CN114410475B - High-density chlorella culture method - Google Patents
High-density chlorella culture method Download PDFInfo
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Abstract
The invention belongs to the field of microbial cultivation, and provides a high-density chlorella cultivation method, which comprises the steps of algae seed activation, shake flask cultivation, seeding tank cultivation and main fermentation tankThree steps of cultivation; the invention adopts facultative mixed culture, and the chlorella can provide energy and fix CO through illumination 2 Meanwhile, the organic carbon source is utilized for metabolism, so that the cost can be reduced, and the biomass of the chlorella can be increased. Compared with the density of the conventional heterotrophic chlorella culture solution of about 15 hundred million/mL, the density of the chlorella of the invention can reach 30 hundred million/mL; compared with the conventional heterotrophic culture chlorella protein content of 30-35% (dry weight), the protein chlorella of the invention has the content of 45% (dry weight); the chlorophyll content can reach 7.0mg/g at most.
Description
Technical Field
The invention belongs to the field of microbial cultivation, and particularly relates to a high-density chlorella cultivation method.
Background
Chlorella (Chlorella) belongs to a kind of unicellular eukaryotic microalgae of Chlorella of Chlorophyta, has the characteristic of eukaryotic gene expression, is rich in nutritional ingredients such as protein, amino acid, vitamin, iron, calcium and the like, is considered as a source of extremely valuable food and feed additives (Bricknell and Dalmo, 2005), is easy to reproduce and culture, and is suitable for large-scale production.
Currently, there are three main modes for chlorella culture: photoautotrophic culture, heterotrophic and facultative mixed culture.
The autotrophic culture mode has low cost and is easy to apply in large scale, but the autotrophic culture mode has low yield, is easy to be influenced by external environment, occupies large area, is difficult to harvest in later period, and the like. The traditional microbial fermentation technology is applied to chlorella culture, the culture is independent of illumination, the growth is rapid, the growth period is short, the cell density is high, the biomass is dozens of times of that obtained by photoautotrophic culture, the occupied area is small, and the harvesting is controllable.
Under the heterotrophic culture mode, nutrients are continuously provided in the fermentation process to maintain the optimal substrate concentration, temperature, pH and the like for the growth of chlorella, but the disadvantages of high price of organic carbon sources, large energy dissipation and easy contamination of bacteria in the fermentation industry exist.
The facultative mixed culture is the combination of two modes of autotrophy and heterotrophy.
Disclosure of Invention
Aiming at the defects of autotrophy and heterotrophy in the existing method, the invention provides a high-density chlorella culture method.
The high-density chlorella cultivating method includes the steps of,
s1 algal species activation: placing chlorella algae seeds in a light incubator for culture and activation;
s2 shake flask culture: placing the activated chlorella strain in a first culture bottle for illumination culture, and then transferring the chlorella strain into a shake flask for dark culture to obtain a seed solution;
s3 seed culture: transferring the seed liquid after the shake flask culture to a seed tank for dark culture;
s4 main fermentation tank culture: placing the seed solution after seed culture in a main fermentation tank, supplementing citric acid with concentration of 0.1g/L at 48h, opening valves of the main fermentation tank and an illumination culture tank, and circularly flowing to receive illumination; supplementing 1g/L urea at 60h, closing valves of the main fermentation tank and the illumination culture tank, and carrying out fermentation culture for 84 h;
the main fermentation tank is internally provided with a fermentation culture medium, and the fermentation culture medium at least comprises:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
1mL/L of trace element solution
Glucosamine 2g/L
2g/L amino acid fermentation liquor
The amino acid fermentation liquor contains at least 15wt% of glutamic acid.
In the amino acid fermentation liquor, at least 22 wt% of organic matters, at least 4 wt% of nitrogen and at least 2.5 wt% of potassium are contained.
Specifically, the illumination incubator is provided with an algae culture medium, and the algae culture medium at least comprises:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
The trace element solution is 1 mL/L.
Specifically, in the activation of S1 algae, the illumination intensity is 1500-.
Specifically, in S2 shake flask culture, the activated chlorella strain is placed in a first culture bottle to be cultured until the OD value is more than or equal to 0.6, and then the chlorella strain is transferred to a shake flask to be cultured in a dark place until the OD value is more than or equal to 0.6;
the culture conditions in the first flask were: the illumination intensity is 1500-:
the culture conditions for the culture in the shake flask were: protecting from light at 20-28 deg.C.
More specifically, in the S2 shake flask culture, BG11 medium is placed in a first culture flask, and BG11 medium at least comprises:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
The trace element solution is 1 mL/L.
Specifically, in the S2 shake flask culture, a shake flask culture medium is placed in the shake flask, and the shake flask culture medium at least comprises:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
1mL/L of trace element solution
Glucosamine is 2 g/L.
Specifically, the S3 seed culture is divided into:
first-level seeding tank culture: transferring the seed solution after shake culture to a first-stage seed tank for dark culture until the OD value is more than or equal to 0.6;
secondary seed tank culture: transferring the seed liquid cultured in the first-stage seed tank to a second-stage seed tank for dark culture until the OD value is more than or equal to 0.6;
the culture conditions of the first-stage seeding tank and the second-stage seeding tank are as follows: keeping out of the sun at 20-28 ℃;
the first-stage seeding tank culture and the second-stage seeding tank contain a seeding tank culture medium, and the seeding tank culture medium at least comprises:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
1mL/L of trace element solution
Glucosamine 2 g/L.
Specifically, the illumination intensity in the culture of the S4 main fermentation tank is 1500-.
Specifically, the trace element solution at least comprises:
H 3 BO 3 2.86g/L
MnCl 2 ·4H 2 O1.86g/L
ZnSO 4 ·7H 2 O0.22g/L
Na 2 MoO 4 '2H 2 O0.39g/L
CuSO 4 ·5H 2 O0.08g/L
Co(NO) 2 ·6H 2 O0.05g/L。
since chlorella has an effect on the absorption of various forms of hydrochloride, sulfate, and nitrate, the salts in the above forms are selected as components of the trace element solution.
The culture space of the chlorella is enlarged every time the chlorella is transferred and cultured, so that the density of the chlorella is reduced, and the OD value is reduced through an intuitive reaction, so that the chlorella needs to be cultured until the density of the chlorella is increased again every time the chlorella is transferred and cultured.
The invention has the beneficial effects that:
the invention adopts facultative mixed culture, and the chlorella can provide energy and fix CO through illumination 2 Meanwhile, the organic carbon source is utilized for metabolism, so that the cost can be reduced, and the biomass of the chlorella can be increased.
Compared with the density of the conventional heterotrophic chlorella culture solution of about 15 hundred million/mL, the density of the chlorella of the invention can reach 30 hundred million/mL; compared with the conventional heterotrophic culture chlorella protein content of 30-35% (dry weight), the protein chlorella of the invention has the content of 45% (dry weight); the chlorophyll content of the chlorella can reach 7mg/g at most.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
In the present invention, the OD value is an optical intensity value.
Example 1
The method comprises the following steps:
s1 algal species activation: selecting single chlorella algae seeds by using an inoculating loop to remove single algae cells, inoculating the single chlorella algae seeds into a 12-hole plate, and then putting the single chlorella algae seeds into a light incubator containing an algae seed culture medium for culture and activation; the illumination intensity is 2000lx, the temperature is 25 ℃, the illumination period is 12h/12h, and the culture lasts for 3 d.
S2 shake flask culture: in S2 shake flask culture, firstly placing activated chlorella strain in a first culture bottle containing BG11 culture medium to be cultured until OD value is more than or equal to 0.6, illumination intensity is 2000lx, and temperature is kept at 28 +/-2 ℃; then transferring the mixture to a shake flask containing a shake flask culture medium to be cultured in a dark place until the OD value is more than or equal to 0.6, and keeping the temperature at 28 +/-2 ℃; obtaining the seed liquid.
S3 seed tank culture: first-level seeding tank culture: transferring the seed solution after shake culture to a first-stage seed tank containing a seed tank culture medium, and culturing in a dark place until the OD value is more than or equal to 0.6;
secondary seeding tank culture: transferring the seed liquid cultured in the first seeding tank to a second seeding tank containing a seeding tank culture medium for light-tight culture until the OD value is more than or equal to 0.6;
the culture conditions of the first-stage seeding tank and the second-stage seeding tank are as follows: protecting from light at the temperature of 22 +/-2 ℃;
the culture medium of the seeding tank contains:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
1mL/L of trace element solution
Glucosamine 2 g/L.
S4 main fermentation tank culture: placing the seed solution after light-tight culture in a main fermentation tank containing a fermentation culture medium, supplementing citric acid with the concentration of 0.1g/L at 48h, opening valves of the main fermentation tank and an illumination culture tank, and circularly flowing to receive illumination; at 60h, supplementing urea with the concentration of 1g/L, closing a valve of the illumination culture tank, and carrying out fermentation culture for 84 h;
the algae strain culture medium contains:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
The trace element solution is 1 mL/L.
BG11 culture medium comprises:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
The trace element solution is 1 mL/L.
The shake flask culture medium comprises:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
1mL/L of trace element solution
Glucosamine 2 g/L.
The fermentation medium comprises:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
1mL/L of trace element solution
Glucosamine 2g/L
2g/L amino acid fermentation liquor
The amino acid fermentation liquor contains at least 15wt% of glutamic acid.
The microelement solution at least comprises:
H 3 BO 3 2.86g/L
MnCl 2 ·4H 2 O1.86g/L
ZnSO 4 ·7H 2 O0.22g/L
Na 2 MoO 4 ·2H 2 O0.39g/L
CuSO 4 ·5H 2 O0.08g/L
Co(NO) 2 ·6H 2 O0.05g/L。
s4 Chlorella was taken out after culturing in the main fermenter and tested for its properties, the results are shown in Table 1.
Example 2
The method comprises the following steps:
s1 algal species activation: selecting single chlorella algae seeds by using an inoculating loop to remove single algae cells, inoculating the single chlorella algae seeds into a 12-hole plate, and then putting the single chlorella algae seeds into a light incubator containing an algae seed culture medium for culture and activation; the illumination intensity is 1500lx, the temperature is 28 ℃, the illumination time is 14h/d, and the culture lasts for 4 d.
S2 shake flask culture: s2 shake flask culture, firstly placing the activated chlorella strain in a first culture flask containing BG11 culture medium, culturing at 28 ℃ in the dark until OD value is more than or equal to 0.6, then transferring to a shake flask containing shake flask culture medium, culturing at 28 ℃ in the dark, and obtaining seed liquid after 3d of culture;
s3 seed tank culture: first-level seeding tank culture: transferring the seed solution after shake culture to a first-stage seed tank containing a seed tank culture medium, and culturing in a dark place until the OD value is more than or equal to 0.6;
secondary seeding tank culture: transferring the seed liquid cultured in the first seeding tank to a second seeding tank containing a seeding tank culture medium for light-tight culture until the OD value is more than or equal to 0.6;
the culture conditions of the first-stage seeding tank and the second-stage seeding tank are as follows: protecting from light at the temperature of 24 +/-2 ℃;
the culture medium in the seeding tank contains:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
1mL/L of trace element solution
Glucosamine 2 g/L.
S4 main fermentation tank culture: placing the seed solution after light-tight culture in a main fermentation tank containing a fermentation culture medium, supplementing citric acid with the concentration of 0.1g/L at 48h, opening valves of the main fermentation tank and an illumination culture tank, and circularly flowing to receive illumination; at 60h, supplementing urea with the concentration of 1g/L, closing a valve of the illumination culture tank, and carrying out fermentation culture for 84 h; the temperature is controlled within 26 +/-2 ℃.
The algal species culture medium, BG11 culture medium, shake flask culture medium, fermentation medium, and trace element solution were the same as in example 1.
S4 Chlorella was taken out after culturing in the main fermenter and tested for its properties, the results are shown in Table 1.
Example 3
The method comprises the following steps:
s1 algal species activation: selecting single chlorella algae seeds by using an inoculating loop to remove single algae cells, inoculating the single chlorella algae seeds into a 12-hole plate, and then putting the single chlorella algae seeds into a light incubator containing an algae seed culture medium for culture and activation; the illumination intensity is 2500lx, the temperature is 20 ℃, the illumination time is 8h/d, and the culture lasts for 4 d.
S2 shake flask culture: in S2 shake-flask culture, firstly placing activated chlorella strain in a first culture bottle containing BG11 culture medium, culturing at 20 ℃ in a dark place until OD value is not less than 0.6, transferring the chlorella strain into a shake flask containing shake-flask culture medium at 20 ℃ in a dark place, and culturing for 3 days to obtain seed liquid;
s3 seed tank culture:
first-level seeding tank culture: transferring the seed solution after shake culture to a first-stage seed tank containing a seed tank culture medium, and culturing in a dark place until the OD value is more than or equal to 0.6;
secondary seeding tank culture: transferring the seed liquid cultured in the first seeding tank to a second seeding tank containing a seeding tank culture medium for light-tight culture until the OD value is more than or equal to 0.6;
the culture conditions of the first-stage seeding tank and the second-stage seeding tank are as follows: protecting from light at the temperature of 28 +/-2 ℃;
the culture medium of the seeding tank contains:
NaNO 3 1.5g/L
K 2 HPO 4 0.04g/L
MgSO 4 ·7H 2 O0.075g/L
CaCl 2 ·2H 2 O0.036g/L
EDTA-Na 2 0.001g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02g/L
1mL/L of trace element solution
Glucosamine 2 g/L.
S4 main fermentation tank culture: placing the seed solution after light-tight culture in a main fermentation tank containing a fermentation culture medium, supplementing citric acid with the concentration of 0.1g/L at 48h, opening valves of the main fermentation tank and an illumination culture tank, and circularly flowing to receive illumination; at 60h, supplementing urea with the concentration of 1g/L, closing a valve of the illumination culture tank, and carrying out fermentation culture for 84 h; the temperature is controlled within 22 +/-2 ℃.
The algal species culture medium, BG11 culture medium, shake flask culture medium, fermentation medium, and trace element solution were the same as in example 1.
S4 Chlorella was taken out after culturing in the main fermenter and tested for its properties, the results are shown in Table 1.
In the above examples 1 to 3: the capacity of a first culture bottle in a light culture stage of shake flask culture is 100ml, and the capacity of a shake flask in a dark culture stage is 300 ml; the capacity of the first-level seeding tank for culture is 4000 ml; the capacity of the secondary seeding tank culture is 180L; the capacity of the main fermentor was 3000L.
Comparative example 1
The difference from example 1 is that the S3 main fermentation tank is protected from light all the time, and BG11 medium is used all the time.
Experimental tests and results
The chlorella cultured in the above examples and comparative examples were tested, and the results are shown in table 1, and the test methods are as follows:
and (3) testing the density:
diluting chlorella fermentation liquor by a certain multiple, and then counting cells by using a blood cell counting plate under a microscope.
Protein content testing:
kai type nitrogen determination method in GB/T6432-2018 determination of crude protein in feed.
Chlorophyll content test:
' Liuxin. plant biology experimental guidelines [ M ]. Beijing, Chinese agricultural Press, 2015: 25-27'.
Experimental tests and results
The chlorella cultured in the above examples and comparative examples was tested and the results are shown in table 1.
TABLE 1
| Density (fresh weight) | Protein content (dry weight) | Chlorophyll (dry weight) | |
| Example 1 | 30 hundred million/mL | 45％ | 6.8mg/g |
| Example 2 | 28 hundred million/mL | 44％ | 6.3mg/g |
| Example 3 | 29 hundred million/mL | 44.5％ | 6.9mg/g |
| Comparative example 1 | 15 hundred million/mL | 30％ | 3.2mg/g |
Claims (5)
1. The high-density chlorella culture method is characterized by comprising the following steps,
s1 algal species activation: placing chlorella algae seeds in a light incubator for culture and activation;
s2 shake flask culture: placing the activated chlorella strain in a first culture bottle for illumination culture, and then transferring the chlorella strain into a shake flask for dark culture to obtain a seed solution;
s3 seed culture: transferring the seed liquid after the shake flask culture to a seed tank for dark culture;
s4 main fermentation tank culture: placing the seed liquid after seed culture in a main fermentation tank, supplementing citric acid with concentration of 0.1g/L at 48h, opening valves of the main fermentation tank and an illumination culture tank, and circularly flowing to receive illumination; supplementing 1g/L urea at 60h, closing valves of the main fermentation tank and the illumination culture tank, and carrying out fermentation culture for 84 h;
the main fermentation tank is internally provided with a fermentation culture medium, and the fermentation culture medium at least comprises:
NaNO 3 1.5 g/L
K 2 HPO 4 0.04 g/L
MgSO 4 ·7H 2 O0.075 g/L
CaCl 2 ·2H 2 O0.036 g/L
EDTA-Na 2 0.001 g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02 g/L
1mL/L of trace element solution
Glucosamine 2g/L
2g/L amino acid fermentation liquor
The amino acid fermentation liquor contains at least 15wt% of glutamic acid;
in the S2 shake flask culture, firstly, the activated chlorella strain is placed in a first culture bottle to be cultured until the OD value is more than or equal to 0.6, and then the chlorella strain is transferred into a shake flask to be cultured in a dark place until the OD value is more than or equal to 0.6;
the culture conditions in the first flask were: the illumination intensity is 1500-;
the culture conditions for the culture in the shake flask were: keeping out of the sun at 20-28 ℃;
in the S2 shake flask culture, BG11 medium is placed in a first culture flask, and the BG11 medium at least comprises:
NaNO 3 1.5 g/L
K 2 HPO 4 0.04 g/L
MgSO 4 ·7H 2 O0.075 g/L
CaCl 2 ·2H 2 O0.036 g/L
EDTA-Na 2 0.001 g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02 g/L
1mL/L of trace element solution;
in the S2 shake flask culture, a shake flask culture medium is placed in a shake flask, and the shake flask culture medium at least comprises:
NaNO 3 1.5 g/L
K 2 HPO 4 0.04 g/L
MgSO 4 ·7H 2 O0.075 g/L
CaCl 2 ·2H 2 O0.036 g/L
EDTA-Na 2 0.001 g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02 g/L
1mL/L of trace element solution
2g/L of glucosamine;
the trace element solution at least comprises:
H 3 BO 3 2.86g/L
MnCl 2 ·4H 2 O1.86 g/L
ZnSO 4 ·7H 2 O0.22g/L
Na 2 MoO 4 ·2H 2 O0.39g/L
CuSO 4 ·5H 2 O0.08g/L
Co(NO) 2 ·6H 2 O0.05g/L。
2. the method for cultivating high-density chlorella according to claim 1, wherein the light incubator is provided with an algae culture medium, and the algae culture medium at least comprises:
NaNO 3 1.5 g/L
K 2 HPO 4 0.04 g/L
MgSO 4 ·7H 2 O0.075 g/L
CaCl 2 ·2H 2 O0.036 g/L
EDTA-Na 2 0.001 g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02 g/L
The trace element solution is 1 mL/L.
3. The method for cultivating high-density chlorella according to claim 1, wherein the S1 algae is activated under the conditions of illumination intensity of 1500-.
4. The method for cultivating high-density chlorella according to claim 1, wherein the S3 seed culture comprises:
first-level seeding tank culture: transferring the seed solution after shake culture to a first-stage seed tank for dark culture until the OD value is more than or equal to 0.6;
secondary seed tank culture: transferring the seed liquid cultured in the first-stage seed tank to a second-stage seed tank for dark culture until the OD value is more than or equal to 0.6;
the culture conditions of the first-stage seeding tank and the second-stage seeding tank are as follows: protecting from light at 20-28 deg.C;
the first-stage seeding tank culture and the second-stage seeding tank contain a seeding tank culture medium, and the seeding tank culture medium at least comprises:
NaNO 3 1.5 g/L
K 2 HPO 4 0.04 g/L
MgSO 4 ·7H 2 O0.075 g/L
CaCl 2 ·2H 2 O0.036 g/L
EDTA-Na 2 0.001 g/L
citric acid 0.006g/L
Ferric citrate 0.006g/L
Na 2 CO 3 0.02 g/L
1mL/L of trace element solution
Glucosamine 2 g/L.
5. The method for culturing high-density chlorella according to claim 1, wherein the illumination intensity in the S4 primary fermentor is 1500-.
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| CN104152357A (en) * | 2014-08-06 | 2014-11-19 | 华南理工大学 | High-density culture method for improving chlorophyll and protein content of chlorella at same time |
| CN104357330A (en) * | 2014-11-11 | 2015-02-18 | 甘肃德福生物科技有限公司 | Chlorella autotrophic-heterotrophic mixed culture method |
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