CN105524836A - Method for economically and efficiently cultivating chlorella - Google Patents
Method for economically and efficiently cultivating chlorella Download PDFInfo
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Abstract
The invention relates to the technical field of biology and aims at providing a method for economically and efficiently cultivating chlorella. By utilizing the method, high-density high-quality chlorella can be obtained within a short cultivation period, and requirements on equipment and operating personnel are lowered; the method is suitable for large-scale cultivation of chlorella. The method includes following steps: (1), heterotrophically cultivating chlorella; (2), mixotrophically cultivating chlorella, wherein a mode for treating a mixotrophic cultivation medium is adding mother liquid of the culture medium into a cultivation container, boiling before adding cold boiled water for fixing volume and cooling, diluting chlorella obtained in the step (1) before inoculating to the mixotrophic cultivation medium in an open condition for stirring cultivation. A semi-boiling mode of treating the mixotrophic cultivation medium is adopted, so that treatment time of the cultivation medium can be reduced remarkably, growth velocity of chlorella can be accelerated, and cultivation period can be shortened.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of method of cultivating chlorella.
Background technology
Chlorella is that one grows unicell green alga rapidly, and photosynthetic efficiency is high, can stabilizing carbon dioxide efficiently, also rich in proteins, polysaccharide, lipid, VITAMIN, mineral substance and pigment etc.And, the chlorella growth factor contained in chlorella, effectively can improve body's immunity, to improving the immunizing power of human body and promoting that biological growth has good effect, be a kind of Biological resources that there is Important Economic and be worth, have huge application and potentiality to be exploited.
Chlorella can utilize luminous energy and inorganic carbon source to carry out photoautotrophy; Also can, under no light condition, one or more organism be utilized to carry out heterotrophic growth as the energy and carbon source; Also can, under illumination condition, organic carbon source be utilized to carry out mixotrophic growth.What current chlorella large scale culturing was more is open or closed pond culture system, is namely autotrophy mode.This culture systems cost and running expense lower, the frustule of unit mass can obtain higher photosynthetic pigments and protein content.But cell density is low, growth cycle is long, thus cause cell harvesting and post-treatment cost very high, but also exist easily pollute, the shortcoming such as condition is wayward, unstable product quality.Chlorella heterotrophy is cultivated can obtain very high cell density, but the apparatus expensive that Heterotrophic culture needs, energy consumption is high, and toxigenic capacity is high.And cultivate the frond obtained, although fat content is high, compared with cultivating with autotrophy, the content of its protein and pigment obviously declines, and affects chlorella quality.
Therefore, while realizing high-density culture, keep its high-quality, CN1837351A provides a kind of method of culturing chlorella with high-density and high-quality.This cultural method comprises bio-reactor high Density Heterotrophic, the dilution of high-density algae liquid and light autotrophy and cultivates three phases.The i.e. series system of first heterotrophism autotrophy again, while raising cell density, quality reaches simple light autotrophy cultivation level.And cultivate through autotrophy, dry cell weight have also appeared the phenomenon of decline, although improve the quality of chlorella, has had a strong impact on the output of chlorella.The method of the cultivation chlorella that CN104357330A provides, is included in mechanical agitating fermentation tank Heterotrophic Mass Cultures of Chlorella, the dilution of algae liquid and light autotrophy and cultivates chlorella three phases.Culture condition is optimized, cultivates to realize chlorella high-density and high-quality.But its culturing process is all aseptically carried out, Heterotrophic culture carries out in mechanical agitating fermentation tank, and adopt feed supplement feeding culture form.Need the culture device of specialty and strict complicated operation, be not suitable for the scale evaluation of chlorella.
Summary of the invention
The object of this invention is to provide a kind of method that economical and efficient cultivates chlorella, utilize the method can obtain high-density and high-quality chlorella in shorter culture cycle, and reduce the requirement to equipment and operator, be suitable for the scale evaluation of chlorella.
For achieving the above object, the technical solution adopted in the present invention is: a kind of economical and efficient cultivates the method for chlorella, comprises the following steps:
(1) chlorella heterotrophy is cultivated;
(2) chlorella raises together with cultivation: processing mode of raising together with substratum used is: in culture vessel, first add substratum mother liquor, boiled rear interpolation cold water constant volume, cooling; Under opening condition, be inoculated in after the chlorella of step (1) gained is diluted and raise together with substratum, carry out stir culture; Chlorella inoculum density OD
680for 0.2-0.7, culture temperature is 25-30 DEG C, and intensity of illumination is 3500-7000lx, and cultivation duration is 4-6 days.
Preferred: described in raise together with substratum be the liquid nutrient medium optimized on BG11 basis, comprise following component: glucose 2-10g/L, NaNO
31.5g/L, K
2hPO
43H
2o0.04g/L, MgSO
47H
2o0.075g/L, CaCl
27H
2o0.036g/L, Na
2-EDTA0.001g/L, Na
2cO
30.02g/L, citric acid 0.006g/L, ferric ammonium citrate 0.006g/L.
Preferred: described in raise together with glucose concn in substratum be 4g/L.
Preferred: it is 3500lx that described step (2) chlorella raises together with intensity of illumination in cultivation.
Preferred: described step (1) chlorella heterotrophy is cultivated and is: chlorella is inoculated in Heterotrophic culture base and carries out shaking table cultivation; Chlorella inoculum density OD
680for 1-5, culture temperature is 27-30 DEG C, and rotating speed is 150-200r/min, and cultivation duration is 2-4 days.
Preferred: described Heterotrophic culture base comprises following component: glucose 30-40g/L, KNO
37-10g/L, KH
2pO
41.25g/L, MgSO
47H
2o1g/L, Na
2-EDTA0.5g/L, H
3bO
30.1142g/L, CaCl
22H
2o0.111g/L, FeSO
47H
2o0.0498g/L, ZnSO
47H
2o0.0882g/L, MnCl
24H
2o0.0142g/L, MoO
30.0071g/L, CuSO
45H
2o0.0157g/L, Co (NO
3)
25H
2o0.0049g/L.
Preferred: the initial pH of described Heterotrophic culture base is 6.0-6.5.
Preferred: in described Heterotrophic culture base, glucose concn is 40g/L, KNO
3concentration is 7g/L.
Preferred: the inoculum density OD that described chlorella heterotrophy is cultivated
680be 2.5.
The present invention has following beneficial effect:
(1) Heterotrophic culture combines with Combined hardening model process by the present invention, first utilizes Heterotrophic culture to carry out expanding species, then adopts the mode of raising together with to carry out amplification culture, can realize the object obtaining high-density and high-quality chlorella in shorter culture cycle.The training method of raising together with can not only improve the quality of chlorella, can also improve biomass.Comprehensive benefit is significantly better than first heterotrophism autotrophy or the training method of directly raising together with again.
(2) stage of raising together with, at opening condition, namely carries out under non-sterile conditions, reduces the requirement to equipment and operator, is suitable for the scale evaluation of chlorella.
(3) raise together with substratum and adopt half boiled processing mode, the medium treatment time can be reduced significantly, and accelerate the growth velocity of chlorella, shorten culture cycle further.
(4) raise together with the substratum that the stage adopts improvement, fill a prescription simple and easy to get, and glucose consumption is few, cost is low, but can significantly improve culture effect, improves comprehensive benefit.
Accompanying drawing explanation
Fig. 1 is the impact on chlorella growth of heterotrophism, mixotrophism and autotrophy;
Fig. 2 is the impact on chlorella quality of heterotrophism, mixotrophism and autotrophy;
Fig. 3 is the chlorella growth graphic representation of the BG11 substratum of different glucose concn in embodiment 2;
Fig. 4 is the chlorella growth graphic representation of different culture media in embodiment 2;
Fig. 5 is the chlorella growth graphic representation of different vaccination density in embodiment 2;
Fig. 6 is the chlorella growth graphic representation of different glucose concn under static conditions in embodiment 4;
Fig. 7 is the chlorella growth graphic representation of different glucose concn under agitation condition in embodiment 4.
Embodiment
Embodiment 1
Cultivate the determination of chlorella nutritional mode
One, experimental technique
Adopt autotrophy respectively, raise together with (i.e. mixotrophism), heterotrophism three kinds of nutritional modes cultivate chlorella.
Two, results and analysis
Dry weight is measured when experiment starts; In experimentation, every day measures OD
680and biomass; At the end of experiment, measure dry weight, chlorophyll content and protein content.The results are shown in Figure 1 and Fig. 2.
From Fig. 1 and Fig. 2, compare with Combined hardening model with autotrophy, the cell density of Heterotrophic culture is high, but represent the protein content of frustule quality and chlorophyll content lower.Cultivate after 4 days, the cell density of raising together with reaches the highest, i.e. OD
680value is 7.02, is 14 times of autotrophy; And after raising together with cultivation, the protein content of frustule and chlorophyll content and autotrophy are cultivated and are on close level.
When this example table understands Heterotrophic culture, chlorella growth speed is the fastest; Raising together with, is the best nutritional mode obtaining chlorella high-biomass, high Contents of Photosynthetic Pigments and higher protein content.And raise together with the actual environment more meeting micro algae growth, educable micro-algae kind is more compared to heterotrophism, is suitable for large-scale production.
Therefore the present invention improves cultivation quality to shorten culture cycle, adopts first Heterotrophic culture to carry out expanding species, then raises together with the cultural method of cultivation.
Embodiment 2
The optimization of chlorella heterotrophy stage culture condition
One, experimental technique
The impact of 1.1 different culture medias
Different culture media is adopted to carry out shaking table cultivation, totally 8 groups respectively; Culture temperature is 28 DEG C, and rotating speed is 200r/min.Actual conditions is in table 1.
Table 1 experimental design table
| Substratum | Glucose (g/L) | SODIUMNITRATE (g/L) | Saltpetre (g/L) | Urea (g/L) |
| BG11 low sugar | 4 | 1.5 | - | - |
| Sugar 1 in BG11 | 10 | 1.5 | - | - |
| Sugar 2 in BG11 | 20 | 3 | - | - |
| The high sugar of BG11 | 40 | 7 | - | - |
| Sugar 1 in SK | 10 | - | 1.25 | - |
| Sugar 2 in SK | 30 | - | 9.28 | - |
| The high sugar of SK | 40 | - | 7 | - |
| Endor | 28 | - | - | 4.8 |
The impact of 1.2 different vaccination density
By chlorella respectively with density OD
6800.5,1,1.5,2,2.5,5,11 totally 7 groups, be inoculated in the high sugar of SK (glucose 40g/L, saltpetre 7g/L) substratum and carry out shaking table cultivation; Culture temperature is 28 DEG C, and rotating speed is 200r/min.
Two, results and analysis
Every day measures OD
680, measure dry weight at the end of experiment, and calculate sugared transformation efficiency.The results are shown in Figure 3-5.
As can be seen from Fig. 3,4, the growth performance of chlorella in SK substratum is obviously better than BG11 substratum, and can obtain the highest biomass in the high sugar (glucose 40g/L) of SK.As can be seen from Figure 5, inoculum density is higher, and lag phase is shorter.
This example table understands that the heterotrophism stage is applicable to selecting SK high glucose medium, and inoculum density OD
680be 2.5.And the mode that shaking table is cultivated does not need the fermentation equipment that specialty is expensive, effectively reduces costs and operation easier, be applicable to suitability for industrialized production.
Embodiment 3
Chlorella raises together with the determination of stage processing mode
One, experimental technique
What use component was identical raises together with substratum, adopts different processing modes, be respectively sterilizing experimental group, boiled experimental group and half boiled experimental group to substratum; The substratum of sterilizing experimental group adopts the processing mode of high pressure steam sterilization; The processing mode that the substratum of boiled experimental group adopts direct related culture vessel boiled together; The processing mode of half boiled experimental group substratum is: in culture vessel, first add substratum mother liquor, boiled rear interpolation cold water constant volume, cooling.
Then chlorella is inoculated in substratum, cultivates.Sterilizing experimental group and half boiled experimental group adopt the training method stirred; Boiled experimental group is then be provided with two groups, and wherein one group is stir culture, and one group is quiescent culture.
Wherein sterilizing experimental group aseptically operates; And boiled experimental group and half boiled experimental group are all at opening condition, namely operate under non-sterile conditions.Chlorella inoculum density OD
680be 0.5, culture temperature is 27 DEG C, and intensity of illumination is 3500lx, and cultivating duration is 5 days.
Two, results and analysis
Experiment start most and at the end of measure OD
680, dry weight, and calculate sugar to cell transformation rate.The results are shown in Table 2.
Table 2 Different treatments is on the impact of chlorella growth
| Processing mode | Dry weight (g/L) | OD 680 | Sugar is to cell transformation rate (g/g) |
| Aseptic | 1.46 | 5.49 | 0.37 |
| Half is boiled | 1.965 | 6.113 | 0.49 |
| Boiled (stirring) | 1.738 | 5.931 | 0.43 |
| Boiled (leaving standstill) | 0.74 | 1.94 | 0.18 |
As can be seen from Table 2, the substratum stir culture of boiled process is obviously better than quiescent culture.Under being stir culture equally, aseptic experiment group gained chlorella dry weight, OD
680and sugar to cell transformation rate all lower than all the other two groups; And half boiled experimental group is better than the result of boiled experimental group.
Therefore compared to the strict complicated aseptically process mode of operation, and the boiled processing mode that the operating time is longer; Half boiled processing mode not only the treatment time short, it is more convenient to operate, and the chlorella growth speed of cultivating is high.This example table understands that half boiled processing mode can reduce the medium treatment time significantly, and accelerates the growth velocity of chlorella, shortens culture cycle further.
Embodiment 4
Chlorella raises together with the optimization of stage culture condition
One, experimental technique
The impact of 1.1 glucose concn
In substratum glucose concn be respectively 0,1,2,4,10,15,20g/L, totally 7 groups are carried out quiescent culture; In substratum glucose concn be respectively 2,4,10,20g/L, totally 4 groups are carried out stir culture.
The impact of 1.2 light intensity and glucose concn
In substratum glucose concn be respectively 2,4,10g/L, intensity of illumination is respectively 3500,7000,14000lx, totally 9 groups are carried out stir culture.
Two, results and analysis
Every day measures OD
680, remaining sugar concentration (DNS method measure reducing sugar), measure dry weight at the end of experiment, and calculate sugar to cell transformation rate.The results are shown in Figure 6,7 and table 3,4.
The different glucose concn of table 3 is on the impact of chlorella growth performance
Table 4 different light intensity and glucose concn cultivate at the end of the growth performance of chlorella
As can be seen from Fig. 3,4 and table 2, stir culture successful is better than quiescent culture; And when glucose sugar concentration is 4g/L, chlorella growth speed is the fastest, and showed obvious growth vigor the 3rd day that cultivates time, at the end of cultivation, OD
680with dry weight also apparently higher than other experimental group.Show that the stage of raising together with is applicable to adopting agitation condition, and substratum glucose sugar concentration is 4g/L, this and conventional wisdom think that the more high better idea of glucose concn is just in time contrary.As can be seen from Table 3, when glucose concn is identical, intensity of illumination is that 3500lx and 7000lx growth performance is better, and when illumination is 14000lx, each performance all has obvious decline.Consider energy consumption and growth, employing illumination is the experimental group better performances of 3500lx.
Claims (9)
1. economical and efficient cultivates a method for chlorella, it is characterized in that: comprise the following steps:
(1) chlorella heterotrophy is cultivated;
(2) chlorella raises together with cultivation: processing mode of raising together with substratum used is: in culture vessel, first add substratum mother liquor, boiled rear interpolation cold water constant volume, cooling; Then, under opening condition, be inoculated in after the chlorella of step (1) gained is diluted and raise together with substratum, carry out stir culture; Chlorella inoculum density OD
680for 0.2-0.7, culture temperature is 25-30 DEG C, and intensity of illumination is 3500-7000lx, and cultivation duration is 4-6 days.
2. a kind of economical and efficient according to claim 1 cultivates the method for chlorella, it is characterized in that: described in raise together with substratum and comprise following component: glucose 2-10g/L, NaNO
31.5g/L, K
2hPO
43H
2o0.04g/L, MgSO
47H
2o0.075g/L, CaCl
27H
2o0.036g/L, Na
2-EDTA0.001g/L, Na
2cO
30.02g/L, citric acid 0.006g/L, ferric ammonium citrate 0.006g/L.
3. a kind of economical and efficient according to claim 2 cultivates the method for chlorella, it is characterized in that: described in raise together with glucose concn in substratum be 4g/L.
4. a kind of economical and efficient according to claim 3 cultivates the method for chlorella, it is characterized in that: it is 3500lx that described step (2) chlorella raises together with intensity of illumination in cultivation.
5. a kind of economical and efficient according to claim 1 cultivates the method for chlorella, it is characterized in that: described step (1) chlorella heterotrophy is cultivated and is: chlorella is inoculated in Heterotrophic culture base and carries out shaking table cultivation; Chlorella inoculum density OD
680for 1-5, culture temperature is 27-30 DEG C, and rotating speed is 150-200r/min, and cultivation duration is 2-4 days.
6. a kind of economical and efficient according to claim 5 cultivates the method for chlorella, it is characterized in that: described Heterotrophic culture base comprises following component: glucose 30-40g/L, KNO
37-10g/L, KH
2pO
41.25g/L, MgSO
47H
2o1g/L, Na
2-EDTA0.5g/L, H
3bO
30.1142g/L, CaCl
22H
2o0.111g/L, FeSO
47H
2o0.0498g/L, ZnSO
47H
2o0.0882g/L, MnCl
24H
2o0.0142g/L, MoO
30.0071g/L, CuSO
45H
2o0.0157g/L, Co (NO
3)
25H
2o0.0049g/L.
7. a kind of economical and efficient according to claim 6 cultivates the method for chlorella, it is characterized in that: the initial pH of described Heterotrophic culture base is 6.0-6.5.
8. a kind of economical and efficient according to claim 7 cultivates the method for chlorella, it is characterized in that: in described Heterotrophic culture base, glucose concn is 40g/L, KNO
3concentration is 7g/L.
9. a kind of economical and efficient according to claim 8 cultivates the method for chlorella, it is characterized in that: the inoculum density OD that described chlorella heterotrophy is cultivated
680be 2.5.
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| CN106237379A (en) * | 2016-08-29 | 2016-12-21 | 董晓 | A kind of chlorella bioid modification size controlled activity glass raw powder's production technology |
| CN106867907A (en) * | 2017-03-07 | 2017-06-20 | 四川大学 | A kind of overcompensation cultural method for improving heterotrophic microalgae protein content |
| CN109402025A (en) * | 2018-12-12 | 2019-03-01 | 河西学院 | A kind of both culturing microalgae prevention and cure of pollution method |
| CN110468025A (en) * | 2019-09-06 | 2019-11-19 | 王习羽 | It is a kind of suitable for heterotrophism and the micro algae culturing device and cultural method of mixotrophic cultivation |
| CN114958611A (en) * | 2021-02-23 | 2022-08-30 | 高丽大学校产学协力团 | Microalgae cultivation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106237379A (en) * | 2016-08-29 | 2016-12-21 | 董晓 | A kind of chlorella bioid modification size controlled activity glass raw powder's production technology |
| CN106867907A (en) * | 2017-03-07 | 2017-06-20 | 四川大学 | A kind of overcompensation cultural method for improving heterotrophic microalgae protein content |
| CN109402025A (en) * | 2018-12-12 | 2019-03-01 | 河西学院 | A kind of both culturing microalgae prevention and cure of pollution method |
| CN110468025A (en) * | 2019-09-06 | 2019-11-19 | 王习羽 | It is a kind of suitable for heterotrophism and the micro algae culturing device and cultural method of mixotrophic cultivation |
| CN114958611A (en) * | 2021-02-23 | 2022-08-30 | 高丽大学校产学协力团 | Microalgae cultivation method |
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Application publication date: 20160427 |