CN103146582A - High-throughput screening method of oil-rich microalgae - Google Patents
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Abstract
The invention discloses a high-throughput screening method of oil-rich microalgae, relates to the field of microalgae biological energy, and aims at solving the problems that an existing microalgae organic solvent extraction method consumes time and labor and requires a large quantity of reagents and samples, a toxic reagent does harm to human body and environment, and the content of oil is hard to measure with high throughput during microalgae screening. The method comprises the following steps of: (1) fetching microalgae and performing enrichment culture for 7-14 days; (II) repeating for twice to three times; (III) after inoculation, culturing for 7-14 days; (IV) diluting and spraying, and culturing until algae colonies appear; (V) choosing large pure algae colonies with a high growth speed, and culturing for 7-14 days; (VI) repeating steps (IV) to (V) for 3-4 times; and (VII) performing ultrasonic breaking, adding a Nile red solution, and selecting an algae strain with the highest fluorescence intensity to obtain an algae strain with a high oil content. The method disclosed by the invention avoids complicated traditional oil detection steps, and overcomes the problem of bottleneck that a quick high-throughput screening method is unavailable in large-scale screening of algae seeds.
Description
Technical field
The present invention relates to little algae bioenergy field.
Background technology
In recent years, along with the fossil oil reserves reduce day by day, the exhaustion of fossil oil and the problems such as Global warming, environmental pollution non-renewable and that burning causes forced countries in the world to seek green that can substitute fossil fuels, the new forms of energy of Sustainable development.Biomass energy can utilize photosynthesis that the form of sun power with chemical energy stored in vivo, is environment-friendly type renewable energy source the most general on the earth.In numerous biomass energies, biofuel has been subject to paying close attention to widely for fastest developing speed, most widely used in the world green novel energy source.
The major ingredient of biofuel is fatty acid methyl ester, and the performance close with petrifaction diesel arranged, and has good combustionproperty, the feature of environmental protection and recyclability.Its main raw material derives from Vegetable oil lipoprotein (soybean oil, rapeseed oil, Semen Maydis oil, curcas oil, plam oil and peanut wet goods), animal grease (tallow fatty acid) and discarded edible oil (sewer oil).Yet the bottleneck of existing biofuel development is that cost of material is high, the supply wretched insufficiency, and strengthen biofuel production will certainly with other farm crop contentions soil, cause the staple food supply problem.Therefore, need the glyceride stock source of seeking low cost, extensive, sustainable supply badly.
Compare with the raw material of other biological diesel oil, with microalgae grease production biofuel, obvious advantage is arranged.The oleaginousness of little algae is high, growth cycle is short, the grease productive rate will be far above common produce oil crop.Little algae is cultivated and is not conflicted with agriculture production, can carry out large-scale production in the non-agricultural waters such as saltings, beach, Desert Area and saline-alkali water, seawater and waste water.Little algae is extracted grease except being used for, and also can develop high value added product (activated protein, active polysaccharide, natural pigment, biogas, feed, surfactant etc.), in order to reduce the cost of little algae biofuel.
The top priority of little algae biofuel large-scale production is that the screening fat content is high, growth is rapid and the algae strain of strong stress resistance, and the training method of little algae plays important effect in the seed selection of rich oil algae kind.The training method of little algae mainly is divided into autotrophy, heterotrophism and three kinds of modes of mixed culture.It is take the inorganic carbide compound as carbon source that autotrophy is cultivated, and utilizes the photosynthesis of little algae chemical energy to be converted into the nutritional mode of bioenergy.It is simple to operate, cost is lower, but the cell density that obtains is low, photosynthetic efficiency is low, and when scale is cultivated, microalgae harvesting is difficult, is difficult to satisfy the needs of large-scale production.It is to utilize organic carbon source as the energy and carbon source unglazed according under condition that heterotrophism is cultivated, can carry out the high-density culture of algae and greatly improve the grease productive rate of little algae, this has reduced difficulty and the extraction cost of gathering accordingly, but heterotrophism is cultivated production cost and energy consumption is all higher.Mixed culture refers to that little algae utilizes inorganics and organism as the training method of carbon source, can utilize simultaneously the ability of algae photoautotrophy and heterotrophic growth, it is reported that its specific growth rate approximates the summation of autotrophy and heterotrophism cultivation, can realize the mutual supplement with each other's advantages that autotrophy is cultivated and heterotrophism is cultivated, therefore mixed culture is a kind of than better suited training method.In addition, traditional organic solvent extraction method measures that microalgae grease content exists that time and effort consuming, required reagent and sample size are large, toxic agent can to problems such as human body and environment work the mischief, be difficult to satisfy the needs of high throughput assay fat content in the oil-rich microalgae screening process.
Summary of the invention
The objective of the invention is the rich oil algae kind that to carry out mixed culture in order to screen, and the existing little algae organic solvent extraction method of solution exists, and time and effort consuming, required reagent and sample size are large, toxic agent can work the mischief to human body and environment, be difficult to satisfy the problem of the needs of high throughput assay fat content in little algae screening, and a kind of high-throughput screening method of oil-rich microalgae is provided.
The high-throughput screening method of a kind of oil-rich microalgae of the present invention is to realize by following steps:
One, get little algae sample and joined in culture medium A enrichment culture 15～30 days, get algae liquid; Two, get the algae liquid that step 1 obtains, by volume percentage composition is that 10% inoculum size is inoculated in culture medium A, cultivates 7～14 days in temperature is the illumination box of 25 ℃; Three, repeating step is 2 2～3 times, gets algae liquid A; Four, get the algae liquid A that step 3 obtains, by volume percentage composition is that 10% inoculum size is inoculated in substratum B, cultivates 7～14 days in temperature is the incubator of 25 ℃, gets algae liquid B; Five, get algae liquid B that step 4 obtains with sterilized water by 10
-1, 10
-2, 10
-3, 10
-4, 10
-5With 10
-6Multiple dilute, with 10
-5～10
-6Algae liquid after multiple dilution is evenly coated on solid medium C flat board, is cultured to algae to occur and fall in 25 ℃ of incubators; Six, in gnotobasis, picking list algae falls, and is transferred in substratum B, cultivates 7～14 days in temperature is the incubator of 25 ℃; Seven, repeating step five to step 6 3～4 times, is namely completed the separation of algae strain, obtains little algae liquid; Eight, get little algae liquid that 3～10mL step 7 obtains, carry out fragmentation with ultrasonic wave, add Nile red solution in the algae liquid after fragmentation, after mixing, measure the fluorescence intensity of algae liquid, select the highest algae strain of fluorescence intensity, namely obtain the algae strain of high fat content;
Wherein, the culture medium A described in step 1 and step 2 is comprised of 0.1～1.5 part of SODIUMNITRATE, 0.018～0.09 part of sal epsom, 0.02～0.08 part of dipotassium hydrogen phosphate, 0.009～0.054 part of calcium chloride, 0.01～0.06 part of sodium carbonate, 0.003～0.012 part of citric acid, 0.003～0.012 part of ferric ammonium citrate, 0.0005～0.01 part of ethylenediamine tetraacetic acid (EDTA) and 0.005～0.01 part of trace element by weight;
Substratum B described in step 4 and step 6 is comprised of 0.1～1.5 part of SODIUMNITRATE, 0.018～0.09 part of sal epsom, 0.02～0.08 part of dipotassium hydrogen phosphate, 0.009～0.054 part of calcium chloride, 0.01～0.06 part of sodium carbonate, 0.003～0.012 part of citric acid, 0.003～0.012 part of ferric ammonium citrate, 0.0005～0.01 part of ethylenediamine tetraacetic acid (EDTA), 0.005～0.01 part of trace element and 5～50 parts of glucose by weight;
Solid medium C described in step 5 is comprised of 0.1～1.5 part of SODIUMNITRATE, 0.018～0.09 part of sal epsom, 0.02～0.08 part of dipotassium hydrogen phosphate, 0.009～0.054 part of calcium chloride, 0.01～0.06 part of sodium carbonate, 0.003～0.012 part of citric acid, 0.003～0.012 part of ferric ammonium citrate, 0.0005～0.01 part of ethylenediamine tetraacetic acid (EDTA), 0.005～0.01 part of trace element, 5～50 parts of glucose and 5～20 parts of agar by weight;
The volume ratio of the algae liquid described in step 8 and Nile red solution is 3:1000.
The present invention comprises following beneficial effect:
The present invention adopts a kind of method of fluorescent dye, has avoided miscellaneous conventional grease detecting step, has overcome algae kind Large-scale Screening and has lacked bottleneck problem quick, high-throughout screening method.Table 1 is the comparison of organic solvent extraction method and fluorescent method, as seen from table, method of the present invention have required sample size little, measure quick, consuming time less, to advantages such as human and environment nontoxicitys.
Table 1 organic solvent extraction method and fluorescent method comparison sheet
Description of drawings
Fig. 1 is the fluorescent dye the selection result of the 89 little algae lipid contents of strain.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the high-throughput screening method of a kind of oil-rich microalgae of present embodiment is to realize by following steps:
One, get little algae sample and joined in culture medium A enrichment culture 15～30 days, get algae liquid; Two, get the algae liquid that step 1 obtains, by volume percentage composition is that 10% inoculum size is inoculated in culture medium A, cultivates 7～14 days in temperature is the illumination box of 25 ℃; Three, repeating step is 2 2～3 times, gets algae liquid A; Four, get the algae liquid A that step 3 obtains, by volume percentage composition is that 10% inoculum size is inoculated in substratum B, cultivates 7～14 days in temperature is the incubator of 25 ℃, gets algae liquid B; Five, get algae liquid B that step 4 obtains with sterilized water by 10
-1, 10
-2, 10
-3, 10
-4, 10
-5With 10
-6Multiple dilute, with 10
-5～10
-6Algae liquid after multiple dilution is evenly coated on solid medium C flat board, is cultured to algae to occur and fall in 25 ℃ of incubators; Six, in gnotobasis, picking list algae falls, and is transferred in substratum B, cultivates 7～14 days in temperature is the incubator of 25 ℃; Seven, repeating step five to step 6 3～4 times, is namely completed the separation of algae strain, obtains little algae liquid; Eight, get little algae liquid that 3～10mL step 7 obtains, carry out fragmentation with ultrasonic wave, add Nile red solution in the algae liquid after fragmentation, after mixing, measure the fluorescence intensity of algae liquid, select the highest algae strain of fluorescence intensity, namely obtain the algae strain of high fat content;
Wherein, the culture medium A described in step 1 and step 2 is comprised of 0.1～1.5 part of SODIUMNITRATE, 0.018～0.09 part of sal epsom, 0.02～0.08 part of dipotassium hydrogen phosphate, 0.009～0.054 part of calcium chloride, 0.01～0.06 part of sodium carbonate, 0.003～0.012 part of citric acid, 0.003～0.012 part of ferric ammonium citrate, 0.0005～0.01 part of ethylenediamine tetraacetic acid (EDTA) and 0.005～0.01 part of trace element by weight;
Substratum B described in step 4 and step 6 is comprised of 0.1～1.5 part of SODIUMNITRATE, 0.018～0.09 part of sal epsom, 0.02～0.08 part of dipotassium hydrogen phosphate, 0.009～0.054 part of calcium chloride, 0.01～0.06 part of sodium carbonate, 0.003～0.012 part of citric acid, 0.003～0.012 part of ferric ammonium citrate, 0.0005～0.01 part of ethylenediamine tetraacetic acid (EDTA), 0.005～0.01 part of trace element and 5～50 parts of glucose by weight;
Solid medium C described in step 5 is comprised of 0.1～1.5 part of SODIUMNITRATE, 0.018～0.09 part of sal epsom, 0.02～0.08 part of dipotassium hydrogen phosphate, 0.009～0.054 part of calcium chloride, 0.01～0.06 part of sodium carbonate, 0.003～0.012 part of citric acid, 0.003～0.012 part of ferric ammonium citrate, 0.0005～0.01 part of ethylenediamine tetraacetic acid (EDTA), 0.005～0.01 part of trace element, 5～50 parts of glucose and 5～20 parts of agar by weight;
The volume ratio of the algae liquid described in step 8 and Nile red solution is 3:1000.
Present embodiment adopts a kind of method of fluorescent dye, has avoided miscellaneous conventional grease detecting step, has overcome algae kind Large-scale Screening and has lacked bottleneck problem quick, high-throughout screening method.Table 1 is the comparison of organic solvent extraction method and fluorescent method, as seen from table, the method for present embodiment have required sample size little, measure quick, consuming time less, to advantages such as human and environment nontoxicitys.
Table 1 organic solvent extraction method and fluorescent method comparison sheet
Embodiment two: what present embodiment was different from embodiment one is: the little algae sample described in step 1 is that one or more in chlorella, grid algae, diatom, Crypthecodinium cohnii, flat algae, Dunaliella salina, spirulina, chrysophyceae are mixed by any ratio.Other is identical with embodiment one.
Embodiment three: what present embodiment was different from embodiment one or two is: the culture medium A described in step 1 and step 2 is comprised of 1 part of SODIUMNITRATE, 0.075 part of sal epsom, 0.04 part of dipotassium hydrogen phosphate, 0.036 part of calcium chloride, 0.02 part of sodium carbonate, 0.006 part of citric acid, 0.006 part of ferric ammonium citrate, 0.001 part of ethylenediamine tetraacetic acid (EDTA) and 0.00541 part of trace element by weight.Other is identical with embodiment one or two.
Embodiment four: what present embodiment was different from one of embodiment one to three is: the substratum B described in step 4 and step 6 is comprised of 0.5 part of SODIUMNITRATE, 0.075 part of sal epsom, 0.04 part of dipotassium hydrogen phosphate, 0.036 part of calcium chloride, 0.02 part of sodium carbonate, 0.006 part of citric acid, 0.006 part of ferric ammonium citrate, 0.001 part of ethylenediamine tetraacetic acid (EDTA), 0.00541 part of trace element and 10 parts of glucose by weight.Other is identical with one of embodiment one to three.
Embodiment five: what present embodiment was different from one of embodiment one to four is: the solid medium C described in step 5 is comprised of 0.5 part of SODIUMNITRATE, 0.075 part of sal epsom, 0.04 part of dipotassium hydrogen phosphate, 0.036 part of calcium chloride, 0.02 part of sodium carbonate, 0.006 part of citric acid, 0.006 part of ferric ammonium citrate, 0.001 part of ethylenediamine tetraacetic acid (EDTA), 0.00541 part of trace element, 10 parts of glucose and 15 parts of agar by weight.Other is identical with one of embodiment one to four.
Embodiment six: what present embodiment was different from one of embodiment one to five is: described trace element is the boric acid of 0.00286 weight part, the Manganous chloride tetrahydrate of 0.00181 weight part, the Sodium orthomolybdate of 0.00039 weight part, the zinc sulfate of 0.000222 weight part, the copper sulfate of 0.000079 weight part and the Jing Ti/Bao Pian COBALT NITRATE CRYSTALS/FLAKES of 0.000049 weight part.Other is identical with one of embodiment one to five.
Embodiment seven: what present embodiment was different from one of embodiment one to six is: the little algae sample source described in step 1 is in natural water (fresh water, seawater and salt water etc.) or soil.Other is identical with one of embodiment one to six.
By following verification experimental verification beneficial effect of the present invention:
The high-throughput screening method of this test oil-rich microalgae is realized according to the following steps: one, get little algae sample, joined in culture medium A enrichment culture 15～30 days; Two, get the algae liquid that step 1 obtains, by volume percentage composition is that 10% inoculum size is inoculated in culture medium A, cultivates 7～14 days in temperature is the illumination box of 25 ℃; Three, repeating step is 2 2～3 times, gets algae liquid A; Four, get the algae liquid A that step 3 obtains, by volume percentage composition is that 10% inoculum size is inoculated in substratum B, cultivates 7～14 days in temperature is the incubator of 25 ℃, gets algae liquid B; Five, get algae liquid B that step 4 obtains with sterilized water by 10
-1, 10
-2, 10
-3, 10
-4, 10
-5With 10
-6Multiple dilute, with 10
-5～10
-6Algae liquid after multiple dilution is evenly coated on solid medium C flat board, is cultured to algae to occur and fall in 25 ℃ of incubators; Six, in gnotobasis, picking growth comparatively fast, larger single algae falls, and is transferred in substratum B, cultivated 7～14 days in temperature is the incubator of 25 ℃; Seven, repeating step five to step 6 3～4 times, is namely completed the separation of algae strain, obtains little algae liquid; Eight, get little algae liquid that step 7 obtains, carry out fragmentation with ultrasonic wave, add Nile red solution in the algae liquid after fragmentation, after mixing dyeing, measure the fluorescence intensity of algae liquid, select the highest algae strain of fluorescence intensity, namely obtain the algae strain of 89 plant height fat contents;
Culture medium A described in this testing sequence one and step 2 mark by weight is comprised of 1 part of SODIUMNITRATE, 0.075 part of sal epsom, 0.04 part of dipotassium hydrogen phosphate, 0.036 part of calcium chloride, 0.02 part of sodium carbonate, 0.006 part of citric acid, 0.006 part of ferric ammonium citrate, 0.001 part of ethylenediamine tetraacetic acid (EDTA) and 0.00541 part of trace element;
Substratum B described in step 4 and step 6 mark by weight is comprised of 0.5 part of SODIUMNITRATE, 0.075 part of sal epsom, 0.04 part of dipotassium hydrogen phosphate, 0.036 part of calcium chloride, 0.02 part of sodium carbonate, 0.006 part of citric acid, 0.006 part of ferric ammonium citrate, 0.001 part of ethylenediamine tetraacetic acid (EDTA), 0.00541 part of trace element and 10 parts of glucose;
Solid medium C described in step 5 mark by weight is comprised of 0.5 part of SODIUMNITRATE, 0.075 part of sal epsom, 0.04 part of dipotassium hydrogen phosphate, 0.036 part of calcium chloride, 0.02 part of sodium carbonate, 0.006 part of citric acid, 0.006 part of ferric ammonium citrate, 0.001 part of ethylenediamine tetraacetic acid (EDTA), 0.00541 part of trace element, 10 parts of glucose and 15 parts of agar;
The volume ratio of the algae liquid described in step 8 and Nile red solution is 3:1000.
The fluorescent dye the selection result of little algae lipid content of the algae strain of the high fat content that this test obtains as shown in Figure 1, fluorescence intensity is 2 strain grid algaes higher than 600 algae strain, fluorescence intensity is that 400～600 algae strain has 1 chlorella, 3 strain grid algaes show that these algae strains have higher fat content and good exploitation prospect.
Claims (6)
1. the high-throughput screening method of an oil-rich microalgae, the high-throughput screening method that it is characterized in that oil-rich microalgae is to realize by following steps: one, get little algae sample and joined in culture medium A enrichment culture 15～30 days, get algae liquid; Two, get the algae liquid that step 1 obtains, by volume percentage composition is that 10% inoculum size is inoculated in culture medium A, cultivates 7～14 days in temperature is the illumination box of 25 ℃; Three, repeating step is 2 2～3 times, gets algae liquid A; Four, get the algae liquid A that step 3 obtains, by volume percentage composition is that 10% inoculum size is inoculated in substratum B, cultivates 7～14 days in temperature is the incubator of 25 ℃, gets algae liquid B; Five, get algae liquid B that step 4 obtains with sterilized water by 10
-1, 10
-2, 10
-3, 10
-4, 10
-5With 10
-6Multiple dilute, with 10
-5～10
-6Algae liquid after multiple dilution is evenly coated on solid medium C flat board, is cultured to algae to occur and fall in 25 ℃ of incubators; Six, in gnotobasis, picking list algae falls, and is transferred in substratum B, cultivates 7～14 days in temperature is the incubator of 25 ℃; Seven, repeating step five to step 6 3～4 times, is namely completed the separation of algae strain, obtains little algae liquid; Eight, get little algae liquid that 3～10mL step 7 obtains, carry out fragmentation with ultrasonic wave, add Nile red solution in the algae liquid after fragmentation, after mixing, measure the fluorescence intensity of algae liquid, select the highest algae strain of fluorescence intensity, namely obtain the algae strain of high fat content;
Wherein, the culture medium A described in step 1 and step 2 is comprised of 0.1～1.5 part of SODIUMNITRATE, 0.018～0.09 part of sal epsom, 0.02～0.08 part of dipotassium hydrogen phosphate, 0.009～0.054 part of calcium chloride, 0.01～0.06 part of sodium carbonate, 0.003～0.012 part of citric acid, 0.003～0.012 part of ferric ammonium citrate, 0.0005～0.01 part of ethylenediamine tetraacetic acid (EDTA) and 0.005～0.01 part of trace element by weight;
Substratum B described in step 4 and step 6 is comprised of 0.1～1.5 part of SODIUMNITRATE, 0.018～0.09 part of sal epsom, 0.02～0.08 part of dipotassium hydrogen phosphate, 0.009～0.054 part of calcium chloride, 0.01～0.06 part of sodium carbonate, 0.003～0.012 part of citric acid, 0.003～0.012 part of ferric ammonium citrate, 0.0005～0.01 part of ethylenediamine tetraacetic acid (EDTA), 0.005～0.01 part of trace element and 5～50 parts of glucose by weight;
Solid medium C described in step 5 is comprised of 0.1～1.5 part of SODIUMNITRATE, 0.018～0.09 part of sal epsom, 0.02～0.08 part of dipotassium hydrogen phosphate, 0.009～0.054 part of calcium chloride, 0.01～0.06 part of sodium carbonate, 0.003～0.012 part of citric acid, 0.003～0.012 part of ferric ammonium citrate, 0.0005～0.01 part of ethylenediamine tetraacetic acid (EDTA), 0.005～0.01 part of trace element, 5～50 parts of glucose and 5～20 parts of agar by weight;
The volume ratio of the algae liquid described in step 8 and Nile red solution is 3:1000.
2. the high-throughput screening method of a kind of oil-rich microalgae according to claim 1 is characterized in that the little algae sample described in step 1 is that one or more in chlorella, grid algae, diatom, Crypthecodinium cohnii, flat algae, Dunaliella salina, spirulina, chrysophyceae are mixed by any ratio.
3. the high-throughput screening method of a kind of oil-rich microalgae according to claim 1, is characterized in that the culture medium A described in step 1 and step 2 is comprised of 1 part of SODIUMNITRATE, 0.075 part of sal epsom, 0.04 part of dipotassium hydrogen phosphate, 0.036 part of calcium chloride, 0.02 part of sodium carbonate, 0.006 part of citric acid, 0.006 part of ferric ammonium citrate, 0.001 part of ethylenediamine tetraacetic acid (EDTA) and 0.00541 part of trace element by weight.
4. the high-throughput screening method of a kind of oil-rich microalgae according to claim 1, is characterized in that the substratum B described in step 4 and step 6 is comprised of 0.5 part of SODIUMNITRATE, 0.075 part of sal epsom, 0.04 part of dipotassium hydrogen phosphate, 0.036 part of calcium chloride, 0.02 part of sodium carbonate, 0.006 part of citric acid, 0.006 part of ferric ammonium citrate, 0.001 part of ethylenediamine tetraacetic acid (EDTA), 0.00541 part of trace element and 10 parts of glucose by weight.
5. the high-throughput screening method of a kind of oil-rich microalgae according to claim 1, is characterized in that the solid medium C described in step 5 is comprised of 0.5 part of SODIUMNITRATE, 0.075 part of sal epsom, 0.04 part of dipotassium hydrogen phosphate, 0.036 part of calcium chloride, 0.02 part of sodium carbonate, 0.006 part of citric acid, 0.006 part of ferric ammonium citrate, 0.001 part of ethylenediamine tetraacetic acid (EDTA), 0.00541 part of trace element, 10 parts of glucose and 15 parts of agar by weight.
6. according to claim 1, the high-throughput screening method of 3,4 or 5 described a kind of oil-rich microalgae, it is characterized in that described trace element is the boric acid of 0.00286 weight part, the Manganous chloride tetrahydrate of 0.00181 weight part, the Sodium orthomolybdate of 0.00039 weight part, the zinc sulfate of 0.000222 weight part, the copper sulfate of 0.000079 weight part and the Jing Ti/Bao Pian COBALT NITRATE CRYSTALS/FLAKES of 0.000049 weight part.
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| CN104568881B (en) * | 2014-12-30 | 2017-02-22 | 浙江大学 | Method for screening microalgae unicells which grow fast and are high in grease content through fluorescence microscope |
| JP2016124998A (en) * | 2015-01-06 | 2016-07-11 | 国立大学法人東京工業大学 | Algae fat extraction method, and ultrasonic processor |
| CN110628646A (en) * | 2019-10-11 | 2019-12-31 | 哈尔滨工业大学 | Method for improving yield of oil and fat of growing microalgae in sewage and in-situ flocculation recovery efficiency |
| CN114672421A (en) * | 2022-03-02 | 2022-06-28 | 陕西海斯夫生物工程有限公司 | Method for cultivating and screening microalgae with high tocopherol content |
| CN115011484A (en) * | 2022-07-25 | 2022-09-06 | 哈尔滨工业大学 | Method for removing heavy metal hexavalent chromium and promoting microalgae grease production under low-temperature acidic condition |
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