CN104830691A - Culture method of chlorella - Google Patents

Culture method of chlorella Download PDF

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CN104830691A
CN104830691A CN201510202667.4A CN201510202667A CN104830691A CN 104830691 A CN104830691 A CN 104830691A CN 201510202667 A CN201510202667 A CN 201510202667A CN 104830691 A CN104830691 A CN 104830691A
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chlorella
culture
cultural method
hpo
heterotrophic
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卢碧林
付艳
祁亮
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Yangtze University
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor

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Abstract

The invention discloses a culture method of chlorella, wherein the culture method comprises the steps: carrying out heterotrophic culture on the chlorella, in the middle period of an exponential growth period, filtering out a part of chlorella, supplementing a part of nutrients, and continuing culture. Compared with the prior art, the culture method has the following advantages that in the heterotrophic culture process, a part of chlorella are separated, and a certain volume of culture liquid is supplemented, so that the vitality of cultured chlorella cells is ensured, the chlorella culture productivity is improved greatly, the utilization rate of a carbon source is improved, and scale heterotrophic production is facilitated.

Description

The cultural method of chlorella
Technical field
The present invention relates to biological technical field, be specifically related to the cultural method of a kind of chlorella.
Background technology
Chlorella is the general natural disposition unicell green alga of Chlorophyta Chlorella, is a kind of spherical unicellular algae, diameter 3 ~ 8 μm, be one of life the earliest on the earth, before appearing at more than 20 hundred million years, gene does not change all the time, a kind of efficient photosynthetic plant, with photoautotrophy growth and breeding.Chlorella is rich in lubricant component owing to having and has growth advantageous advantage rapidly, and replacing fossil oil with chlorella as raw material production biofuel, is one of developing direction of micro-algae bioenergy.
The main training method of chlorella has autotrophy, raises together with and heterotrophism.Current pilot scale culture many employings autotrophy, but autotrophy exists, and culture density is low, growth cycle is long, production efficiency is low, floor space is large, the disadvantageous effects such as difficulty of gathering.And employing is cheap, suitable and widely used organic carbon source glucose is under the culture condition of sole carbon source, the Heterotrophic culture of chlorella does not affect by the natural environment and climate such as illumination, temperature condition, and growth velocity is fast, and culture density is high, production process easily controls, and productive rate can significantly improve.
Current existing Heterotrophic culture mode is mainly batch culture, stream adds batch culture and intermittent type fed batch cultivation.Wherein batch culture is the training method the most generally adopted.But it is longer that batch culture exists lag phase in culturing process, cultivate the aging growth of frustule after reaching stationary phase and slow down, and cultivate the higher wasting problem of glucose concn in latter stage, cultivation productive rate is reduced.
Summary of the invention
In view of this, the object of the invention be to synthesize that a kind of culture cycle is short, high yield, chlorella that utilization of carbon source rate is high cultural method.
A cultural method for chlorella, its step comprises: carry out Heterotrophic culture to chlorella, in mid-term exponential phase of growth, filter out some beads algae, then supplementary portion divides nutrition and water, continues to cultivate.
Compared with prior art; the present invention has the following advantages: in Heterotrophic culture process; by cutting out partial chlorella; and supplement the nutrient solution of certain volume; thus ensure that the vigor of frustule, shorten culture cycle, substantially increase the cultivation productive rate of chlorella simultaneously; and improve the utilization ratio of carbon source, be conducive to mass-producing heterotrophism and produce.
Accompanying drawing explanation
Fig. 1 is the chlorella growth graphic representation of aerated culture under quiescent culture, aerated culture and interpolation glucose condition respectively.
Fig. 2 is under interpolation glucose, aeration condition, chlorella growth graphic representation in early stage exponential phase of growth, mid-term and later stage batch filtration sampling feed supplement situation.
Embodiment
The invention provides the cultural method of a kind of chlorella, its step comprises: carry out Heterotrophic culture to chlorella, in mid-term exponential phase of growth, filter out some beads algae, then supplementary portion divides nutrition and water, continues to cultivate.
Preferably, at middle exponential growth, the operation that every day carries out " filter out some beads algae, then supplementary portion divides nutrition and water, continue cultivate " once, until culture cycle terminates.
Preferably, the composition of described Heterotrophic culture liquid comprises: glucose 20g/L, NaNO 31.5g/L, K 2hPO 30.3g/L, K 2hPO 30.2g/L, MgSO 47H 2o 0.1g/L, CaCl 22H 2o 0.05g/L, C 6h 8o 70.01g/L, FeC 6h 5oNH 4oH 0.01g/L, EDTANa 20.05g/L, A5 liquid microelement 1mL/L, all the other are water, and wherein A5 solution formula is H 3bO 32.86g/L, MnCl 24H 2o 1.86g/L, ZnSO 47H 2o 0.22g/L, Na 2moO 42H 2o 0.39g/L, CuSO 45H 2o 0.08g/L and Co (NO 3) 26H 2o 0.05g/L.
Preferably, described Heterotrophic culture liquid pH value is 7.2 ~ 8.0.
Preferably, described extra-nutrition thing composition comprises in every 1000mL nutrient solution and adds glucose 4-5g, NaNO 30.3g, K 2hPO 30.05g, K 2hPO 30.01g, MgSO 47H 2o 0.02g, CaCl 22H 2o0.01g, C 6h 8o 70.001g, FeC 6h 5oNH 4oH 0.001g, EDTANa 20.01g, A5 liquid microelement 0.5mL.
Preferably, in described Heterotrophic culture process, the inoculum size of chlorella is the volume accounting of nutrient solution 10%.
Preferably, culture temperature 25--28 DEG C in described Heterotrophic culture process, blowing air amount 1.0L/min, add the silicone antifoam agent of 0.05%.
Preferably, described mid-term exponential phase of growth is the 3rd day to the 4th day from inoculation culture.
Preferably, filter out the chlorella of 1/2 at every turn.
Preferably, in continuation culturing process, within every 3 days, injecting the antibiotics penicillin sodium salt 1mL of 300mg/L, every 15 days is a production cycle
In addition, dry algae powder is obtained to filtering the chlorella that obtains through cleaning, centrifugal, drying.
Embodiment 1
Common quiescent culture
In the culturing bottle of 1000mL, quiescent culture under common BG11 substratum, culture condition is:
Chlorella inoculum density is 0.155g/L; The composition of substratum comprises: NaNO 31.5g/L, K 2hPO 30.3g/L, K 2hPO 30.2g/L, MgSO 47H 2o 0.1g/L, CaCl 22H 2o 0.05g/L, C 6h 8o 70.01g/L, FeC 6h 5oNH 4oH 0.01g/L, EDTANa 20.05g/L, A5 liquid microelement 1mL/L, all the other are water, and wherein A5 solution formula is H 3bO 32.86g/L, MnCl 24H 2o 1.86g/L, ZnSO 47H 2o 0.22g/L, Na 2moO 42H 2o 0.39g/L, CuSO 45H 2o 0.08g/L and Co (NO 3) 26H 2o 0.05g/L; Culture temperature 25--28 DEG C.Result display as shown in Figure 1.
Ventilation autotrophy is cultivated
In the culturing bottle of 1000mL, autotrophy of ventilating under common BG11 substratum is cultivated, and culture condition is:
Chlorella inoculum density is 1.65g/L; The composition of Heterotrophic culture base comprises: NaNO 31.5g/L, K 2hPO 30.3g/L, K 2hPO 30.2g/L, MgSO 47H 2o 0.1g/L, CaCl 22H 2o 0.05g/L, C 6h 8o 70.01g/L, FeC 6h 5oNH 4oH 0.01g/L, EDTANa 20.05g/L, A5 liquid microelement 1mL/L, all the other are water, and wherein A5 solution formula is H 3bO 32.86g/L, MnCl 24H 2o1.86g/L, ZnSO 47H 2o 0.22g/L, Na 2moO 42H 2o 0.39g/L, CuSO 45H 2o 0.08g/L and Co (NO 3) 26H 2o 0.05g/L; Culture temperature 25--28 DEG C, blowing air amount 1.0L/min, add the silicone antifoam agent of 0.05%.Result display as shown in Figure 1.
Additional carbon glucose ventilation Heterotrophic culture
In the culturing bottle of 1000mL, additional carbon source glucose ventilation Heterotrophic culture under common BG11 substratum, culture condition is:
Chlorella inoculum density is 0.1693g/L; The composition of Heterotrophic culture base comprises: glucose 20g/L, NaNO 31.5g/L, K 2hPO 30.3g/L, K 2hPO 30.2g/L, MgSO 47H 2o 0.1g/L, CaCl 22H 2o 0.05g/L, C 6h 8o 70.01g/L, FeC 6h 5oNH 4oH 0.01g/L, EDTANa 20.05g/L, A5 liquid microelement 1mL/L, all the other are water, and wherein A5 solution formula is H 3bO 32.86g/L, MnCl 24H 2o 1.86g/L, ZnSO 47H 2o 0.22g/L, Na 2moO 42H 2o 0.39g/L, CuSO 45H 2o 0.08g/L and Co (NO 3) 26H 2o 0.05g/L; Culture temperature 25--28 DEG C, blowing air amount 1.0L/min, add the silicone antifoam agent of 0.05%.Result display as shown in Figure 1.
As shown in Figure 1, adopt the chlorella of common quiescent culture, at the 12nd day, still chlorella was in exponential phase of growth; Adopt the chlorella that ventilation autotrophy is cultivated, the chlorella of the 10th day ventilation autotrophy cultivation enters stationary phase, cultivates end subsequently; The chlorella of employing additional carbon glucose ventilation Heterotrophic culture, the chlorella cultivating the cultivation of the 2nd day ventilation autotrophy and Heterotrophic culture after starting enters exponential phase of growth, and the chlorella of the 6th day ventilation Heterotrophic culture enters stationary phase, cultivates end subsequently.
Embodiment 2
Additional carbon glucose ventilation Heterotrophic culture, filters fluid infusion in early stage exponential phase and cultivates.
In the culturing bottle of 1000mL, additional carbon source glucose ventilation Heterotrophic culture under common BG11 substratum, culture condition is:
Chlorella inoculum density is 0.1693g/L; The composition of Heterotrophic culture base comprises: glucose 20g/L, NaNO 31.5g/L, K 2hPO 30.3g/L, K 2hPO 30.2g/L, MgSO 47H 2o 0.1g/L, CaCl 22H 2o 0.05g/L, C 6h 8o 70.01g/L, FeC 6h 5oNH 4oH 0.01g/L, EDTANa 20.05g/L, A5 liquid microelement 1mL/L, all the other are water, and wherein A5 solution formula is H 3bO 32.86g/L, MnCl 24H 2o 1.86g/L, ZnSO 47H 2o 0.22g/L, Na 2moO 42H 2o 0.39g/L, CuSO 45H 2o 0.08g/L and Co (NO 3) 26H 2o 0.05g/L; Culture temperature 25--28 DEG C, blowing air amount 1.0L/min, add the silicone antifoam agent of 0.05%;
Started at the 2nd day, filter out the chlorella of 1/2 every day, then supplement equivalent nutritive medium, continue to cultivate, wherein, the composition of described extra-nutrition thing comprises: glucose 4-5g, NaNO 30.3g, K 2hPO 30.05g, K 2hPO 30.01g, MgSO 47H 2o 0.02g, CaCl 22H 2o 0.01g, C 6h 8o 70.001g, FeC 6h 5oNH 4oH 0.001g, EDTANa 20.01g, A5 liquid microelement 0.5mL.In continuation culturing process, within every 3 days, injecting the antibiotics penicillin sodium salt 1mL of 300mg/L, every 15 days is a production cycle.Result display as shown in Figure 2.
Additional carbon glucose ventilation Heterotrophic culture, filters fluid infusion in mid-term exponential phase and cultivates.
This experiment is substantially identical with the experiment of " additional carbon glucose ventilate Heterotrophic culture, filters fluid infusion cultivate in early stage exponential phase ", and difference is, at the 3rd day ~ the 4th day, filters out the chlorella of 1/2 every day, then supplements equivalent nutritive medium, continues cultivation.Result display as shown in Figure 2.
Additional carbon glucose ventilation Heterotrophic culture, filters fluid infusion at late exponential phase and cultivates.
This experiment is substantially identical with the experiment of " additional carbon glucose ventilate Heterotrophic culture, filters fluid infusion cultivate in early stage exponential phase ", and difference is, starts, filter out the chlorella of 1/2 every day at the 5th day, then supplements equivalent nutritive medium, continues cultivation.Result display as shown in Figure 2.
As shown in Figure 2, additional carbon glucose ventilation Heterotrophic culture, filter fluid infusion in early stage exponential phase and cultivate, in nutrient solution, the biomass of chlorella is stabilized between 1.9-2.1g/L; Filter fluid infusion in mid-term exponential phase to cultivate, in nutrient solution, biomass a few days ago slightly the declining in sampling feed supplement of chlorella, is then stabilized in about 4.6g/L; Filter fluid infusion at late exponential phase to cultivate, in nutrient solution, the biomass of chlorella is in the decline a few days ago had by a relatively large margin of sampling feed supplement, is then stabilized in about 3.8-4.1g/L.
The cultural method of chlorella provided by the invention, in Heterotrophic culture process, is filtered by silk and takes out part frustule, then supplement the nutrient solution of certain volume, makes frustule continue to cultivate in Heterotrophic culture bottle.Adopt the method, substantially increase the cultivation productive rate of chlorella, ensure that the vigor of frustule, improve the utilization ratio of carbon source simultaneously, be conducive to mass-producing heterotrophism and produce; In addition, by filtering and sampling, remain the nutritive ingredient in nutrient solution, save production cost, avoid waste and environmental pollution, the easy optimal control of culture condition.
The present invention is not limited to above-mentioned embodiment, and for those skilled in the art, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications are also considered as within protection scope of the present invention.The content be not described in detail in this specification sheets belongs to the known prior art of professional and technical personnel in the field.

Claims (10)

1. a cultural method for chlorella, its step comprises: carry out Heterotrophic culture to chlorella, in mid-term exponential phase of growth, filter out some beads algae, then supplementary portion divides nutrition and water, continues to cultivate.
2. the cultural method of chlorella as claimed in claim 1, is characterized in that: at middle exponential growth, the operation that every day carries out " filter out some beads algae, then supplementary portion divides nutrition and water, continue to cultivate " once, until culture cycle terminates.
3. the cultural method of chlorella as claimed in claim 1, is characterized in that: the composition of described Heterotrophic culture liquid comprises: glucose 20g/L, NaNO 31.5g/L, K 2hPO 30.3g/L, K 2hPO 30.2g/L, MgSO47H 2o 0.1g/L, CaCl 22H 2o 0.05g/L, C 6h 8o 70.01g/L, FeC 6h 5oNH 4oH0.01g/L, EDTANa 20.05g/L, A5 liquid microelement 1mL/L, all the other are water, and wherein A5 liquid microelement composition comprises H 3bO 32.86g/L, MnCl 24H 2o 1.86g/L, ZnSO 47H 2o 0.22g/L, Na 2moO 42H 2o 0.39g/L, CuSO 45H 2o 0.08g/L and Co (NO 3) 26H 2o 0.05g/L.
4. the cultural method of chlorella as claimed in claim 1, is characterized in that: described Heterotrophic culture liquid pH value is 7.2 ~ 8.0.
5. the cultural method of chlorella as claimed in claim 1, is characterized in that: the composition of described extra-nutrition thing comprises in every 1000mL nutrient solution and adds glucose 4-5g, NaNO 30.3g, K 2hPO 30.05g, K 2hPO 30.01g, MgSO 47H 2o 0.02g, CaCl 22H 2o 0.01g, C 6h 8o 70.001g, FeC 6h 5oNH 4oH 0.001g, EDTANa 20.01g, A5 liquid microelement 0.5mL.
6. the cultural method of chlorella as claimed in claim 1, is characterized in that: in described Heterotrophic culture process, the inoculum size of chlorella is the volume accounting of nutrient solution 10%.
7. the cultural method of chlorella as claimed in claim 1, is characterized in that: culture temperature 25--28 DEG C in described Heterotrophic culture process, blowing air amount 1.0L/min, add the silicone antifoam agent of 0.05%.
8. the cultural method of chlorella as claimed in claim 1, is characterized in that: described mid-term exponential phase of growth is the 3rd day to the 4th day from inoculation culture.
9. the cultural method of chlorella as claimed in claim 1, is characterized in that: filter out the chlorella of 1/2 at every turn.
10. the cultural method of chlorella as claimed in claim 1, is characterized in that: in continuation culturing process, within every 3 days, injecting the antibiotics penicillin sodium salt 1mL of 300mg/L, every 15 days is a production cycle.
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Cited By (7)

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Publication number Priority date Publication date Assignee Title
CN105524836A (en) * 2016-02-29 2016-04-27 通威股份有限公司 Method for economically and efficiently cultivating chlorella
CN106771111A (en) * 2016-12-05 2017-05-31 唐肖近 A kind of chronotoxicity microplate analysis method
CN106867909A (en) * 2017-04-13 2017-06-20 河南师范大学 A kind of cultural method of very thin Euglena
CN106916750A (en) * 2015-12-28 2017-07-04 国家开发投资公司 A kind of method for preventing and/or suppressing chrysophyceae pollution in chlorella cultivating system
CN109797104A (en) * 2019-02-22 2019-05-24 中国科学院水生生物研究所 A kind of chlorella Chlorella zofingiensis heterotrophism high-density cultivation method
CN112111406A (en) * 2020-10-26 2020-12-22 安徽天邦生物技术有限公司 Culture medium for large-scale culture of chlorella and culture method thereof
CN115791716A (en) * 2022-09-19 2023-03-14 河北科技大学 Method for detecting biological toxicity of Chlorella pyrenoidosa by fluorescence

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106916750A (en) * 2015-12-28 2017-07-04 国家开发投资公司 A kind of method for preventing and/or suppressing chrysophyceae pollution in chlorella cultivating system
CN106916750B (en) * 2015-12-28 2020-05-19 国投生物科技投资有限公司 Method for preventing and/or inhibiting algae pollution in chlorella culture system
CN105524836A (en) * 2016-02-29 2016-04-27 通威股份有限公司 Method for economically and efficiently cultivating chlorella
CN106771111A (en) * 2016-12-05 2017-05-31 唐肖近 A kind of chronotoxicity microplate analysis method
CN106867909A (en) * 2017-04-13 2017-06-20 河南师范大学 A kind of cultural method of very thin Euglena
CN109797104A (en) * 2019-02-22 2019-05-24 中国科学院水生生物研究所 A kind of chlorella Chlorella zofingiensis heterotrophism high-density cultivation method
CN112111406A (en) * 2020-10-26 2020-12-22 安徽天邦生物技术有限公司 Culture medium for large-scale culture of chlorella and culture method thereof
CN115791716A (en) * 2022-09-19 2023-03-14 河北科技大学 Method for detecting biological toxicity of Chlorella pyrenoidosa by fluorescence
CN115791716B (en) * 2022-09-19 2023-07-21 河北科技大学 Method for detecting plowing biotoxicity by using chlorella pyrenoidosa fluorescence

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Application publication date: 20150812