Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the present invention is further described in detail with reference to the following specific examples, which should be noted that the examples are only specific descriptions of the present invention and should not be construed as limiting the present invention.

The chlorella pyrenoidosa used in the present invention is isolated from chlorella pyrenoidosa pieces or extracted from aquatic organisms in the waters of Jiangsu/nearby. However, the method of the present invention is not limited to the Chlorella pyrenoidosa pieces derived from Chlorella pyrenoidosa tablets exemplified in the present invention or Chlorella pyrenoidosa extracted from aquatic organisms in Jiangsu/nearby waters.

The method of the invention can be applied to other non-processed products⁶⁰CO-gamma ray mutagenesis and CO₂Domesticated chlorella pyrenoidosa, but not limited to chlorella pyrenoidosa.

Example 1

(1) Sterilizing BG11 culture medium at 121 deg.C for 30min, cooling to room temperature, packaging into 600mL round bottom glass bottles, inoculating microalgae, and performing first stage culture;

BG11 medium consisted of the following components: NaNO₃ 1500mg/L，K₂HPO₄·3H₂O 40mg/L，MgSO₄·7H₂O 75mg/L，Na₂CO₃ 20mg/L，CaCl₂27mg/L, 6mg/L citric acid monohydrate, 6mg/L ferric ammonium citrate, Na₂EDTA 1mg/L, trace element A₅1mL of solution, wherein, A₅The composition consists of the following components: h₃BO₃ 2.86mg/L，MnCl₂·4H₂O 1.81mg/L，ZnSO₄·7H₂O 0.222mg/L，CuSO₄·5H₂O 0.079mg/L，CoCl₂·6H₂O 0.050mg/L，Na₂MoO₄·2H₂O 0.39mg/L；

In this example, microalgae species are obtained by⁶⁰CO-gamma ray mutagenesis and tolerance of a volume fraction of 60% CO₂The chlorella pyrenoidosa is subjected to high concentration gradient CO₂Domesticating, wherein the concentration of inoculated microalgae is 450 mg/L.

The culture conditions of the whole culture process are as follows: introducing 15% by volume of CO₂As a carbon source, the gas supply rate was 0.1 v/v/m; the temperature is 27 ℃, the pH is 6.0 to 8.0, the illumination is 8500Lx, and the light-dark period is 16h to 8 h; wherein the gas supply rate is 0.1v/v/m, which means that 0.1 volume of gas is introduced into each volume of culture medium per minute, and the whole culture process comprises a first stage culture and a second stage culture.

After 9 days of culture, the nitrogen source and the phosphorus source in the culture medium are completely consumed, namely the content of the nitrogen source and the phosphorus source can not be detected by the instrument, and the first-stage culture is finished.

(2) In the second stage of culture, the phosphorus source is added into the first stage culture medium once, and added into the post-culture medium PO₄ ^3-The concentration of P is 70mg/L and the addition form is K₂HPO₄·3H₂O, and adding a certain amount of iron source in the form of FeCl₃·6H₂O, added in one time and added into the post-culture medium³⁺The concentration of (2) is 1.2-2.4mg/L, the culture process enters a nitrogen-deficiency, phosphorus-enrichment and iron-enrichment stage, and the culture is continued for 12 days.

Example 2

In this example, the microalgae species were not passed through⁶⁰CO-gamma-ray mutagenesis but with a high concentration gradient of CO₂The concentration of the inoculated microalgae of the domesticated chlorella pyrenoidosa is 450 mg/L.

The other steps of this example are the same as example 1.

Comparative example 1

In the second stage of culture, the phosphorus source is added into the culture medium once and added into PO in the post-culture medium₄ ^3-The concentration of P is 70mg/L and the addition form is K₂HPO₄·3H₂O, but no iron source is added, so that the culture process enters nitrogen deficiencyPhosphorus phase, continue culturing for 12 days.

The other steps of this comparative example were the same as example 1.

Comparative example 2

In the second stage of culture, adding phosphorus source into the culture medium, and adding PO into the post-culture medium₄ ^3-The concentration of P is 5mg/L and the addition form is K₂HPO₄·3H₂O, but not adding an iron source, leading the culture process to enter a nitrogen deficiency and phosphorus limitation stage, and continuing to culture for 12 days.

The other steps of this comparative example were the same as example 1.

Comparative example 3

In the second stage of culture, no phosphorus source or iron source is added into the culture medium, so that the culture process enters a nitrogen and phosphorus deficiency stage and continues to culture for 12 days.

The other steps of this comparative example were the same as example 1.

Data determination

The biomass, oil content, oil production efficiency and carbon fixation rate of the microalgae of examples 1-2 and comparative examples 1-3 were measured at total days of culture of 3, 9, 13, 21, respectively.

The biomass measuring method comprises the following steps: measuring biomass by a dry weight method, filtering a certain volume of algae liquid by using a filter membrane with a constant weight and a pore diameter of 0.45 mu m, placing the filter membrane in a drying oven at 105 ℃ until drying, then placing the filter membrane in a dryer to cool to room temperature, and calculating the weight difference before and after weighing to obtain the size of the biomass.

The method for measuring the oil content and the oil production efficiency comprises the following steps:

the calculated Fatty Acid Methyl Ester (FAMEs) content is taken as the oil content, and the FMAEs yield is taken as the oil production efficiency. Simple and rapid methods for FAMEs analysis were used. First, lipids are methyl esterified to fatty acid methyl esters. 20mg of dry algae powder is added into a glass tube with a cover and a volume of 10mL, 2mL of freshly prepared methyl esterification reagent (acetyl chloride/methanol, volume ratio of acetyl chloride to methanol is 1:9) is added, the cover is added and screwed, and then the mixture is placed in a water bath at 80 ℃ for reaction for 2.5 hours. After the reaction was complete, it was cooled to room temperature and 1mL of 1% aqueous NaCl solution and 2mL of n-hexane containing methyl benzoate (approximately 0.36mg/mL n-hexane) were added, with methyl benzoate as an internal standard. After shaking and centrifuging, layering, recovering the upper layer containing FAMEs to a GC sample tube, drying by anhydrous sodium sulfate, and then determining.

The total amount and composition of FAMEs was determined using a gas chromatograph Agilent 6890 using a flame ionization detector and a DB-FFAP model capillary column (30 m.times.0.25 mm.times.0.25 μm). The gas chromatography was run under the following conditions: the sample volume is 1 mu L; the split ratio is 1: 10; the air flow rate is 450mL/min, the H is 240 mL/min, and the carrier gas (N2) is 45 mL/min; the temperature of a sample inlet is 250 ℃; the temperature of the detector is 300 ℃; the initial furnace temperature was 140 ℃ and held for 2min, followed by a 10 ℃/min ramp to 240 ℃ and a 2min hold at 240 ℃. The FAMEs content was calculated from the peak area of each fatty acid methyl ester in the sample and the peak area of the internal standard. The yield of FAMEs was calculated as follows:

where t0 represents the initial time at the time of inoculation and t1 represents the nth day of culture.

CO₂The fixed rate calculation method comprises the following steps: analyzing the content of carbon in the algae by using an element analyzer, and calculating CO according to the following formula₂Fixed rate:

where t0 represents the initial time at the time of inoculation and t1 represents the nth day of culture.

The biomass, oil content, oil production efficiency and carbon fixation rate of microalgae under the two-stage culture conditions are shown in table 1. The biomass, oil content, oil production efficiency and carbon fixation rate of microalgae under the second stage culture conditions in examples 1-2 and comparative example 3 are shown in table 2.

TABLE 1 Biomass, oil content, oil production efficiency and carbon fixation rate of microalgae in example 1 and comparative examples 1-3 under two-stage culture conditions

TABLE 2 Biomass, oil content, oil production efficiency and carbon fixation rate of microalgae in examples 1-2 and comparative example 3 under the second stage culture conditions

In Table 1, N-P + represents: the culture medium type is nitrogen-deficient and phosphorus-rich, namely, after the first stage is finished, adding rich phosphorus source but not adding iron source into the culture medium, entering the stage of nitrogen deficiency and phosphorus-rich, corresponding to the culture of the second stage of the comparative example 1; N-Plim denotes: the culture medium type is nitrogen deficiency and phosphorus limitation, namely, after the first stage is finished, a small amount of phosphorus source is added, but no iron source is added, and the stage enters a nitrogen deficiency and phosphorus limitation stage, which corresponds to the second stage culture of the comparative example 2; N-P-represents: the type of the medium was nitrogen and phosphorus deficient, which means that after the first stage was completed, the medium was fed to the stage of nitrogen and phosphorus deficiency without adding a phosphorus source and an iron source, corresponding to the second stage of the culture of comparative example 3. N-P + Fe + represents: the culture medium type is nitrogen-deficient, phosphorus-enriched and iron-enriched, which means that after the first stage is finished, a phosphorus source and an iron source are added into the culture medium, and the culture enters a nitrogen-deficient, phosphorus-enriched and iron-enriched stage, corresponding to the culture of the second stage of the example 1; ND means no detection.

The nitrogen and phosphorus deficiency stage is an extension of the first stage in practice because no phosphorus source or iron source is added in the second stage.

As can be seen from Table 1, in the second stage of 4 days of culture, the nitrogen and phosphorus deficiency stage of comparative example 1 has biomass, oil content, oil production efficiency and CO in comparison with the nitrogen and phosphorus deficiency stage of comparative example 3 when the total number of days of culture is 13 days₂The fixed rates are 1.2, 2.3, and 1.9 times the latter, respectively; this indicates that the nitrogen-deficient and phosphorus-rich stage can improve the oil production efficiency and CO of the chlorella pyrenoidosa₂The rate is fixed.

Second stage culture for 4 daysThe total days of nutrient supply was 13 days, the nitrogen and phosphorus deficient iron rich phase of example 1 compared to the nitrogen and phosphorus deficient phase of comparative example 3, the biomass, oil content, oil production efficiency and CO of example 1₂The fixed rates were biomass, oil content, oil production efficiency and CO of comparative example 3, respectively₂1.5, 1.2, 3.3, and 1.9 times the fixed rate. This indicates that the nitrogen-deficient, phosphorus-rich and iron-rich stage can improve the oil production efficiency and CO of the chlorella pyrenoidosa₂The rate is fixed.

Example 1 compared to comparative example 1, the biomass, oil content, oil production efficiency and CO of example 1₂Fixed rate average ratio to biomass, oil content, oil production efficiency and CO of comparative example 1₂The high fixing rate shows that the phosphorus element and the iron element can synergistically improve the biomass, the oil content, the oil production efficiency and the CO of the chlorella pyrenoidosa₂The rate is fixed.

The second stage culture is carried out for 12 days, namely the total days of the culture is 21 days, the biomass in the stage of nitrogen deficiency, phosphorus and iron deficiency is 6100.00mg/L, the oil content is 44.21 percent, and CO is₂The carbon fixing rate is 481.85mg/L/day, and the oil production efficiency is 177.30 mg/L/day. The chlorella pyrenoidosa cultured by the method disclosed by the invention has high oil content, oil production efficiency and carbon fixation rate.

In the second stage of culture for 4 days or 12 days, that is, 13 days or 21 days, the biomass, oil content, oil production efficiency and CO of example 1 are compared with those of comparative examples 1 to 3₂The fixed rates are all compared with the biomass, oil content, oil production efficiency and CO of the comparative examples 1-3₂The high fixing rate shows that the phosphorus element and the iron element can synergistically improve the biomass, the oil content, the oil production efficiency and the CO of the chlorella pyrenoidosa₂The rate is fixed.

As can be seen from Table 2, in the second stage of culture for 4 days or 12 days, i.e., 13 days or 21 days in total, the nitrogen-deficient, phosphorus-enriched and iron-enriched stage of example 1 was⁶⁰The chlorella pyrenoidosa induced by CO-gamma ray is subjected to a 2-stage of nitrogen deficiency, phosphorus and iron enrichment without being subjected to⁶⁰The chlorella pyrenoidosa induced by CO-gamma ray is compared with the comparative example 3 at the stage of nitrogen deficiency and phosphorus deficiency⁶⁰CO-gamma ray mutagenized protein core globulesCompared with the algae, the biomass, the oil content, the carbon fixing rate and the oil production efficiency of the chlorella pyrenoidosa are higher in the example 1 and the example 2, which shows that the phosphorus element and the iron element can synergistically act to improve the biomass, the oil content, the carbon fixing rate and the oil production efficiency of the chlorella pyrenoidosa no matter whether the chlorella pyrenoidosa passes through or not⁶⁰And (3) performing CO-gamma ray mutagenesis.