-;
and B component: 0.5-0.6 g of zinc chloride, 0.5-0.6 g of cobalt chloride, 0.2-0.25 g of ammonium molybdate and 0.5-0.6 g of copper sulfate; the component A and the component B are put into 1 ton of water to be fully dissolved, then 2.0-2.5 g of vitamin B1 and B12 are added respectively, and the mixture is stirred uniformly to prepare a culture solution.
5. The method for culturing chlorella according to claim 4, wherein: the culture solution also comprises a component C: 2-5 g of chlorella powder, putting the component A, the component B and the component C into 1 ton of water, fully and uniformly mixing, then adding 2.0-2.5 g of vitamin B1 and B12 respectively, uniformly stirring, performing ultrasonic treatment, and filtering to obtain the culture solution.
6. The method for culturing chlorella according to claim 5, wherein: the preparation method of the chlorella powder comprises the following steps: drying chlorella at 50-60 deg.C, wherein the water content of the dried chlorella is lower than 5%, and pulverizing into 200-mesh 300-mesh powder;
and/or, the ultrasound conditions are: 50-60 ℃ for 6-8 hours.
7. The method for culturing chlorella according to claim 1, wherein: in the step (2), the water used for preparing the culture solution is seawater or fresh water, and the seawater or fresh water is the seawater or fresh water obtained after precipitation and filtration;
and/or, in the step (2), the culture solution is used after being heated and sterilized, wherein the heating temperature is 85-100 ℃, and the heating time is 5-6 minutes, or the culture solution is heated to be boiled.
8. The method for culturing chlorella according to claim 1, wherein: in the step (3), the algae species with strong vitality and vigorous growth are selected and inoculated in the morning in sunny weather.
9. The method for culturing chlorella according to claim 1, wherein: in the step (4), the temperature is reduced from 25-30 ℃ to 0-5 ℃ in a step-type cooling mode from the first stage to the second stage, and the cooling rate is 1-2 ℃/h;
and/or in the step (4), in the culture process, stirring the culture solution, continuously changing the positions of the algae, balancing the illumination, nutrients and water temperature, and observing the algae solution and preventing pollution;
and/or, in the step (4), in the culture process of the chlorella, whether the color of the chlorella liquid is normal, whether the precipitation and the wall attachment phenomenon exist or not and whether the liquid surface has a mycoderm or not are often observed; if protozoa live under the microscope, they can be killed with 1mg/L formalin.
10. The method for culturing chlorella according to claim 1, wherein: in the step (5), during the collection, 0.3-0.5% of alum powder is dissolved in the culture solution, and after the chlorella is settled in the water bottom, the water on the upper layer is removed, so that the concentrated solution of the chlorella can be obtained.
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| CN103114041A (en) * | 2012-12-19 | 2013-05-22 | 盐城工学院 | Method for rapidly cultivating chlorella |
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