CN102174409A - Method for quickly accumulating grease through mixotrophic sterile culture of microalgae - Google Patents
Method for quickly accumulating grease through mixotrophic sterile culture of microalgae Download PDFInfo
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- CN102174409A CN102174409A CN2011100431653A CN201110043165A CN102174409A CN 102174409 A CN102174409 A CN 102174409A CN 2011100431653 A CN2011100431653 A CN 2011100431653A CN 201110043165 A CN201110043165 A CN 201110043165A CN 102174409 A CN102174409 A CN 102174409A
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Abstract
The invention relates to an algae organism culture technology, in particular to a method for quickly growing chlorella and accumulating a large amount of biological grease through mixotrophic culture. In the method, two-stage culture is adopted, namely at the first stage, high biomass is obtained through the autotrophic aerated culture of the chlorella, and at the second stage, an aerated heterotrophic culture mode of adding organic matters is adopted, the single nutrition mode of the chlorella is changed, and the grease is quickly accumulated on the basis of a large amount of biomass; meanwhile, a novel air sterilization method is adopted to effectively solve the problem of bacterial contamination of culture solution, and culture cost is reduced because a filter membrane is not needed; and algae powder grease is extracted by an alkali-catalyzed anhydrous diethyl ether-petroleum ether extraction method. The method has the advantages that: the whole process is easy to operate, experiment cost and production cost are low, the culture period is short, the grease accumulation concentration of microalgae can be quickly improved, and the extracted grease has high content and purity and is not required to be decolorized; and the method is suitable for preparing microalgae grease in a laboratory or in the industry, and has a good application prospect.
Description
Technical field
The present invention relates to the algae bio culture technique, relate in particular to the method that a kind of mixotrophism is cultivated rapid growth of chlorella and a large amount of accumulation bio-oils.
Background technology
In recent years, the global energy demand constantly enlarges, and the renewable and clean energy resource of seeking to substitute oil is to be badly in need of the major issue that solves at present.The characteristics that algae has the photosynthetic efficiency height, adaptive capacity to environment is strong, growth cycle is short, biological yield is high, fat content is abundant, so algae is the good material of preparation bio oil fuel.But the subject matter of producing little algae biofuel at present is that the cultivation cost is higher.Have only the seed selection that at first solves growth fast and the little algae kind of high fat content, optimize culture condition and technology, the improvement mass production conditions, the high efficiency separation that improves the target product lipid could effectively reduce production costs.
Chlorella vulgaris (Chlorella vulgaris) is a kind of green alga that utilizes the organic carbon source heterotrophism to cultivate, and the speed of growth is following faster than illumination condition, is similar to the metabolism growth of bacterium, transforms the heterophytic chlorella that can obtain high fat content by heterotrophism.The large scale culturing chlorella is mainly adopted the outdoor culture systems of open pond formula and various semi closed and airtight culture systems up to now, owing to still adopt illumination autotrophy mode, the cost height, production efficiency is on the low side.Carry out heterotrophism with organism as the sole carbon source of chlorella and cultivate, have the illumination of need not, cell proliferation is fast, the concentration height, and production system is easy to realize automatic control, can utilize advantages such as existing fermentation equipment.But the very easily microbiological contamination of the nutrient solution of frustule heterotrophic growth, the ventilation of fermentation equipment at present can't thoroughly be removed airborne other assorted bacterium through the degerming of filter membrane simple filtration device, and filter membrane needs periodic replacement, and cost is higher.
Summary of the invention
The invention provides method and the grease extracting method of a kind of mixotrophism sterile culture high-density chlorella, its preparation process is:
A. adopt chlorella autotrophy aerated culture to obtain: in 50mL improvement SE liquid nutrient medium, to cultivate chlorella cells than high-biomass, progressively move to 250mL again, 500mL, aseptic ventilation enlarged culturing in the triangular flask of 3000mL, 25～27 ℃ of temperature, incubation time 4-5 days.
Wherein, described improvement SE liquid culture based component is: contain NaNO in every 1000mL substratum
30.25g, CaCl
2.2H
2O 0.025g, MgSO
47H
2O 0.075g, K
2HPO
40.075g, KH
2PO
40.175g, NaCl 0.025g, FeCl
36H
2O 0.005g, Fe-EDTA 2mL, A5 trace element mixed solution 1mL.
Wherein, described aseptic ventilation enlarged culturing method is: with the copper-bath degerming of magnetic force air compressor pump feeding 1%, remove airborne micro-sulfuric acid copper component through sterilized water again, last sterile air feeds the 3000mL triangular flask provides cell required CO
2And suspend and stirring.
Wherein, described aerated culture is: magnetic force air compressor pump ventilation flow rate 10-15L/min, 22～27 ℃ of culture temperature, illumination 120 μ molm
-2S
-1
B. adopt the organic ventilation heterotrophism of interpolation to cultivate chlorella and obtain oil and fat accumulation fast: adding organism is the aseptic glucose solution of concentration 40g/L, and carries out the shading aerated culture, 22～27 ℃ of culture temperature.
Wherein, described aseptic glucose solution is sterilized through 80 ℃ of vapor pressures, sterilization time 20 minutes.
Wherein, the ventilation flow rate of described shading aerated culture is 20-25L/min.
C. the chlorella dry algae powder adopts the anhydrous diethyl ether-sherwood oil method of base catalysis to extract grease: get an amount of bead algae powder in the 5mL centrifuge tube, fully grind the back and add 3mL anhydrous diethyl ether-sherwood oil mixed solution, suitably vibration lixiviate under 40 ℃ of conditions, the static 30min of potassium hydroxide solution of adding 10%, the centrifugal 10min of mixing solutions 4000rpm is collected supernatant liquor in the centrifuge tube of constant weight, 60 ℃ of water-baths boil off unnecessary solvent, and weighing is also calculated fat content.
Wherein, the volume ratio of described anhydrous diethyl ether-sherwood oil mixed solution is 1: 2.
The positively effect that method provided by the invention is compared with art methods is as follows:
(1). the aseptic aerated culture chlorella of described mixotrophism method, the germ of bringing in the time of effectively removing ventilation and other microorganisms.The effect that ventilation not only provides cell suspension to stir, and effective carbon source is provided in culturing process, making chlorella a large amount of fast breedings in inorganic medium, this moment, chlorella formed growth vigor rapidly, also was unfavorable for varied bacteria growing.
(2). described subordinate phase is added a small amount of glucose and is carried out chlorella heterotrophy and secretly cultivate, and can improve the microalgae grease accumulation rapidly, and fat content increases greatly in the frustule.By mixotrophism sterile culture chlorella, every liter of algae liquid can get chlorella powder and be about 3.77g.Cultivate the chlorella that obtains under the autotrophy condition fully, every liter of algae liquid gained algae powder only is 0.123g.
(3). the aseptic aerated culture chlorella of described mixotrophism method, dense thick, its colour changed into yellow of frustule in the nutrient solution, frustule chlorophyll disappear, and cell volume increases, and fat content increases greatly in the frustule, is 4.8 times of fat content under the autotrophy condition.It is dark green to cultivate the chlorella color that obtains fully under the autotrophy condition, and cell quantity is more relatively but fat content is low.
(4). because little algae contains higher chlorophyll, the color with oil color of extracting darker, the processing of need decolouring had both increased cost, lost grease again.The anhydrous diethyl ether of described base catalysis-sherwood oil method is extracted chlorella algae powder oil and fat method, the fat content height of extraction, and color and luster is good, the processing of can decolouring.
Description of drawings
Fig. 1 is the method flow diagram that the little algae of mixotrophism sterile culture provided by the invention accumulates an embodiment of oil and fat method fast.
Fig. 2 is the aseptic ventilation mixed culture chlorella device synoptic diagram of the little algae of a mixotrophism sterile culture provided by the invention embodiment accumulating oil and fat method fast.
Fig. 3 is the 3L chlorella cultivation figure of the little algae of a mixotrophism sterile culture provided by the invention embodiment accumulating oil and fat method fast.
Fig. 4 is autotrophy and dry cell weight under the mixotrophism condition and the fat content comparison diagram of the little algae of a mixotrophism sterile culture provided by the invention embodiment accumulating oil and fat method fast.
Fig. 5 is the grease obtained spirogram that contains of Different Extraction Method of the little algae of a mixotrophism sterile culture provided by the invention embodiment accumulating oil and fat method fast.
Embodiment
The little algae of embodiment 1 mixotrophism sterile culture provided by the invention accumulates the method flow diagram (with reference to figure 1) of oil and fat method fast
1. first section ventilation of little algae autotrophy cultural method is: described little algae can separate from environment, also can buy to domestic and international algae kind storehouse.Present embodiment is example with the chlorella, cultivates chlorella cells, 25～27 ℃ of temperature, illumination 120 μ molm in improvement SE liquid nutrient medium
-2S
-1, incubation time 4-5 days.Described improvement SE liquid nutrient medium composition characteristics is: contain NaNO in described every 1000mL substratum
30.25g, CaCl
2.2H
2O 0.025g, MgSO
47H
2O 0.075g, K
2HPO
40.075g, KH
2PO
40.175g, NaCl 0.025g, FeCl
36H
2O 0.005g, Fe-EDTA 2mL, A5 trace element mixed solution 1mL.
2. the aseptic ventilation enlarged culturing of described chlorella method is: with reference to shown in Figure 2, copper-bath degerming with magnetic force air compressor pump feeding 1%, remove airborne micro-sulfuric acid copper component through sterilized water again, last sterile air feeds the 3000mL triangular flask provides cell required CO
2And suspend and stirring described magnetic force air compressor pump ventilation flow rate 10-15L/min, 22～27 ℃ of culture temperature, illumination 120 μ molm
-2S
-1
3. described chlorella is added organic ventilation heterotrophism training mode for second section: adding organism is aseptic 40g/L glucose solution, and carries out the shading aerated culture, 22～27 ℃ of culture temperature.The described glucose of adding is sterilized through 80 ℃ of vapor pressures, sterilization time 20 minutes, described magnetic force air compressor pump ventilation flow rate 20-25L/min.
4. the preparation of described bead algae powder: ventilation is dark cultivated after 5 days, the centrifugal collection of 5500-7500rpm algae mud, and-50 ℃ of lyophilize algae powder to constant weights make dry algae powder.
5. the organic solvent mixed solution method of described base catalysis extraction chlorella algae powder oil and fat method is: get an amount of bead algae powder in centrifuge tube, fully grind the back and add an amount of anhydrous diethyl ether-sherwood oil mixed solution, suitably vibration lixiviate under 40 ℃ of conditions, the potassium hydroxide solution of adding 10%, with the centrifugal collection supernatant liquor of mixing solutions, 60 ℃ of water-baths boil off unnecessary solvent, and weighing is also calculated fat content.The volume ratio of described anhydrous diethyl ether-sherwood oil mixed solution is 1: 2.The grease percentage composition calculates with following formula: fat content (%)=(total lipid content/frustule dry weight) * 100%.
The greasy concrete steps of accumulation are as follows fast for embodiment 2. present embodiment mixed once nutrition sterile culture chlorellas:
1. first section of chlorella ventilation autotrophy cultural method step: described chlorella comes from environment and through the plate streaking separation and purification.At 50m L improvement SE liquid nutrient medium enrichment culture frustule, cultivate after 4-5 days earlier, the frustule of enrichment is lined on the flat board of SE solid medium, wherein the agar amount 2.0%, 25～27 ℃ of culture temperature, illumination 120 μ molm
-2S
-1Grow green single algae on 2 all left and right sides solid plates and fall, picking list algae drop on SE solid medium flat board rule repeatedly separate 2 times after, obtain pure strain.Described improvement SE liquid nutrient medium composition characteristics is: contain NaNO in described every 1000mL substratum
30.25g, CaCl
2.2H
2O 0.025g, MgSO
47H
2O 0.075g, K
2HPO
40.075g, KH
2PO
40.175g, NaCl 0.025g, FeCl
36H
2O 0.005g, Fe-EDTA 2mL, A
5Trace element mixed solution 1mL.
2. the aseptic ventilation enlarged culturing of described chlorella step is: the chlorella cells that culture purified is good in 50mL SE liquid nutrient medium is to logarithmic phase, and cell density is 1.5-2.5 * 10
8Individual/mL.Progressively be extended to aseptic 250mL again according to 10% inoculum size, illumination shaking culture in the 500mL triangular flask, 100 rev/mins of shaking speed, 22～27 ℃ of culture temperature, illumination 120 μ molm
-2S
-1Chlorella is adopted aseptic aerated culture in the 3000mL triangular flask, its step is for using magnetic force air compressor pump delivery air (in figure 2
Shown in), air is fed 1% copper-bath degerming (in figure 2
Shown in), remove airborne micro-sulfuric acid copper component (in figure 2 through sterilized water again
Shown in), last sterile air feeds the 3000mL triangular flask provides cell required CO
2Reach suspension and stir (in the reference drawing
Shown in), described magnetic force air compressor pump ventilation flow rate 15L/min, 22～27 ℃ of culture temperature, illumination 120 μ molm
-2S
-1
3. described chlorella is added organic ventilation heterotrophism culturing step for second section: the autotrophy aerated culture is after 4 days, adding concentration in the 3000mL triangular flask is the glucose solution of 40g/L, the described glucose of adding is sterilized sterilization time 20 minutes through 80 ℃ of vapor pressures.Carry out the shading aerated culture, described magnetic force air compressor pump ventilation flow rate 25L/min, 27 ℃ of culture temperature after adding glucose solution.Behind second section organic ventilation heterotrophism of interpolation of described chlorella, dense thick, its colour changed into yellow of frustule, frustule chlorophyll disappears, and cell volume increases, and fat content increases greatly (in figure 3 in the frustule
Shown in).And the chlorella color that cultivation obtains under the complete autotrophy condition is dark green, and cell quantity is more relatively but fat content is low (in figure 3
Shown in).
4. the preparation of described bead algae powder: after chlorella is secretly cultivated 5 days, 7000 rev/mins of centrifugal 15 minutes collection algae mud of rotating speed are in culture dish,-50 ℃ of lyophilize algae powder make dry algae powder to constant weight, and the algae dried bean noodles is 3.77g/L heavily, are 30.65 times (with reference to figure 4) under the autotrophy condition.
5. the organic solvent mixed solution method of described base catalysis extraction chlorella grease step is: get 0.3g algae powder in the 5mL centrifuge tube, fully grind, the adding volume ratio is 3mL anhydrous diethyl ether-sherwood oil mixed solution of 1: 2, extract lixiviate under 40 ℃ of water bath condition, during to add 0.5mL concentration be 10% the potassium hydroxide solution mixing that suitably vibrates.After treating that 3h extracts end, with mixing solutions at 4000 rev/mins of centrifugal 10min, collect supernatant liquor in the centrifuge tube of constant weight, in 60 ℃ of water-baths, boil off unnecessary solvent rapidly, calculate fat content according to following formula: fat content (%)=(total lipid content/frustule dry weight) * 100%.Present embodiment method gained chlorella color with oil color is limpid faint yellow (with reference to the 2nd sample pipe among the figure 5), and fat content is 51.08%, is 4.84 times of fat content under the autotrophy condition.
1. described potassium hydroxide-methyl alcohol method is extracted the grease step: get 0.3g bead algae powder and add a little distilled water ,-20 ℃ are descended in freezing and 40 ℃ of water to dissolve 5min, twice repeatedly.Algae powder with the freeze-thaw method processing, 1M potassium hydroxide-the methyl alcohol mixed liquor that adds 4mL, 75 ℃ of water-bath 10min add the 2mL normal hexane, mixing, the centrifugal 10min of 4000r/min, get supernatant liquor, add normal hexane washing residue, merge supernatant liquor, 60 ℃ of solvent flashings are weighed, and calculate fat content according to following formula: total fat percentage composition (%)=(total lipid content/frustule dry weight) * 100%.The described potassium hydroxide of present embodiment-methyl alcohol method is extracted the chlorella color with oil color and is limpid light green (with reference to the 1st sample pipe among the figure 5), and fat content is 28.57%.
2. described chloroform-methanol method is extracted the grease step: get 0.3g bead algae powder and add a little distilled water ,-20 ℃ are descended in freezing and 40 ℃ of water to dissolve 5min, twice repeatedly.Algae powder with the freeze-thaw method processing, add 1: 2 3mL chloroform-methanol mixed solution of volume ratio, fully concussion 2min adds 1mL chloroform concussion mixing again, adds 1.5mL distilled water, the centrifugal 10min of 4000r/min, get the sodium chloride solution that supernatant liquor adds equal-volume 0.1%, mixing, the centrifugal 10min of 4000r/min, get 60 ℃ of volatilizations of supernatant liquor and remove chloroform and weigh, calculate fat content.The described chloroform-methanol method of present embodiment is extracted the chlorella color with oil color and is blackish green (with reference to the 3rd sample pipe among the figure 5), and fat content is 46.98%.
3. described normal hexane-Ethanol Method is extracted the grease step: get 0.3g bead algae powder and add a little distilled water ,-20 ℃ are descended in freezing and 40 ℃ of water to dissolve 5min, twice repeatedly.Algae powder with freeze-thaw method is handled adds 3mL normal hexane-alcohol mixeding liquid of 1: 2 of volume ratio, and following extraction step is as each step as described in 2 in the present embodiment.The described normal hexane of present embodiment-Ethanol Method is extracted the chlorella color with oil color and is blue-greenish colour (with reference to the 4th sample pipe among the figure 5), and fat content is 19.09%.
Claims (9)
1. the method for a mixotrophism sterile culture high-density chlorella, this method adopt two-part mixotrophism to cultivate, and promptly first section is adopted chlorella ventilation autotrophy cultural method, and second section is adopted and add organic ventilation heterotrophism training mode.
2. a chlorella dry algae powder extracts greasy method, and this method adopts the anhydrous diethyl ether-sherwood oil method of base catalysis to extract algae powder grease.
3. according to the described chlorella ventilation of claim 1 autotrophy cultural method, it is characterized in that: in 50mL improvement SE liquid nutrient medium, cultivate chlorella cells, progressively move to 250mL again, 500mL, aseptic ventilation enlarged culturing in the triangular flask of 3000mL, 25～27 ℃ of temperature, incubation time 4-5 days.
4. according to the described improvement of claim 3 SE liquid nutrient medium, its composition characteristics is: contain NaNO in described every 1000mL substratum
30.25g, CaCl
2.2H
2O 0.025g, MgSO
47H
2O 0.075g, K
2HPO
40.075g, KH
2PO
40.175g, NaCl 0.025g, FeCl
36H
2O 0.005g, Fe-EDTA 2mL, A
5Trace element mixed solution 1mL.
5. according to the described aseptic ventilation enlarged culturing method of claim 3, it is characterized in that: with the copper-bath degerming of magnetic force air compressor pump feeding 1%, remove airborne micro-sulfuric acid copper component through sterilized water again, last sterile air feeds the 3000mL triangular flask provides cell required CO
2And suspend and stirring described magnetic force air compressor pump ventilation flow rate 10-15L/min, 22～27 ℃ of culture temperature, illumination 120 μ molm
-2S
-1
6. add organic ventilation heterotrophism training mode according to the described chlorella of claim 1, it is characterized in that: it is aseptic 40g/L glucose solution that described chlorella heterotrophy is cultivated the interpolation organism, and carries out the shading aerated culture, 22～27 ℃ of culture temperature.
7. according to the described chlorella ventilation of claim 6 heterotrophism training mode, it is characterized in that: the described glucose of adding is sterilized through 80 ℃ of vapor pressures, sterilization time 20 minutes, described magnetic force air compressor pump ventilation flow rate 20-25L/min.
8. extract bead algae powder grease according to the anhydrous diethyl ether-sherwood oil method of the described base catalysis of claim 2, it is characterized in that: get 0.3g bead algae powder in the 5mL centrifuge tube, fully grind the back and add 3mL anhydrous diethyl ether-sherwood oil mixed solution, suitably vibration lixiviate under 40 ℃ of conditions, the static 30min of potassium hydroxide solution of adding 10%, the centrifugal 10min of mixing solutions 4000rpm is collected supernatant liquor in the centrifuge tube of constant weight, and 60 ℃ of water-baths boil off unnecessary solvent, and weighing is also calculated fat content.
9. the anhydrous diethyl ether of described base catalysis-Petroleum ether extraction chlorella algae powder oil and fat method according to Claim 8, it is characterized in that: the volume ratio of described anhydrous diethyl ether-sherwood oil mixed solution is 1: 2.
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Cited By (9)
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|---|---|---|---|---|
| CN102505026A (en) * | 2011-11-08 | 2012-06-20 | 浙江大学 | Method for extracting microalgae oil membrane substrate in situ |
| CN103060308A (en) * | 2013-01-14 | 2013-04-24 | 安徽省农业科学院水产研究所 | Mutagenesis and screening method of high-yield chlorella new germplasm by means of chromosome doubling |
| CN107699493A (en) * | 2017-11-17 | 2018-02-16 | 新奥科技发展有限公司 | A kind of both culturing microalgae method |
| CN107937276A (en) * | 2017-12-12 | 2018-04-20 | 中国石油大学(华东) | A kind of method that carbon dioxide and acetic acid mixing regulation and control promote the growth of chlorella carbon sequestration |
| TWI678416B (en) * | 2017-03-07 | 2019-12-01 | 國立臺灣海洋大學 | Closed-type liquid culture system and method thereof for microorganisms |
| WO2020038296A1 (en) * | 2018-08-20 | 2020-02-27 | 梁云 | Microbial oil and extraction method therefor |
| CN111266000A (en) * | 2020-01-20 | 2020-06-12 | 北京航空航天大学 | Treatment of CO-containing microalgae2Method for producing flue gas |
| CN114015493A (en) * | 2021-09-26 | 2022-02-08 | 上海师范大学 | Method for extracting grease from algae |
| CN115029248A (en) * | 2022-06-21 | 2022-09-09 | 昆明理工大学 | Method for improving microalgae lipid yield by utilizing recycled wastewater |
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| CN102505026A (en) * | 2011-11-08 | 2012-06-20 | 浙江大学 | Method for extracting microalgae oil membrane substrate in situ |
| CN102505026B (en) * | 2011-11-08 | 2014-04-16 | 浙江大学 | Method for extracting microalgae oil membrane substrate in situ |
| CN103060308A (en) * | 2013-01-14 | 2013-04-24 | 安徽省农业科学院水产研究所 | Mutagenesis and screening method of high-yield chlorella new germplasm by means of chromosome doubling |
| TWI678416B (en) * | 2017-03-07 | 2019-12-01 | 國立臺灣海洋大學 | Closed-type liquid culture system and method thereof for microorganisms |
| CN107699493A (en) * | 2017-11-17 | 2018-02-16 | 新奥科技发展有限公司 | A kind of both culturing microalgae method |
| CN107937276A (en) * | 2017-12-12 | 2018-04-20 | 中国石油大学(华东) | A kind of method that carbon dioxide and acetic acid mixing regulation and control promote the growth of chlorella carbon sequestration |
| CN107937276B (en) * | 2017-12-12 | 2021-05-28 | 中国石油大学(华东) | Method for promoting carbon sequestration growth of chlorella by mixing and regulating carbon dioxide and acetic acid |
| WO2020038296A1 (en) * | 2018-08-20 | 2020-02-27 | 梁云 | Microbial oil and extraction method therefor |
| CN111266000A (en) * | 2020-01-20 | 2020-06-12 | 北京航空航天大学 | Treatment of CO-containing microalgae2Method for producing flue gas |
| CN114015493A (en) * | 2021-09-26 | 2022-02-08 | 上海师范大学 | Method for extracting grease from algae |
| CN114015493B (en) * | 2021-09-26 | 2024-02-20 | 上海师范大学 | Method for extracting grease from algae |
| CN115029248A (en) * | 2022-06-21 | 2022-09-09 | 昆明理工大学 | Method for improving microalgae lipid yield by utilizing recycled wastewater |
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Application publication date: 20110907 |