CN107937276B - Method for promoting carbon sequestration growth of chlorella by mixing and regulating carbon dioxide and acetic acid - Google Patents
Method for promoting carbon sequestration growth of chlorella by mixing and regulating carbon dioxide and acetic acid Download PDFInfo
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Abstract
The invention relates to CO2A method for regulating and promoting carbon fixation growth of chlorella by mixing with acetic acid, which aims to solve the problems of high cost, relatively low biomass yield, easy infectious microbe contamination and low efficiency of fixing carbon dioxide in the culture process using acetic acid as a regulating and controlling means; and in the presence of CO2CO present during autotrophic cultivation as sole carbon source2Poor supplement effect, difficult pH control, insufficient algae cell morphology and the like. The method utilizes CO in a combined manner2The chlorella carbon fixation agent is mixed with acetic acid to regulate and promote carbon fixation growth of chlorella, so that biomass yield of chlorella and plumpness of chlorella cells can be obviously improved, carbon fixation efficiency of chlorella is improved, production cost is reduced, and probability of contamination of bacteria is reduced.
Description
Technical Field
The invention relates to the technical field of bioenergy and microalgae carbon sequestration, in particular to a method for promoting carbon sequestration growth of chlorella by mixing and regulating carbon dioxide and acetic acid.
Background
With the rapid development of the world economy, the use of a large amount of traditional fossil-based energy causes a large amount of environmental problems, and generates a large amount of greenhouse gases, which causes the aggravation of global climate change, the rise of air temperature, the deterioration of ecological environment, such as sea level rise, flooding, frequent occurrence of extreme weather, change of ecological system, and the like. Meanwhile, in spring and winter, China frequently has severe haze weather and severe air pollution, and the method also draws wide attention of governments and people. Therefore, the development of clean renewable energy and carbon emission reduction technologies has become a hot spot of current research. At present, CO2The contribution to the greenhouse effect is the largest, and the greenhouse effect accounts for more than 68 percent of the greenhouse effect gas emission of the whole atmosphere. Thus, CO2The emission reduction and control become the current concern of governments, business industries and academic circles of all countries in the worldOne of the focal points of (a).
In recent years, various carbon fixation methods such as physical, chemical, and biological methods have been developed. Among them, the physical and chemical methods belong to artificial carbon sequestration, the treatment cost is high and the sealing capacity is limited. The biological carbon sequestration method using photosynthesis of microalgae is considered to be an effective biological carbon sequestration method. The microalgae has the advantages of high oil content, high photosynthetic efficiency, short growth period and the like, and the microalgae has low requirements on environmental conditions and strong adaptability, and many microalgae can tolerate high-concentration CO2. The microalgae carbon sequestration culture technology can effectively fix CO2Can also reduce the emission of CO from microalgae2Organically combined with the production of biofuel from microalgae. It is estimated that about 1.82 tons of CO per ton of microalgal biomass can be fixed2Thus, increasing microalgal biomass production means increasing CO2The fixed amount of the carbon is fixed, so the carbon fixing culture mode of the microalgae is particularly important. Among these, chlorella is one of the most common and most widely used species of microalgae.
Chlorella (Chlorella) is a spherical unicellular freshwater alga of Chlorella genus, and has a diameter of 3-8 μm. Chlorella appears about 20 billion years ago, is one of the earliest lives on the earth, is a high-efficiency photosynthetic organism, mainly grows and breeds by photoautotrophy, and is extremely wide in distribution. Under natural conditions, chlorella individuals are small, and the chlorella is artificially cultured and massively propagated. The protein, fat and carbohydrate content in cells are high, and the fish feed has multiple vitamins, can be eaten and used as bait, and has good development prospect in the fields of food, medicine, genetic engineering, liquid fuel and the like.
The common culture mode for producing chlorella mainly utilizes acetic acid as a growth regulator for regulation and control culture, the acetic acid can be used as a pH regulator and a carbon source at the same time, the algae cells obtained by the culture mode are full in shape and easy to separate and collect, but the production mode has relatively low biomass yield, high production cost, easy contamination of mixed bacteria (the acetic acid belongs to an organic carbon source and is easy to be utilized by microorganisms) and low efficiency of fixing carbon dioxide. While using CO alone2Regulating and controlling microalgae to carry out carbon fixation culture in a CO mode2The supplement effect is betterPoor pH and difficulty in stable control, and the obtained algae cells are not full enough in shape, so that the difficulty in microalgae collection is increased.
Disclosure of Invention
Aiming at the problems, the invention provides CO2A method for regulating and promoting carbon fixation growth of chlorella by mixing with acetic acid, which aims to solve the problems of high cost, relatively low biomass yield, easy infectious microbe contamination and low efficiency of fixing carbon dioxide in the culture process using acetic acid as a regulating and controlling means; and in the presence of CO2CO present during autotrophic cultivation as sole carbon source2Poor supplement effect, difficult pH control, insufficient algae cell morphology and the like. The method utilizes CO in a combined manner2The chlorella is mixed with acetic acid to regulate and promote the growth of chlorella, the biomass yield and carbon fixation rate of chlorella can be obviously improved, the plumpness of chlorella cells is improved, the production cost is reduced, and the probability of mixed bacteria pollution is reduced.
In order to achieve the purpose, the technical scheme of the invention is that CO2The method for regulating and promoting carbon sequestration growth of chlorella by mixing with acetic acid comprises the following steps: CO is simultaneously utilized during the culture period2And acetic acid to perform regulated culture on the chlorella. That is, the process of the present invention uses CO2And acetic acid are combined to be used as a regulation and control mode and a culture means, and are used for simultaneously regulating the pH of the chlorella growth environment and supplementing a carbon source.
Further, said simultaneous utilization of CO2And acetic acid to perform regulated culture on the chlorella comprises the following steps: during the culture period, CO is introduced during the day2Culturing, and culturing with acetic acid at night. Namely, introducing CO in the growth culture of chlorella in the daytime2And use of Na2CO3/CO2Regulating pH value with a buffer system, and performing photoautotrophic culture; acetic acid is fed at night to regulate pH value, and the acetic acid is used as carbon source for heterotrophic culture.
The day time refers to a time period when the chlorella can use solar energy as energy for photosynthesis, namely, the time period when natural sunlight can provide enough energy for the chlorella to carry out photosynthesis; the evening refers to a time period when the chlorella cannot utilize solar energy as energy for photosynthesis, namely, a time period when natural sunlight cannot provide enough energy for the chlorella to carry out photosynthesis.
Alternatively, said simultaneous utilization of CO2And acetic acid to perform regulated culture on the chlorella comprises the following steps: in the culture period, CO is introduced for the first 2/3 periods2Culturing, and adding acetic acid for culturing at post 1/3 period.
In the present invention, the culture period of chlorella is 4 to 12 days (greatly different depending on the amount of inoculation), preferably 8 to 11 days, and more preferably 10 days.
Wherein the first culture mode is that CO is introduced in the daytime during the culture period2The culture is preferably carried out by feeding acetic acid at night.
Further, said simultaneous utilization of CO2And acetic acid to carry out regulated culture on the chlorella comprises the following steps:
(1) liquid culture of chlorella strain: adjusting the pH of the algae liquid culture medium to 6.5-7.5, wherein the illumination intensity is 13200Lux, the culture temperature is 25 +/-1 ℃, and the algae liquid culture medium is subcultured once every 10 days;
(2) carbon sequestration culture of chlorella: introducing CO into algae liquid culture medium in daytime25% strength air and CO2Mixing gas, and adding acetic acid at night to maintain pH of the algae solution at 6.5-7.5, preferably about 6.5.
In addition, in the liquid culture in the step (1), the liquid culture can be performed by static culture or shaking culture in a horizontal shaking table at 100-120 rpm.
Alternatively, the step (1) adopts chlorella algae seed solid culture: adding agar 1.5 wt% into the algae liquid culture medium, adjusting pH to 6.5-7.5, sterilizing, pouring into flat plate, performing streak culture on the flat plate with illumination intensity of 13200Lux and culture temperature of 25 + -1 deg.C, and subculturing once every 1 month.
Furthermore, the algae liquid culture medium adopts BG-11 culture medium. The BG-11 medium comprises the following components: NaNO3 1～2g/L，K2HPO4·3H2O 0.03～0.05g/L，MgSO4·7H2O 0.07～0.08g/L，CaCl2·7H2O 0.035～0.038g/L，Na2-EDTA 0.0005～0.0015g/L，Na2CO30.01-0.03 g/L, 0.005-0.007 g/L citric acid, 0.005-0.007 g/L ferric ammonium citrate, H3BO3 2.5～3mg/L，MnCl2·4H2O 1.5～2mg/L，ZnSO4·7H2O 0.1～0.3mg/L，Na2MoO4·2H2O 0.3～0.4mg/L，CuSO4·5H2O 0.07～0.08mg/L，CoCl2·6H2O 0.03～0.05mg/L。
Further, the BG-11 medium comprises the following components: NaNO3 1.5g/L，K2HPO4·3H2O 0.04g/L，MgSO4·7H2O 0.075g/L，CaCl2·7H2O 0.036g/L，Na2-EDTA 0.001g/L，Na2CO30.02g/L, citric acid 0.006g/L, ferric ammonium citrate 0.006g/L, H3BO3 2.86mg/L，MnCl2·4H2O 1.86mg/L，ZnSO4·7H2O 0.222mg/L，Na2MoO4·2H2O 0.39mg/L，CuSO4·5H2O 0.079mg/L，CoCl2·6H2O 0.04mg/L。
Further, the algae liquid culture medium or BG-11 culture medium is sterilized at 121 ℃ for 20min before use.
Further, CO is introduced into the algae liquid culture medium or BG-11 culture medium2When in use, CO is introduced in a mode of aeration through the nano microporous aeration pipe2。
Furthermore, when the algae liquid culture medium or BG-11 culture medium is supplemented with acetic acid, acetic acid is fed in a feeding mode, and the feeding speed is controlled to be based on the condition that the pH value of the culture medium is controlled to be 6.5-7.5.
The method of the invention has the following advantages:
the invention passes CO through specific steps and parameter conditions2Autotrophic culture as a carbon source and heterotrophic culture by taking acetic acid as the carbon source and a pH regulator are combined, and the problems of high cost, relatively low biomass yield, easy infectious microbe contamination and low efficiency of fixing carbon dioxide in the culture process by taking the acetic acid as a regulation and control means are solved; andin the presence of CO2CO present during autotrophic cultivation as sole carbon source2Poor supplement effect, difficult stable control of pH, insufficient algae cell morphology and the like.
The method utilizes CO in a combined manner2The chlorella carbon fixation agent is mixed with acetic acid to regulate and promote the carbon fixation growth of chlorella, thereby obviously improving the biomass yield of chlorella and the fullness of chlorella cells, improving the carbon fixation efficiency, reducing the production cost and reducing the probability of mixed bacteria pollution.
Drawings
FIG. 1 is a graph showing the variation of OD750 values (750nm absorbance) with days of chlorella cultured by the method of the present invention by CO passage during the day and chlorella obtained from a control group2Acetic acid was added overnight.
FIG. 2 is a graph showing the change of OD750 values (750nm absorbance) of chlorella cultured by the method of the present invention with respect to chlorella obtained from a control group in days, wherein the culturing method is performed by introducing CO for the first 2/3 cycles2And acetic acid is fed at the later 1/3 period.
FIG. 3 is a schematic view of the aeration mode of the nano-microporous aeration pipe used in the chlorella culture process.
FIG. 4 is a photograph showing the result of inverted fluorescence microscopy of chlorella obtained by culturing chlorella directly with BG-11 medium.
FIG. 5 shows the introduction of CO into BG-11 medium2And (4) carrying out an inverted fluorescence microscope microscopic examination result photo of the chlorella obtained by culturing.
FIG. 6 is a photograph showing the result of inverted fluorescence microscopy of Chlorella cultured in BG-11 medium with acetic acid.
FIG. 7 shows daytime CO injection in BG-11 medium2And (5) adding acetic acid at night to culture the chlorella, and carrying out microscope examination on the chlorella by using an inverted fluorescence microscope to obtain a result picture.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Examples
Culture of chlorella
A. Selecting chlorella algae, and originating from bioengineering and technology center of China university of Petroleum (east China).
B. Culturing chlorella strains:
(a) liquid culture conditions of chlorella strains are as follows: adopting BG-11 culture medium (formula shown in Table 1), adjusting pH to 6.5-7.5, light intensity to 13200Lux, culture temperature to 25 + -1 deg.C, standing for culture or placing in horizontal shaking table for shaking culture at 100-120 rpm, and subculturing once every 10 days; or
(b) Solid culture conditions of chlorella algae species: adding agar 1.5 wt% into BG-11 culture medium, adjusting pH to about 7.0, sterilizing, pouring into flat plate, performing streak culture on the flat plate with illumination intensity of 13200Lux and culture temperature of 25 + -1 deg.C, and subculturing once every 1 month.
C. Carbon sequestration culture of chlorella: with CO2In autotrophic culture using carbon source, BG-11 medium was continuously aerated with CO25% strength air and CO2Culturing the mixed gas in a nano-pore aeration pipe aeration mode; in heterotrophic culture using acetic acid as a carbon source, acetic acid is fed to BG-11 medium to maintain the pH of the algal solution at about 6.5; by using CO2Mixing with acetic acid to regulate chlorella growth, introducing CO in BG-11 culture medium during daytime25% strength air and CO2Mixing gas, wherein the aeration mode is a nano-pore aeration pipe aeration mode; acetic acid was added at night to maintain the pH of the algal solution at about 6.5 for cultivation.
Wherein CO is introduced into the culture medium or BG-112When in use, CO is introduced in a mode of aeration through the nano microporous aeration pipe2As shown in fig. 3.
TABLE 1 BG-11 Medium formulation
(it is stated that the total volume of the culture medium is up to 1000mL, agar is added to the solid culture medium in an amount of 1.5% by weight, and the solid culture medium is sterilized at 121 ℃ for 20min)
Second, determination of algal cell count
1mL of algae solution (after 3 days, the algae solution needs to be diluted by a culture medium, so that the reading of the cuvette does not exceed 1) is placed in the cuvette, the absorbance is measured at the wavelength of 750nm, and the number of the algae cells is positively correlated with OD 750.
The results are shown in FIGS. 1 and 2.
The cultivation method used in FIG. 1 is daytime CO supply2Acetic acid is added at night, wherein
Control group: chlorella vulgaris No. 1, naturally growing without addition of CO2And acetic acid; introducing CO into Chlorella vulgaris 225% strength air and CO2Mixing the gas; adding acetic acid into Chlorella vulgaris No. 3;
experimental groups: introducing CO into Chlorella vulgaris 4 in daytime25% strength air and CO2Mixing the gas, and adding acetic acid at night to maintain the pH of the algae solution at about 6.5.
The experimental results show that in CO2CO is introduced during daytime in mixed culture with acetic acid2The culture effect of acetic acid fed at night is better than that of CO fed alone2Or the growth rate of acetic acid fed independently is high and the effect is good.
The cultivation method used in FIG. 2 is that the CO is introduced for the first 2/3 cycles2Post 1/3 periodic feeding of acetic acid, wherein
Control group: chlorella vulgaris No. 1, naturally growing without addition of CO2And acetic acid; introducing CO into Chlorella vulgaris 225% strength air and CO2Mixing the gas; adding acetic acid into Chlorella vulgaris No. 3;
experimental groups: introducing CO at 2/3 period before Chlorella vulgaris No. 425% strength air and CO2Mixing the gas, and adding acetic acid to maintain the pH of the algae solution at about 6.5 after 1/3.
The experimental result shows that 2/3 periods are filled with CO before the chlorella is used2The culture scheme of post 1/3 cycle flowing acetic acid is more than that of direct CO flowing2The growth rate obtained by the culture was low, but higher than that of acetic acid fed alone.
Thirdly, taking a picture by an inverted fluorescence microscope
Morphological observation of algal cells: and (3) sucking 10 mu L of algae liquid by using a liquid transfer machine, dripping the algae liquid to the center of the glass slide, firstly contacting one side of the glass cover with a sample on the glass slide, then slowly putting down the glass cover to ensure that the glass cover is completely covered on the glass slide, taking the prepared water sealing piece to an inverted fluorescence microscope for observation, and observing by using a 100-time oil lens.
The results are shown in FIGS. 4-7. FIG. 4 is a photograph showing the result of inverted fluorescence microscopy of chlorella cultured directly with BG-11 medium, in which the concentration and number of chlorella cells are low and the form of chlorella cells is not full. FIG. 5 shows the introduction of CO into BG-11 medium2The result photo of the inverted fluorescence microscopy of the chlorella obtained by culturing is that the concentration and the number of the algae cells are obviously much higher than those in the picture 4, but most of the algae cells are not full in shape and have larger difference. FIG. 6 is a photograph showing the result of an inverted fluorescence microscope examination of chlorella cultured in BG-11 medium with acetic acid added thereto, and it is seen from the photograph that the concentration and number of algal cells are slightly higher and the morphology of algal cells is relatively full compared with those in FIG. 4. FIG. 7 shows daytime CO injection in BG-11 medium according to the invention2The result photo of the inverted fluorescence microscope microscopic examination of the chlorella cultured by adding acetic acid at night shows that compared with the picture 4, the concentration and the number of the chlorella cells are obviously much higher, and the forms of the chlorella cells are full and uniform, thereby meeting the requirements of industrial production.
Fourthly, measuring the yield and the growth rate of the biomass
Taking 50ml of cultured algae liquid, centrifuging 6000g for 20min, discarding supernatant, putting the remaining biomass into an oven for drying at 80 ℃, weighing the mass of the obtained biomass, and further calculating the mass of the biomass contained in 1L of algae liquid. The growth rate is the mass of the biomass obtained at the end divided by the number of days of cultivation. The results are shown in Table 2.
TABLE 2 Chlorella biomass production and growth rate
Daytime CO (carbon monoxide)2Acetic acid was added overnight.
As shown in Table 2, in the chlorella growth culture, CO was introduced by day2The scheme of adding acetic acid in a flowing way at night is used for culturing than that of introducing CO alone2Higher biomass yields and growth rates are obtained with or without acetic acid alone.
In addition, the chlorella is administered with CO for 2/3 cycles before use2The culture scheme of adding acetic acid in post 1/3 period is more than that of directly introducing CO2The biomass yield obtained by cultivation is low and slightly better than direct fed acetic acid, so the method is not an optimal scheme.
Summary of the experiments
The invention uses the culture medium to culture chlorella in the presence of CO2In the autotrophic culture process as a unique carbon source, the OD750 value of chlorella can reach 9.477, the biomass yield reaches 3.56g/L, the growth rate is 0.395g/L/d, the growth rate is high, the number of algae cells is large, but the general shape of the algae cells is small, the shape is not full, the centrifugal collection is not facilitated, and the difficulty of microalgae collection is increased; in the mixotrophic culture process using acetic acid as a carbon source, the OD750 value of chlorella can reach 7.938, the biomass yield reaches 2.196g/L, the growth rate is 0.244g/L/d, the growth rate is slow, but the algae cell morphology is full and easy to collect; in the utilization of CO2In the mixed regulation of acetic acid, the CO is introduced by daytime2The regulation and control mode of adding acetic acid at night has the advantages that the OD750 value of chlorella can reach 10.233, the biomass yield reaches 3.722g/L, the growth rate is 0.413g/L/d, the growth rate is high, the cell shape of chlorella is plump, the industrial production requirement is met, and the optimal scheme is provided.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (6)
1. A method for regulating and promoting carbon sequestration growth of chlorella by mixing carbon dioxide and acetic acid is characterized by comprising the following steps: CO is simultaneously utilized during the culture period2And acetic acid to perform regulated culture on the chlorella,
the method comprises the following steps: (1) liquid culture of chlorella strain: adjusting the pH of the algae liquid culture medium to 6.5-7.5, wherein the illumination intensity is 13200Lux, the culture temperature is 25 +/-1 ℃, and the algae liquid culture medium is subcultured once every 10 days;
(2) carbon sequestration culture of chlorella: introducing CO into the algae liquid culture medium in daytime25% strength air/CO2Mixing the gases and using Na2CO3/CO2Regulating pH value with a buffer system, and performing photoautotrophic culture; adding acetic acid into the algae liquid culture medium at night to regulate the pH value to be 6.5-7.5, and performing heterotrophic culture by taking the acetic acid as a carbon source;
the algal solution culture medium adopts a BG-11 culture medium, and the BG-11 culture medium comprises the following components: NaNO31～2g/L，K2HPO4·3H2O 0.03～0.05g/L，MgSO4·7H2O 0.07～0.08g/L，CaCl2·7H2O 0.035～0.038g/L，Na2-EDTA0.0005～0.0015g/L，Na2CO30.01-0.03 g/L, 0.005-0.007 g/L citric acid, 0.005-0.007 g/L ferric ammonium citrate, H3BO32.5～3mg/L，MnCl2·4H2O 1.5～2mg/L，ZnSO4·7H2O 0.1～0.3mg/L，Na2MoO4·2H2O 0.3～0.4mg/L，CuSO4·5H2O 0.07～0.08mg/L，CoCl2·6H2O 0.03～0.05mg/L。
2. The method of claim 1, wherein the CO-utilizing is performed simultaneously with CO2And acetic acid to perform regulated culture on the chlorella comprises the following steps: in the culture period, CO is introduced for the first 2/3 periods2Culturing, and adding acetic acid for culturing at post 1/3 period.
3. The method according to claim 1 or 2, wherein the cultivation period is 4 to 12 days.
4. The method according to claim 1 or 2, characterized in that CO is being utilized2When the chlorella is regulated and controlled to be cultured, CO is introduced into the culture medium in an aeration mode of the nano microporous aeration pipe2。
5. The method according to claim 1, wherein the liquid culture in step (1) is performed by static culture or shaking culture in a horizontal shaking table at 100-120 rpm.
6. The method of claim 1, wherein step (1) is carried out by solid culture of chlorella species: adding agar 1.5 wt% into the algae liquid culture medium, adjusting pH to 6.5-7.5, sterilizing, pouring into flat plate, performing streak culture on the flat plate with illumination intensity of 13200Lux and culture temperature of 25 + -1 deg.C, and subculturing once every 1 month.
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