CN101265449A - Fast high-density culture method for algae cell - Google Patents
Fast high-density culture method for algae cell Download PDFInfo
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- CN101265449A CN101265449A CNA200710010593XA CN200710010593A CN101265449A CN 101265449 A CN101265449 A CN 101265449A CN A200710010593X A CNA200710010593X A CN A200710010593XA CN 200710010593 A CN200710010593 A CN 200710010593A CN 101265449 A CN101265449 A CN 101265449A
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Abstract
The invention relates to high-density cultivation of algae, particularly a method for quickly cultivating algae cells with high density. The method comprises the steps of adding a high density culture medium to a photo-bioreactor, inoculating the algae cells in the photo-bioreactor, and lighting in the right and the left directions. The invention realizes the quick proliferation of the algae in the short run and shortens cultivation time through rational disposition of nitrogen phosphorus nutrient concentration, carbon source, light intensity, effective acceptance of transformed luminous light, and agitation degree of algae liquid. The photo-bioreactor can make the most of transformed luminous energy, effectively ensure the algae liquid inside a system is distributed uniformly, avoid the dead angle problem in the photo-reactor, ensure the full gas exchange, reduce the growth of adherent cells, and greatly improve the cultivation rate of micro algae. The method in the invention is suitable for mass production of the micro algae with high density, and provides economic benefit for comprehensive exploitation and utilization of the micro algae.
Description
Technical field
The present invention relates to the algae high-density culture, is a kind of fast high-density culture method for algae cell specifically, can be used for cultivating algae, produces related products or aquaculture bait etc. is provided.
Background technology
Algae can utilize the carbonic acid gas in the gas to carry out the light autotrophy, and nutritive substances such as rich in proteins, fat and VITAMIN have advantages such as breeding is fast, environmental compatibility is strong, the bait of Chang Zuowei used for aquiculture.Simultaneously, the complicacy of algae heredity and biochemical composition singularity and pathways metabolism determines its potential nutrition and pharmacy value, and the mankind can therefrom develop the bioactive ingredients of the special high added value of a large amount of structures.
It is the frustule poor growth that the subject matter that exists is cultivated in inferior heart-shaped flat algae mass-producing, and doubling time length has restricted the widespread use of inferior heart-shaped flat algae in each field.The algae large-scale artificial is cultivated open cultivation and the closed photo bioreactor two class modes of mainly containing at present.Open cultivation is simple relatively, investment is low; But it is low that this mode is cultivated the productive rate of little algae, has serious biological pollution, restriction algae culture and development of resources.Complicated closed photo bioreactor is cultivated algae and is easy to control biological pollution, and productive rate is high relatively, but cost is relatively also high.
Therefore, the exploitation high-density culture medium and make up the cheap apparatus of optical-biological reaction efficiently become the exploitation algae resource the key core technology.
Summary of the invention
A kind of fast high-density culture method for algae cell that the object of the present invention is to provide, the present invention will optimize substratum and bioreactor coupling, can reach the purpose of algae high-density culture, and it is short to cultivate required time, can reduce investment significantly, it is feasible to reach the algae resource exploiting economy.
For achieving the above object, the technical solution used in the present invention is:
A kind of fast high-density culture method for algae cell,
Add high-density culture medium in bioreactor, microalgae cell is inoculated in the bioreactor, inoculum density is 0.1 * 10
6～1 * 10
6Cells/mL, average intensity are 4000～6000 1x, adopt left and right sides both direction daylighting, and temperature is controlled at 15～30 ℃, and the dark time ratio of light is 10-24h: 14-0h, and every liter of substratum air flow is 0.05～1L/min;
Described high-density culture medium is to make by add Different Nutrition salt after the natural sea-water sterilization cooling of sand filtration, and the addition of nutritive salt is NaNO
34～1360mg/L, NaH
2PO
42H
2O 12～100mg/L, Tris 0～1000mg/L, CH
3COOH 0～2ml, EDTA-Na 20～60mg/L, H
3BO
325～50mg/L, FeCl
36H
2O 1～5mg/L, MnCl
24H
2O 0～0.5mg/L, ZnCl
20～0.05mg/L, CoCl
26H
2O 0～0.05mg/L, CuSO
45H
2O 0～0.05mg/L.
The gas that feeds is that air or air and carbonic acid gas (comprise the CO in the atmosphere
2And the CO in factory's discharging gas
2) gas mixture, the volume ratio of carbonic acid gas and air is: 0.05～10%; Little algae comprises the algae from fresh water or seawater, is preferably green alga or chrysophyceae.
Described high-density culture medium is to make by add Different Nutrition salt after the natural sea-water sterilization cooling of sand filtration, and the addition of nutritive salt is preferably NaNO
3807mg/L, NaH
2PO
42H
2O 55mg/L, Tris830mg/L, CH
3COOH 0.6ml, EDTA-Na 45mg/L, H
3BO
333.6mg/L, FeCl
36H
2O1.3mg/L, MnCl
24H
2O 0.36mg/L, ZnCl
20.021mg/L, CoCl
26H
2O 0.02mg/L, CuSO
45H
2O 0.02mg/L, (NH
4)
6M
O7O
244H
2O 0.009mg/L.
Described bioreactor is connected to form through main line and cultivation unit by source of the gas;
Wherein cultivate the unit and be a plurality of parallel connections more than 1 or 1; Cultivate the unit and make, cultivate the unitary left and right sides and be provided with light source (light source is sunlight and source of artificial light) by transparent material (transparent material is that Glass tubing and plastics bag comprise polypropylene, polyvinyl chloride or polypropylene etc.); Cultivate unitary bottom and be provided with the gas pipe that is made of porous material, the gas pipe lower end is provided with the inlet mouth that communicates with outer pipeline;
Described source of the gas is gas cylinder and a compression pump parallel with one another, and they are connected with main line by gas control valve and gas meter respectively.
Described porous material can be glass envelope, sieve plate, karang or gas stone etc., is preferably sieve plate.
The present invention compared with prior art has following advantage:
The present invention realizes algae fast breeding in a short time by reasonable disposition nitrogen and phosphorous nutrient concentration, carbon source, light intensity, effective stirring degree that transforms luminous energy, algae liquid that receives, and shortens incubation time.Bioreactor of the present invention can make full use of conversion luminous energy, the effective algae liquid nutrient distribution uniformity of security system inside is avoided problems in the photoreactor, the problem includes: the dead angle problem, and gaseous interchange (compensation carbonic acid gas) fully, reduce the cell attachment growth, greatly improved little algae culture efficiency.The present invention is suitable for the scale production of little algae high-density, for the utilization of little algae resource synthetic development provides economic benefit.
Description of drawings
Fig. 1 is a bioreactor anterior view synoptic diagram of the present invention;
Fig. 2 sees synoptic diagram for bioreactor of the present invention side;
Annotate: 1. gas cylinder; 2. compression pump; 3. gas control valve; 4. gas meter; 5. bioreactor; 6. light source; 7 is gas pipe; (arrow is represented air flow line)
Fig. 3 is the growth kinetics collection of illustrative plates of different substratum and training method flat algae cell.
Embodiment
Below by specific embodiment method of the present invention and result are described, the patent that cell cultures of the present invention has been declared with reference to the applicant (application number 03110981.0, denomination of invention: a kind of marine green algae two-step approach biophotolysis water hydrogen production process) carry out, other reagent are business-like reagent.
Described bioreactor is connected to form through main line and cultivation unit 5 by source of the gas;
Described cultivation unit 5 is a plurality of parallel connections; Cultivate unit 5 and make, cultivate the unitary left and right sides and be provided with light source 6 by transparent material; The bottom of cultivating unit 5 is provided with the gas pipe of being made by sieve plate 7, and the gas pipe lower end is provided with the inlet mouth that communicates with outer pipeline;
Described source of the gas is a gas cylinder 1 and compression pump 2 parallel with one another, and they are connected with main line by gas control valve 3 and gas meter 4 respectively.
Inferior heart-shaped flat algae is provided by Dalian, Liaoning Province aquatic products institute, and through the dull and stereotyped purifying in this laboratory.With the 110 ℃ of high pressure steam sterilization 15min of natural sea-water through sand filtration, Different Nutrition salt is added in the cooling back, preparation Kang Wei side substratum (NaNO
3100mg/L, NaH
2PO
4H
2O 20mg/L, EDTA-Na 45mg/L, H
3BO
333.6mg/L, FeCl
36H
2O 1.3mg/L, MnCl
24H
2O 0.36mg/L, ZnCl
20.021mg/L, CoCl
26H
2O 0.02mg/L, CuSO
45H
2O 0.02mg/L, (NH
4) 6M
O7O
244H
2O 0.009mg/L).
Fall from the single-cell algae of agar plate picking,, obtain seed cell through amplification culture step by step.Cell inoculation density is 0.6 * 10
6Cells/mL, average intensity are 5000 1x, adopt left and right sides both direction daylighting, and temperature is controlled at 25 ℃, and the dark time ratio of light is 14h: 10h.
Require nitrogen, phosphorus, Tris-HAc are mixed with 10 substratum by different ratios according to even experimental design, wherein the nitrogen volumetric molar concentration is 0.05～16mM, the phosphorus volumetric molar concentration is 0.01～1mM, Tris-HAc concentration from 1.65～40mM (in Tris, Tris: HAc=10mM: 1ml), micronutrient levels is all with Kang Wei side's substratum in each substratum.The result who cultivated 10 days is carried out frustule counting, and get the algae liquid that 200ml stirs centrifugal, clean, dry, weigh.The result is as follows:
| The substratum numbering | NO 3-N concentration (mM) | PO 4-P concentration (mM) | Tris-HAc concentration (mM) | |
Dry weight mg/ |
| 1 | 0.05 | 0.08 | 16.5 | 1.24 | 0.642 |
| 2 | 0.1 | 1 | 6.6 | 1.25 | 0.727 |
| 3 | 0.4 | 0.06 | 40 | 1.46 | 0.729 |
| 4 | 0.8 | 0.7 | 13.2 | 2.04 | 1.213 |
| 5 | 1.2 | 0.04 | 4.1 | 2.45 | 1.198 |
| 6 | 2 | 0.4 | 30 | 3.01 | 1.704 |
| 7 | 4 | 0.02 | 10.7 | 4.72 | 1.902 |
| 8 | 6 | 0.2 | 1.65 | 5.16 | 1.938 |
| 9 | 8 | 0.01 | 20 | 5.44 | 2.078 |
| 10 | 16 | 0.1 | 8.3 | 4.82 | 1.840 |
| Kang Weifang | 1.2 | 0.17 | 0 | 2.38 | 0.913 |
7～No. 10 culture medium culturing effects all obviously are better than other substratum as seen from the above table, be that nitrogen concentration can effectively promote flat algae cell fast breeding in 4～16mM scope, its obtain frustule quantity be under Kang Wei side's culture condition more than 1.98 times, be up to 2.3 times.Utilize 10 kinds of substratum to obtain the flat algae dry cell weights its medium component is optimized, under the situation of disregarding trace element, calculate by the Matlab statistical software and to optimize substratum and be: NO
3 --N 9.50mM, PO
4 3--P 0.34mM, Tris-HAc 6.7mM.
Inferior heart-shaped flat algae is provided by Dalian, Liaoning Province aquatic products institute, and through the dull and stereotyped purifying in this laboratory.With the 110 ℃ of high pressure steam sterilization 15min of natural sea-water through sand filtration, nutritive salt is added in the cooling back, preparation Kang Wei side substratum (NaNO
3100mg/L, NaH
2PO
4H
2O 20mg/L, EDTA-Na 45mg/L, H
3BO
333.6mg/L, FeCl
36H
2O 1.3mg/L, MnCl
24H
2O 0.36mg/L, ZnCl
20.021mg/L, CoCl
26H
2O 0.02mg/L, CuSO
45H
20 0.02mg/L, (NH
4)
6MO
7O
244H
2O 0.009mg/L) and optimize substratum (NaNO
3807mg/L, NaH
2PO
42H
2O 55mg/L, Tris 830mg/L, CH
3COOH 0.6ml, EDTA-Na 45mg/L, H
3BO
333.6mg/L, FeCl
36H
2O 1.3mg/L, MnCl
24H
2O 0.36mg/L, ZnCl
20.021mg/L, CoCl
26H
2O 0.02mg/L, CuSO
45H
2O 0.02mg/L, (NH
4)
6M
O7O
244H
2O 0.009mg/L)).。
Fall from the single-cell algae of agar plate picking,, obtain seed cell through amplification culture step by step.Cell inoculation density is 0.6 * 10
6Cells/mL is divided into two groups with algae liquid, and one group is carried out Kang Wei side's culture media shaking vase and leaves standstill cultivation, regularly shake every day 4～6 times, another group is put into 12 and is cultivated cell arrangement, and transparent material is a Glass tubing, bottom breather porous material is a sieve plate, the optimization substratum that homogeneous design obtains, bubbling air is cultivated, and flow velocity is 200ml/min, average intensity is 5000 1x, adopt left and right sides both direction daylighting, temperature is controlled at 25 ℃, and the dark time ratio of light is 14h: 10h.
As can be seen from Figure 3, cultivated the 15th day, employing is shaken bottle and is left standstill best cultivation, and cell density reaches 2 * 10
6Cells/mL cultivates and adopt bioreactor to optimize the substratum blowing air, and cell density reaches 8 * 10
6Cells/mL.Utilize bioreactor, light self-shielding phenomenon is improved, and the efficiency of light energy utilization improves, and gaseous interchange is unobstructed, shortens the production cycle, reduces production costs.
Claims (7)
1. fast high-density culture method for algae cell is characterized in that:
Add high-density culture medium in bioreactor, microalgae cell is inoculated in the bioreactor, inoculum density is 0.1 * 10
6～1 * 10
6Cells/mL, average intensity are 4000～6000lx, adopt left and right sides both direction daylighting, and temperature is controlled at 15～30 ℃, and the dark time ratio of light is 10-24h: 14-0h, and every liter of substratum air flow is 0.05～1L/min;
Described high-density culture medium is to make by add Different Nutrition salt after the natural sea-water sterilization cooling of sand filtration, and the addition of nutritive salt is NaNO
34～1360mg/L, NaH
2PO
42H
2O 12～100mg/L, Tris 0～1000mg/L, CH
3COOH 0～2ml, EDTA-Na 20～60mg/L, H
3BO
325～50mg/L, FeCl
36H
2O 1～5mg/L, MnCl
24H
2O 0～0.5mg/L, ZnCl
20～0.05mg/L, CoCl
26H
2O 0～0.05mg/L, CuSO
45H
2O 0～0.05mg/L.
2. according to the described fast high-density culture method for algae cell of claim 1, it is characterized in that: the gas of described feeding is the gas mixture of air or air and carbonic acid gas, and the volume ratio of carbonic acid gas and air is: 0.05～10%.
3. according to the described fast high-density culture method for algae cell of claim 1, it is characterized in that: described little algae is green alga or chrysophyceae.
4. according to the described fast high-density culture method for algae cell of claim 1, it is characterized in that: described high-density culture medium is to make by add Different Nutrition salt after the natural sea-water sterilization cooling of sand filtration, and the addition of nutritive salt is NaNO
3807mg/L, NaH
2PO
42H
2O 55mg/L, Tris 830mg/L, CH
3COOH 0.6ml, EDTA-Na 45mg/L, H
3BO
333.6mg/L, FeCl
36H
2O 1.3mg/L, MnCl
24H
2O 0.36mg/L, ZnCl
20.021mg/L, CoCl
26H
2O 0.02mg/L, CuSO
45H
2O 0.02mg/L, (NH
4)
6M
O7O
244H
2O 0.009mg/L.
5. according to the described fast high-density culture method for algae cell of claim 1, it is characterized in that: described bioreactor is connected to form through main line and cultivation unit (5) by source of the gas;
Wherein cultivate unit (5) and can be a plurality of parallel connections more than 1 or 1; Cultivate unit (5) and make, cultivate the unitary left and right sides and be provided with light source (6) by transparent material; The bottom of cultivating unit (5) is provided with the gas pipe (7) that is made of porous material, and the gas pipe lower end is provided with the inlet mouth that communicates with outer pipeline;
Described source of the gas is a gas cylinder (1) and compression pump (2) parallel with one another, and they are connected with main line by gas control valve (3) and gas meter (4) respectively.
6. according to the described fast high-density culture method for algae cell of claim 5, it is characterized in that: described porous material is glass envelope, sieve plate, karang or gas stone.
7. according to the described fast high-density culture method for algae cell of claim 5, it is characterized in that: described porous material is a sieve plate.
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|---|---|---|---|
| CNA200710010593XA CN101265449A (en) | 2007-03-14 | 2007-03-14 | Fast high-density culture method for algae cell |
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|---|---|---|---|
| CNA200710010593XA CN101265449A (en) | 2007-03-14 | 2007-03-14 | Fast high-density culture method for algae cell |
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Family
ID=39988141
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Cited By (12)
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| CN101870953A (en) * | 2009-04-24 | 2010-10-27 | 中国石油化工股份有限公司 | Method for culturing microalgae |
| WO2012019539A1 (en) * | 2010-08-10 | 2012-02-16 | 中国科学院青岛生物能源与过程研究所 | A semi-dry and semi-solid cultivation method for industrialized production of microalgae |
| CN102517218A (en) * | 2011-12-28 | 2012-06-27 | 广东海洋大学 | Method for culturing microalgae on sea surface |
| CN102816687A (en) * | 2012-08-30 | 2012-12-12 | 浙江大学 | Device and method for culturing microalgae for simple flow rising type light bioreactor system |
| CN103224889A (en) * | 2013-05-21 | 2013-07-31 | 青岛蔚蓝生物集团有限公司 | Microalga culture medium and application thereof |
| CN103849550A (en) * | 2014-03-20 | 2014-06-11 | 环境保护部南京环境科学研究所 | Secondary culture apparatus and culture method for alga |
| CN103911290A (en) * | 2014-04-16 | 2014-07-09 | 沈阳化工研究院有限公司 | Close-type microalgae culture method by intermittent gas introduction in dark period |
| CN103911289A (en) * | 2014-04-16 | 2014-07-09 | 沈阳化工研究院有限公司 | Enclosed micro algae culture method in intermittent ventilation mode |
| CN104805016A (en) * | 2015-04-28 | 2015-07-29 | 中国科学院过程工程研究所 | Method for cultivating microalgae by using CO2 |
| WO2016169061A1 (en) * | 2015-04-24 | 2016-10-27 | 亿利资源集团有限公司 | Microalgae culture photobioreactor |
| CN106915804A (en) * | 2015-12-28 | 2017-07-04 | 国家开发投资公司 | The method for preventing and treating chrysophyceae |
| CN109868206A (en) * | 2019-02-01 | 2019-06-11 | 中国科学院水生生物研究所 | High-throughput algae culture and test method |
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2007
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101870953B (en) * | 2009-04-24 | 2013-06-26 | 中国石油化工股份有限公司 | Method for culturing microalgae |
| CN101870953A (en) * | 2009-04-24 | 2010-10-27 | 中国石油化工股份有限公司 | Method for culturing microalgae |
| WO2012019539A1 (en) * | 2010-08-10 | 2012-02-16 | 中国科学院青岛生物能源与过程研究所 | A semi-dry and semi-solid cultivation method for industrialized production of microalgae |
| CN102373156A (en) * | 2010-08-10 | 2012-03-14 | 中国科学院青岛生物能源与过程研究所 | Half-dry solid state cultivation method used for industrial production of microalgae |
| CN102517218A (en) * | 2011-12-28 | 2012-06-27 | 广东海洋大学 | Method for culturing microalgae on sea surface |
| CN102816687A (en) * | 2012-08-30 | 2012-12-12 | 浙江大学 | Device and method for culturing microalgae for simple flow rising type light bioreactor system |
| CN103224889B (en) * | 2013-05-21 | 2015-04-15 | 青岛蔚蓝生物集团有限公司 | Microalga culture medium and application thereof |
| CN103224889A (en) * | 2013-05-21 | 2013-07-31 | 青岛蔚蓝生物集团有限公司 | Microalga culture medium and application thereof |
| CN103849550B (en) * | 2014-03-20 | 2015-09-16 | 环境保护部南京环境科学研究所 | A kind of algae secondary culture apparatus and cultural method thereof |
| CN103849550A (en) * | 2014-03-20 | 2014-06-11 | 环境保护部南京环境科学研究所 | Secondary culture apparatus and culture method for alga |
| CN103911289A (en) * | 2014-04-16 | 2014-07-09 | 沈阳化工研究院有限公司 | Enclosed micro algae culture method in intermittent ventilation mode |
| CN103911290A (en) * | 2014-04-16 | 2014-07-09 | 沈阳化工研究院有限公司 | Close-type microalgae culture method by intermittent gas introduction in dark period |
| WO2016169061A1 (en) * | 2015-04-24 | 2016-10-27 | 亿利资源集团有限公司 | Microalgae culture photobioreactor |
| CN104805016A (en) * | 2015-04-28 | 2015-07-29 | 中国科学院过程工程研究所 | Method for cultivating microalgae by using CO2 |
| CN104805016B (en) * | 2015-04-28 | 2018-02-16 | 中国科学院过程工程研究所 | One kind utilizes CO2The method for cultivating microalgae |
| CN106915804A (en) * | 2015-12-28 | 2017-07-04 | 国家开发投资公司 | The method for preventing and treating chrysophyceae |
| CN106915804B (en) * | 2015-12-28 | 2020-08-04 | 国投生物科技投资有限公司 | Method for preventing and treating golden algae |
| CN109868206A (en) * | 2019-02-01 | 2019-06-11 | 中国科学院水生生物研究所 | High-throughput algae culture and test method |
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Open date: 20080917 |