CN112266938A - Method for improving chlorella pyrenoidosa fermented oil - Google Patents

Method for improving chlorella pyrenoidosa fermented oil Download PDF

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CN112266938A
CN112266938A CN202011121571.2A CN202011121571A CN112266938A CN 112266938 A CN112266938 A CN 112266938A CN 202011121571 A CN202011121571 A CN 202011121571A CN 112266938 A CN112266938 A CN 112266938A
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chlorella pyrenoidosa
culture medium
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李悦明
徐建春
徐炳政
韩萍
董婉
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Qingdao Kehai Biological Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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Abstract

The invention discloses a method for improving chlorella pyrenoidosa fermented oil, and belongs to the technical field of microalgae development. The method comprises the following steps: an activation culture step, namely inoculating the chlorella pyrenoidosa in a culture medium for activation culture to a logarithmic phase and using the chlorella pyrenoidosa as a seed solution; then inoculating the chlorella pyrenoidosa into a shake flask of a culture medium, and placing the chlorella pyrenoidosa in an illumination shaking table for culture; and finally, centrifuging, washing and freeze-drying the microbial cells after the culture is finished to obtain powder for measuring and evaluating the grease concentration, the total lipid content, the fat content and the yield of the chlorella pyrenoidosa biomass. The invention utilizes different culture medium compositions comprising carbon sources, nitrogen sources, carbon-nitrogen ratios, divalent metal ions and different culture conditions such as temperature, initial culture pH values and the like to influence the growth of chlorella cells and fat accumulation, measures and analyzes the fatty acid composition of the microbial oil, and the gas chromatography analysis result shows that the content of unsaturated fatty acid reaches about 73 percent.

Description

Method for improving chlorella pyrenoidosa fermented oil
Technical Field
The invention relates to the technical field of microalgae development, in particular to a method for fermenting oil by using chlorella pyrenoidosa.
Background
Microalgae is a kind of unicellular algae, and has the advantages of fast cell proliferation, short production period, no limitation of seasons and lands, rich sources of required culture medium, and similar oil components produced with vegetable oil, so that the development of oil resources by using microalgae is increasingly favored by people. Microalgae are considered to be a very potential biodiesel feedstock.
The chlorella pyrenoidosa is chlorella belonging to Chlorophyta, belongs to the general unicellular green algae, has extremely strong reproductive capacity, can produce a large amount of protein by utilizing sunlight, has the protein content of about 50 percent and is more than high-protein food such as beef, soybean and the like; also contains 10-30% of unsaturated fatty acid, 10-25% of carbohydrate, 8 essential amino acids, abundant multivitamins and minerals such as iron, zinc, calcium, potassium and the like. In addition, it has been found that chlorella contains a precious growth factor (CGF) which promotes the enhancement of physical constitution of the body, particularly children and the elderly, and enhances immune function.
In the prior art, research is carried out on the utilization of chlorella pyrenoidosa as a biological adsorbent for adsorbing heavy metals, for example, application number 201910856249.5 discloses a preparation method of the biological adsorbent for adsorbing heavy metals, wherein a culture medium with finished volume determination is added into a first conical flask, and then hydrochloric acid is dropwise added; placing the culture medium in a sterilization device for high-temperature sterilization, and cooling the sterilized culture medium; adding the stock solution into the cooled culture medium; after the chlorella pyrenoidosa seed liquid is inoculated, placing the second conical bottle in an illumination incubator for standing culture; shaking the second conical bottle 3 times every day at regular time, transferring the first generation algae liquid into the second generation algae liquid by the inoculation amount with the volume ratio of 1:1 after culturing for 3 days, repeating the transfer for 3 generations, successfully activating, diluting the activated chlorella pyrenoidosa algae liquid, transferring the algae seeds into a heavy metal biological adsorbent after expanding culture, and achieving the purpose that the adsorbent can simultaneously remove various heavy metals in water.
In the prior art, the chlorella pyrenoidosa is used for adsorbing heavy metals, but the application of the chlorella pyrenoidosa in the preparation of biological raw oil is only reported in the prior art.
Disclosure of Invention
The invention aims to provide a method for improving chlorella pyrenoidosa fermentation oil, the method utilizes chlorella pyrenoidosa to produce oil, greatly improves the yield of the chlorella pyrenoidosa oil, is simple and easy to implement, can reduce energy consumption and production cost, and has important application value in the aspect of utilizing oil-producing microorganisms to prepare biological raw oil.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for improving chlorella pyrenoidosa fermented oil sequentially comprises the following steps:
s1, activation culture:
inoculating chlorella pyrenoidosa into a first culture medium for activated culture, and culturing the chlorella pyrenoidosa to a logarithmic phase to be used as a seed solution;
the first culture medium comprises the following components in percentage by weight: glucose 10g/L, KNO3 0.08g/L，K2HPO4·3H2O 1.0g/L，MgSO4·7H2O 0.3g/L，FeSO4·7H20.003g/L of O, 15-10 g/L of vitamin B and 1mL/L of A5 trace element solution;
s2, culturing in a light shaking table:
inoculating the activated chlorella pyrenoidosa into a shake flask filled with a second culture medium, and placing the shake flask in a light shaking table for culture;
s3, and S2, sequentially centrifuging, washing and freeze-drying the microbial cells obtained after the culture in the light shaking table is finished.
In a preferred embodiment of the present invention, in step S1, the inoculum size is 10%, and the culture conditions are: the temperature is 20-30 ℃, the illumination intensity is 1500Lux, the rotating speed of a shaking table is 100-120 r/min, the fermentation culture period is 5-8 d, and the initial pH is 6.5.
In another preferred embodiment of the present invention, in step S2, the mass-to-volume ratio of C/N in the second medium is 16-64: 1.
Further, the mass-volume ratio of C/N in the second culture medium is 48: 1.
Further preferably, in step S2, the components and contents of the second culture medium are: peptone 0.08g/L, MgSO4·7H2O0.004g/L，FeSO4·7H2O 0.004g/L，CaCl2·2H2O0.04 g/L, citric acid 0.05g/L, EDTA-Fe0.001 g/L, NaCl0.0025 g/L, K2HPO4 0.0075g/L，KH2PO40.0175g/L, and 0.1mL of A1 solution; wherein the A1 liquid contains H3BO3 286mg、MnCl2·4H2O 181mg、NaMnO4·2H2O 3.9mg、ZnSO4·7H2O 22mg、CuSO4·5H2O7.9mg。
Further preferably, in step S2, the culture conditions are: the temperature is 25-30 ℃, the rotating speed of a shaking table is 140-160 r/min, the culture lasts for 10-14 days, and the initial pH is 5.3.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention provides a method for improving chlorella pyrenoidosa fermentation oil, which can obviously improve the oil production capacity of chlorella pyrenoidosa and can reasonably select a culture medium comprising a carbon source, a nitrogen source, a carbon-nitrogen ratio and divalent metal ions (Mg)2+And Fe2+) And the effects of culture adjustments such as temperature, initial pH of the medium, etc. on the growth of Chlorella pyrenoidosa cells and fat accumulation, and the fatty acid composition of microbial oils was determined and analyzed. According to detection, when the C/N in the culture medium is 48, the total fat content can reach 50.22%, and the yield is 333 mg/L/d.
The method is simple and easy to operate, can effectively reduce energy consumption and production cost, and has important application value in the aspect of preparing biological raw oil by utilizing oleaginous microorganisms.
Detailed Description
The invention provides a method for improving the fermentation oil of chlorella pyrenoidosa, and in order to make the advantages and technical scheme of the invention clearer and clearer, the invention is described in detail with reference to specific embodiments.
The raw materials required by the invention can be purchased from commercial sources.
Example 1:
a method for improving the yield of oil produced by fermenting chlorella pyrenoidosa comprises the following steps:
1) activation culture: inoculating the chlorella pyrenoidosa cells into a first culture medium, performing activated culture to a logarithmic growth phase, and using the first culture medium as a seed solution, wherein the first culture medium comprises: glucose 10g/L, KNO3 0.08g/L，K2HPO4·3H2O 1.0g/L，MgSO4.7H2O 0.3g/L，FeSO4.7H20.003g/L of O, 15-10 g/L of Vitamin B, and 1mL/L of A5 trace element liquid.
The culture conditions are as follows: the test was carried out in a 250mL triangular flask having a liquid volume of 100 mL. The inoculation amount is 10%, the culture temperature is 25 ℃, the initial pH is 6.5, the illumination intensity is 1500Lux, the rotating speed of a shaking table is 110r/min, and the fermentation culture period is 7 d.
2) Inoculating Chlorella pyrenoidosa into a second culture medium containing different glucose concentrations, wherein glucose is used as a carbon source, the concentration of fixed nitrogen source amino acid is 0.08g/L, and the culture medium contains different C/N ratios, namely 16, 32, 48 and 64. Initial algal cell count 5 x 105The inoculated shake flask of cells/mL is placed in a light incubator at the temperature of 28 ℃ and the rotating speed of 150 r/min. The light intensity was 1500Lux, the initial pH was 6.4 for 12 days of culture. The second culture medium is: the components of the culture medium are (g/L): urea 0.08, MgSO4·7H2O 0.004，FeSO4·7H2O 0.004，CaCl2·2H2O0.04, citric acid 0.05, EDTA-Fe0.001, NaCl0.0025, K2HPO4 0.0075，KH2PO40.0175, and 0.1mL of A1 solution. Wherein the formula of the A1 liquid is H3BO3 286mg，MnCl2·4H2O 181mg，NaMnO4·2H2O 3.9mg，ZnSO4·7H2O 22mg，CuSO4·5H2O 7.9mg。
3) And (3) centrifuging, washing and freeze-drying the microbial cells cultured in the step 2) to obtain powder for measuring and evaluating the dry weight of the biomass, the total lipid content and the fatty acid content.
Example 2:
a method for improving the yield of oil produced by fermenting chlorella pyrenoidosa comprises the following steps:
1) activation culture: inoculating the chlorella pyrenoidosa cells into a first culture medium, performing activated culture to a logarithmic growth phase, and using the first culture medium as a seed solution, wherein the first culture medium comprises: glucose 10g/L, KNO3 0.08g/L、K2HPO4·3H2O 1.0g/L、MgSO4.7H2O0.3g/L、FeSO4.7H20.003g/L of O, 0. 15-10 of Vitamin B and 1mL/L of A5 trace element liquid.
The culture conditions are as follows: the test was carried out in a 250mL triangular flask having a liquid volume of 100 mL. The inoculation amount is 10%, the culture temperature is 25 ℃, the initial pH is 6.5, the illumination intensity is 1500Lux, the rotating speed of a shaking table is 110r/min, and the fermentation culture period is 7 d.
2) Inoculating chlorella pyrenoidosa into culture media with different glucose concentrations, wherein sucrose is used as a carbon source, the concentration of amino acid in a fixed nitrogen source is 0.08g/L, and the culture media contain different C/N ratios of 16, 32, 48 and 64 respectively. Initial algal cell count 5 x 105The shake flask after cell/mL inoculation is placed in a light incubator at the temperature of 28 ℃ and the rotating speed of 150 r/min. The light intensity was 1500Lux, the initial pH was 5.3 for 12 days of culture. The second culture medium is: the components of the culture medium are (g/L): peptone 0.08, MgSO4·7H2O0.004，FeSO4·7H2O 0.004，CaCl2·2H2O0.04, citric acid 0.05, EDTA-Fe0.001, NaCl0.0025, K2HPO4 0.0075，KH2PO40.0175, and 0.1mL of A1 solution. Wherein the formula of the A1 liquid is H3BO3 286mg，MnCl2·4H2O181mg，NaMnO4·2H2O 3.9mg，ZnSO4·7H2O 22mg，CuSO4·5H2O 7.9mg。
3) And (3) centrifuging, washing and freeze-drying the microbial cells cultured in the step 2) to obtain powder for measuring and evaluating the dry weight of the biomass, the total lipid content and the fatty acid content.
Example 3:
a method for improving the yield of oil produced by fermenting chlorella pyrenoidosa comprises the following steps:
1) activation culture: inoculating the chlorella pyrenoidosa cells into a first culture medium, performing activated culture to a logarithmic growth phase, and using the first culture medium as a seed solution, wherein the first culture medium comprises: glucose 10g/L, KNO3 0.08g/L，K2HPO4·3H2O 1.0g/L，MgSO4.7H2O 0.3g/L，FeSO4.7H20.003g/L of O, 1mL/L of Vitamin B15-10 and A5 of trace element liquid.
The culture conditions are as follows: the test was carried out in a 250mL triangular flask having a liquid volume of 100 mL. The inoculation amount is 10%, the culture temperature is 25 ℃, the initial pH is 6.5, the illumination intensity is 1500Lux, the rotating speed of a shaking table is 110r/min, and the fermentation culture period is 7 d.
2) Inoculating Chlorella pyrenoidosa into a second culture medium containing different glucose concentrations, wherein glucose is used as a carbon source, the concentration of fixed nitrogen source amino acid is 0.08g/L, and the culture medium contains different C/N ratios, namely 16, 32, 48 and 64. Initial algal cell count 5 x 105Placing the inoculated shake flask of cells/mL into a light incubator, wherein the culture temperature is 28 ℃, and the rotation speed is 150 r/min. The light intensity was 1500Lux, the initial pH was 4.6 for 12 days of culture. The second culture medium is: the components of the culture medium are (g/L): glycine 0.08, MgSO4·7H2O 0.004，FeSO4·7H2O 0.004，CaCl2·2H2O0.04, citric acid 0.05, EDTA-Fe0.001, NaCl0.0025, K2HPO4 0.0075，KH2PO40.0175, and 0.1mL of A1 solution. Wherein the formula of the A1 liquid is H3BO3286mg，MnCl2·4H2O 181mg，NaMnO4·2H2O 3.9mg，ZnSO4·7H2O 22mg，CuSO4·5H2O 7.9mg。
3) And (3) centrifuging, washing and freeze-drying the microbial cells cultured in the step 2) to obtain powder for measuring and evaluating the dry weight of the biomass, the total lipid content and the fatty acid content.
The results are shown in Table 1, as measured by dry weight of biomass, total lipid content and fatty acid content.
TABLE 1
C/N Biomass Oil content Oil yield Oil yield
16 1.83 24.7 0.63 42.44
32 2.92 25.41 0.74 61.67
48 4.13 29.72 1.23 102.5
64 3.1 36.04 1.12 93.33
As can be seen from Table 1, the biomass gradually increased with increasing C/N and reached a maximum of 4.13g/L at a C/N of 48, and then slowly decreased with increasing C/N. The optimal C/N is 53.6. the results of gas chromatography analysis show that the main fatty acid components of the oil-producing chlorella pyrenoidosa comprise palmitic acid, oleic acid, linoleic acid, linolenic acid and stearic acid, and the contents of the fatty acid components are 23.4%, 32, 42%, 20.48%, 20.62% and 8.35%, respectively. Wherein the content of unsaturated fatty acid reaches about 73%. The composition of the biodiesel is similar to that of vegetable oil, so that the biodiesel can be used as a raw material of vegetable biodiesel.
The parts which are not described in the invention can be realized by taking the prior art as reference.
It should be noted that: any equivalents or obvious modifications thereof which may occur to persons skilled in the art and which are given the benefit of this description are deemed to be within the scope of the invention.

Claims (6)

1. A method for improving chlorella pyrenoidosa fermented oil is characterized by sequentially comprising the following steps:
s1, activation culture:
inoculating chlorella pyrenoidosa into a first culture medium for activated culture, and culturing the chlorella pyrenoidosa to a logarithmic phase to be used as a seed solution;
the first culture medium comprises the following components in percentage by weight: glucose 10g/L, KNO3 0.08g/L，K2HPO4·3H2O 1.0g/L，MgSO4·7H2O 0.3g/L，FeSO4·7H20.003g/L of O, 15-10 g/L of vitamin B and 1mL/L of A5 trace element solution;
s2, culturing in a light shaking table:
inoculating the activated chlorella pyrenoidosa into a shake flask filled with a second culture medium, and placing the shake flask in a light shaking table for culture;
s3, and S2, sequentially centrifuging, washing and freeze-drying the microbial cells obtained after the culture in the light shaking table is finished.
2. The method for improving the chlorella pyrenoidosa fermented oil according to claim 1, wherein the method comprises the following steps: in step S1, the inoculum size was 10%, and the culture conditions were: the temperature is 20-30 ℃, the illumination intensity is 1500Lux, the rotating speed of a shaking table is 100-120 r/min, the fermentation culture period is 5-8 d, and the initial pH is 6.5.
3. The method for improving the chlorella pyrenoidosa fermented oil according to claim 1, wherein the method comprises the following steps: in step S2, the mass-to-volume ratio of C/N in the second culture medium is 16-64: 1.
4. The method for improving the chlorella pyrenoidosa fermented oil according to claim 3, wherein the method comprises the following steps: the mass volume ratio of C/N in the second culture medium is 48: 1.
5. The method for improving the chlorella pyrenoidosa fermented oil according to claim 1, wherein the method comprises the following steps: in step S2, the components and contents of the second culture medium are: peptone 0.08g/L, MgSO4·7H2O 0.004g/L，FeSO4·7H2O 0.004g/L，CaCl2·2H2O0.04 g/L, citric acid 0.05g/L, EDTA-Fe0.001 g/L, NaCl0.0025 g/L, K2HPO40.0075g/L，KH2PO40.0175g/L, and 0.1mL of A1 solution; wherein the A1 liquid contains H3BO3 286mg、MnCl2·4H2O 181mg、NaMnO4·2H2O 3.9mg、ZnSO4·7H2O 22mg、CuSO4·5H2O 7.9mg。
6. The method for improving the chlorella pyrenoidosa fermented oil according to claim 1, wherein the method comprises the following steps: in step S2, the culture conditions were: the temperature is 25-30 ℃, the rotating speed of a shaking table is 140-160 r/min, the culture lasts for 10-14 days, and the initial pH is 5.3.
CN202011121571.2A 2020-10-20 2020-10-20 Method for improving chlorella pyrenoidosa fermented oil Pending CN112266938A (en)

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Application publication date: 20210126