Method for improving biomass accumulation of haematococcus pluvialis

Abstract

The invention discloses a method for improving biomass accumulation of haematococcus pluvialis, and belongs to the technical field of bioengineering. The method specifically comprises the following steps: firstly, culturing a haematococcus pluvialis seed solution by using an A30 culture medium; and then culturing haematococcus pluvialis in an A30 culture medium added with methanol. Finally, algae cells in the culture solution are collected, and the biomass of haematococcus pluvialis is detected. The culture method of the haematococcus pluvialis is simple and effective, and when the methanol concentration is 0.2%, the biomass of the haematococcus pluvialis is increased by 78% compared with that of a single A30 group; by means of the culture method, the biomass of the haematococcus pluvialis in the green algae stage can be remarkably improved.

Description

Technical Field

[0001] The invention relates to a method for improving biomass accumulation of Haematococcus pluvialis, and belongs to the technical field of bioengineering. Background Art

[0002] Astaxanthin (3,3′-dihydroxy-β, β-carotene-4,4′-dione) is a red keto carotenoid, known as a "super antioxidant" for its super high antioxidant capacity. It is widely used in industries such as medicine, cosmetics, food and aquaculture. In addition, the antioxidant capacity of astaxanthin also shows potential in the prevention and treatment of many diseases, such as anti-inflammatory, anti-cancer and anti-cardiovascular diseases. At present, the main sources of astaxanthin are chemical synthesis and biosynthesis. Due to the high cost of chemical synthesis, the low antioxidant capacity of synthetic astaxanthin and its unstable structure, it has been clearly stipulated that chemically synthesized astaxanthin shall not enter the health care product market. The structure of biosynthesized natural astaxanthin is a trans structure with high stability, and its food safety has been widely accepted by relevant departments and consumers. The demand for large-scale production of natural astaxanthin is becoming more and more urgent.

[0003] Haematococcus pluvialis is the main producer of natural astaxanthin. Under certain conditions, the accumulation of astaxanthin in Haematococcus pluvialis can reach up to 5% of its dry weight.

[0004] At present, a strain of Haematococcus pluvialis H7 that is resistant to high temperature and alkali has been discovered. How to increase the biomass accumulation of this strain of Haematococcus pluvialis is a technical problem that needs to be solved urgently, because the increase in biomass accumulation will also increase the production of astaxanthin. Summary of the invention

[0005] In order to improve the biomass accumulation of Haematococcus pluvialis H7, the present invention provides a method for improving the biomass accumulation of Haematococcus pluvialis, which specifically comprises the following steps:

[0006] (1) Cultivate Haematococcus pluvialis H7 in A30 medium until the logarithmic phase.

[0007] (2) The Haematococcus pluvialis H7 obtained by culturing in step (1) is added to A30 medium containing methanol for culturing.

[0008] Preferably, the Haematococcus pluvialis is Haematococcus pluvialis H7, with a preservation number of CGMCC No. 41186 and a taxonomic name of Haematococcus pluvialis.

[0009] Preferably, the formula of A30 culture medium per liter in step (1) comprises: 590-605 mg sodium nitrate, 76-84 mg magnesium sulfate, 16-19 mg potassium dihydrogen phosphate, 9-11 mg sodium carbonate, 4.5-5.5 mg disodium ethylenediaminetetraacetic acid, 2.5-3.5 mg ferric chloride, 0.78-0.82 mg boric acid, 0.28 mg manganese chloride, 0.021 mg ammonium nitrate, 0.1 mg sodium molybdate, 0.079 mg copper sulfate, 0.222 mg zinc sulfate, 0.1 mg vitamin B1, and 0.00025 mg vitamin B12.

[0010] Preferably, the volume of methanol added to the A30 culture medium in step (2) is 0.1% to 0.25% of the volume of the A30 culture medium.

[0011] Preferably, the culture conditions of step (1) and step (2) are: the ambient temperature is 24-26°C, the light intensity is 35 μmol m -2 s -1 , the sterile air ventilation volume is 1vvm.

[0012] Beneficial effects of the present invention

[0013] (1) The present invention can significantly increase the biomass of Haematococcus pluvialis H7 by culturing it in A30 medium supplemented with methanol. This culturing method significantly promotes the growth of Haematococcus pluvialis H7 and improves the biomass accumulation of Haematococcus pluvialis H7. When the methanol concentration is 0.2%, the biomass of Haematococcus pluvialis H7 on the seventh day reaches 1.3 g / L, which is 78% higher than that of the control group. The biomass yield is 0.417 g / L / day, which is 25.23% higher than that of the control group.

[0014] (2) The process of the present invention is simple, easy to operate, efficient, and easy to produce in factories.

[0015] (3) This method creates an optimal environment for the growth of Haematococcus pluvialis by precisely controlling culture conditions (such as temperature, light intensity, ventilation, etc.). This optimization helps to improve the growth rate and quality of the algae.

[0016] (4) The implementation of this method will help improve the production efficiency of Haematococcus pluvialis H7 and is expected to reduce dependence on natural resources and environmental impact, thereby promoting environmental sustainability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The present invention provides a flow chart of a method for improving biomass accumulation of Haematococcus pluvialis H7 by using methanol.

[0018] Figure 2This is a graph showing the effect of different concentrations of methanol on the biomass of Haematococcus pluvialis H7 provided by an embodiment of the present invention.

[0019] Figure 3 3 is a graph showing the effect of different concentrations of methanol on the biomass yield of Haematococcus pluvialis H7 provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the described contents.

[0021] Unless otherwise specified, all reagents used in the present invention are of conventional commercially available analytical grade.

[0022] Preparation before the experiment

[0023] (1) Culture medium preparation

[0024] Prepare A30 medium according to the composition in Table 1.

[0025] Table 1 shows the composition and content of A30 medium

[0026] Serial number name Chemical formula Standard / ton 1 Sodium nitrate <![CDATA[NaNO 3 ]]> 600g 2 Magnesium sulfate heptahydrate <![CDATA[MgSO 4 ·7H 2 Oh]]> 80g 3 Potassium dihydrogen phosphate <![CDATA[KH 2 AFTER 4 ]]> 18g 4 Sodium carbonate <![CDATA[Na 2 CO 3 ]]> 10g 5 Disodium EDTA <![CDATA[Na 2 EDTA]]> 5g 6 Ferric chloride hexahydrate <![CDATA[FeCl 3 ·6H 2 O]]> 3g 7 Boric acid <![CDATA[H 3 BO 3 ]]> 0.8g 8 Manganese chloride (tetrahydrate) <![CDATA[MnCl 2 ·4H 2 O]]> 0.28g 9 Cobalt nitrate (hexahydrate) <![CDATA[Co(NO 3 , 2 ·6H 2 O]]> 0.021g 10 Sodium molybdate (dihydrate) <h2 style=";text-align:left;direction:ltr"><![CDATA[Na <h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> MoO<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> 2H<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> O]]><h2 style=";text-align:left;direction:ltr"> 0.1g 11 Copper sulfate pentahydrate <![CDATA[CuSO 4 ·5H 2 Oh]]> 0.079g 12 Zinc sulfate heptahydrate <![CDATA[ZnSO 4 ·7H 2 Oh]]> 0.222g 13 Vitamin B1 VB1 0.1g 14 Vitamin B12 VB12 0.00025g

[0027] Example 1

[0028] In the case where the methanol concentration provided in the embodiment of the present invention is 0.1%, the biomass of Haematococcus pluvialis H7 is detected and analyzed, and the specific steps are as follows:

[0029] (1) Cultivating seed solution using A30 medium: Using 2.5 L of A30 medium, culture Haematococcus pluvialis H7 in a 3 L round-bottom flask, and wait for Haematococcus pluvialis H7 to grow to the logarithmic growth phase; wherein the culturing environment temperature is 24-26 ° C, the light intensity is 35 μmol m- 2 s- 1 , the ventilation volume of sterile air is 1vvm.

[0030] (2) Cultivating Haematococcus pluvialis using a medium containing methanol: Using A30 medium as the base medium, methanol was added to the A30 medium, and the volume of methanol added was 0.1% of the volume of the A30 medium (for example, 1 mL of methanol was added to 1 L of A30 culture medium), and the Haematococcus pluvialis H7 obtained by culturing in step (1) was added to ensure that the inoculation amount of Haematococcus pluvialis H7 was 5×10 4 / ml; Sterile air is continuously introduced and cultured under a certain temperature and light intensity to obtain the final culture solution. The culture environment temperature is 24-26°C and the light intensity is 35μmol m- 2 s- 1 , passing 0.5% (volume) CO 2of sterile air with a ventilation volume of 1vvm.

[0031] (3) Determine the biomass of Haematococcus pluvialis in the culture solution obtained in step (2) by dry weight method: The biomass of Haematococcus pluvialis H7 was determined on the 1st, 3rd, 5th and 7th days of culture. 10 mL of the culture solution obtained in step (2) was taken in a sterile clean bench and transferred into a centrifuge tube. The culture solution was centrifuged at 3500 r / min for 3 minutes, and the supernatant was discarded. The algae cells were collected and placed in a -80°C refrigerator for overnight freezing. The cells were freeze-dried for 24 hours in a freeze dryer and weighed. The dry weight of the biomass of Haematococcus pluvialis H7 was finally measured. On the 7th day, the biomass was 0.957 g / L, which was 13.2% higher than that of Comparative Example 1 (see Figure 2 The biomass yield was 0.374 g / L / day, which was 12.3% higher than that of Example 1 (see Figure 3 ).

[0032] Example 2

[0033] In the case where the methanol concentration provided in the embodiment of the present invention is 0.15%, the biomass of Haematococcus pluvialis H7 is detected and analyzed, and the specific steps are as follows:

[0034] (1) Cultivating seed solution using A30 medium: Using 2.5 L of A30 medium, culture Haematococcus pluvialis H7 in a 3 L round-bottom flask, and wait for Haematococcus pluvialis H7 to grow to the logarithmic growth phase; wherein the culturing environment temperature is 24-26°C, and the light intensity is 35 μmol m -2 s -1 , the ventilation volume of sterile air is 1vvm.

[0035] (2) Cultivating Haematococcus pluvialis using a medium containing methanol: Using A30 medium as the base medium, methanol was added to the A30 medium, and the volume of methanol added was 0.15% of the volume of the A30 medium (for example, 1.5 mL of methanol was added to 1LA30 culture), and the Haematococcus pluvialis H7 obtained by culturing in step (1) was added to ensure that the inoculation amount of Haematococcus pluvialis H7 was 5×10 4 The culture medium is 24-26°C and the light intensity is 35 μmol m -2 s -1 , the sterile air ventilation volume is 1vvm.

[0036] (3) Determine the biomass of Haematococcus pluvialis in the culture solution obtained in step (2) by dry weight method: The biomass of Haematococcus pluvialis H7 was determined on the 1st, 3rd, 5th and 7th days of culture. 10 mL of the culture solution obtained in step (2) was taken in a sterile clean bench and transferred into a centrifuge tube. The culture solution was centrifuged at 3500 r / min for 3 minutes, and the supernatant was discarded. The algae cells were collected and placed in a -80°C refrigerator for overnight freezing. The cells were freeze-dried for 24 hours in a freeze dryer and weighed. The dry weight of the biomass of Haematococcus pluvialis H7 was finally measured. On the seventh day, the biomass was 1.02 g / L, which was 40.37% higher than that of Comparative Example 1 (see Figure 2 The biomass yield was 0.383 g / L / day, which was 15.01% higher than that of Example 1 (see Figure 3 ).

[0037] Example 3

[0038] When the methanol concentration provided in the embodiment of the present invention is 0.2%, the biomass of Haematococcus pluvialis H7 is detected and analyzed, and the specific steps are as follows:

[0039] (1) Cultivating seed solution using A30 medium: Using 2.5 L of A30 medium, culture Haematococcus pluvialis in a 3 L round-bottom flask, and wait for Haematococcus pluvialis H7 to grow to the logarithmic growth phase; wherein, the culturing environment temperature is 24-26°C, and the light intensity is 35 μmol m -2 s -1 , the ventilation volume of sterile air is 1vvm.

[0040] (2) Cultivating Haematococcus pluvialis using a medium containing methanol: Using A30 medium as the base medium, methanol was added to the A30 medium, and the volume of methanol added was 0.2% of the volume of the A30 medium (for example, 2 mL of methanol was added to 1LA30 culture), and the Haematococcus pluvialis H7 obtained by culturing in step (1) was added to ensure that the inoculation amount of Haematococcus pluvialis H7 was 5×10 4 The culture medium is 24-26°C and the light intensity is 35 μmol m -2 s -1 , the sterile air ventilation volume is 1vvm.

[0041] (3) Determine the biomass of Haematococcus pluvialis in the culture solution obtained in step (2) by dry weight method: The biomass of Haematococcus pluvialis H7 was determined on the 1st, 3rd, 5th and 7th days of culture. 10 mL of the culture solution obtained in step (2) was transferred into a centrifuge tube in a sterile clean bench. The supernatant was discarded after centrifugation at 3500 r / min for 3 minutes. The algae cells were collected and placed in a -80°C refrigerator for overnight freezing. The cells were freeze-dried for 24 hours in a freeze dryer and weighed. Finally, the dry weight of the biomass of Haematococcus pluvialis H7 was measured. On the third day, the biomass was 1.3 g / L, which was 78% higher than that of Comparative Example 1 (see Figure 2 The biomass yield was 0.417 g / L / day, which was 25.23% higher than that of Comparative Example 1 (see Figure 3 ).

[0042] Example 4

[0043] In the case where the methanol provided in the embodiment of the present invention is 0.25%, the biomass of Haematococcus pluvialis is detected and analyzed, and the specific steps are as follows:

[0044] (1) Cultivating seed solution using A30 medium: Using 2.5 L of A30 medium, culture Haematococcus pluvialis in a 3 L round-bottom flask, and wait for Haematococcus pluvialis H7 to grow to the logarithmic growth phase; wherein, the culturing environment temperature is 24-26°C, and the light intensity is 35 μmol m -2 s -1 , the ventilation volume of sterile air is 1vvm.

[0045] (2) Cultivating Haematococcus pluvialis using a medium containing methanol: Using A30 medium as the base medium, methanol was added to the A30 medium, and the volume of methanol added was 0.1% of the volume of the A30 medium (for example, 2.5 mL of methanol was added to 1 L of A30 culture medium). The Haematococcus pluvialis H7 obtained by culturing in step (1) was added to ensure that the inoculation amount of Haematococcus pluvialis H7 was 5×10 4 The culture medium is 24-26°C and the light intensity is 35 μmol m -2 s -1 , the sterile air ventilation volume is 1vvm.

[0046] (3) Determine the biomass of Haematococcus pluvialis in the culture solution obtained in step (2) by dry weight method: Take 10 mL of the culture solution obtained in step (2) and transfer it into a centrifuge tube in a sterile clean bench, centrifuge it at 3500 r / min for 3 minutes, discard the supernatant, collect the algae cells, freeze them in a -80°C refrigerator overnight, freeze them in a freeze dryer for 24 hours, and weigh them, and finally measure the dry weight of the biomass of Haematococcus pluvialis H7. On the seventh day, the biomass was 0.91 g / L, which was 25.23% higher than that of comparative example 1 (see Figure 2 The biomass yield was 0.366 g / L / day, which was 10% higher than that of Comparative Example 1 (see Figure 3 ).

[0047] Comparative Example 1

[0048] As a comparison, in this comparative example, the biomass of Haematococcus pluvialis was detected and analyzed without adding methanol. The specific steps are as follows:

[0049] (1) Cultivating seed solution using A30 medium: Using 2.5 L of A30 medium, culture Haematococcus pluvialis H7 in a 3 L round-bottom flask, and wait for Haematococcus pluvialis H7 to grow to the logarithmic growth phase; wherein the culturing environment temperature is 24-26°C, and the light intensity is 35 μmol m -2 s -1 , the ventilation volume of sterile air is 1vvm.

[0050] (2) Cultivating Haematococcus pluvialis using A30 medium: Using A30 medium as the base medium, the Haematococcus pluvialis H7 obtained by culturing in step (1) was inoculated into a 1 L conical flask photobioreactor to ensure that the inoculation amount of Haematococcus pluvialis H7 was 5×10 4 The culture medium is 24-26°C and the light intensity is 35 μmol m -2 s -1 , the sterile air ventilation volume is 1vvm.

[0051] (3) Determine the biomass of Haematococcus pluvialis in the culture solution obtained in step (2) by dry weight method: Take 10 mL of the culture solution obtained in step (2) and transfer it into a centrifuge tube in a sterile clean bench. Centrifuge it at 3500 r / min for 3 minutes, discard the supernatant, collect the algae cells, freeze them in a -80°C refrigerator overnight, freeze them in a freeze dryer for 24 hours, and weigh them. Finally, the dry weight of the biomass of Haematococcus pluvialis H7 is measured. On the seventh day, its biomass is 0.73 g / L (see Figure 2 ); biomass productivity was 0.333 g / L / day (see Figure 3 ).

[0052] Comparative Example 2

[0053] As a comparison, the difference between this comparative example and Example 3 is that the A30 culture medium is replaced by the BG11 culture medium, and the remaining steps are the same as those in Example 3. After culturing Haematococcus pluvialis H7, biomass accumulation cannot be achieved.

Claims

1. A method for increasing biomass accumulation of Haematococcus pluvialis, characterized in that: The specific steps include: (1) Cultivating Haematococcus pluvialis H7 in A30 medium until the logarithmic phase; (2) adding the Haematococcus pluvialis H7 obtained by culturing in step (1) into A30 medium containing methanol for culturing; The Haematococcus pluvialis is Haematococcus pluvialis H7, with a preservation number of CGMCC No.41186 and a taxonomic name of Haematococcus pluvialis.

2. The method for increasing biomass accumulation of Haematococcus pluvialis according to claim 1, characterized in that: The formula of the A30 culture medium per liter described in step (1) includes: 590-605 mg sodium nitrate, 76-84 mg magnesium sulfate, 16-19 mg potassium dihydrogen phosphate, 9-11 mg sodium carbonate, 4.5-5.5 mg disodium ethylenediaminetetraacetic acid, 2.5-3.5 mg ferric chloride, 0.78-0.82 mg boric acid, 0.28 mg manganese chloride, 0.021 mg ammonium nitrate, 0.1 mg sodium molybdate, 0.079 mg copper sulfate, 0.222 mg zinc sulfate, 0.1 mg vitamin B1, and 0.00025 mg vitamin B12.

3. The method for improving biomass accumulation of Haematococcus pluvialis according to claim 1, characterized in that: In step (2), the volume of methanol added to the A30 culture medium is 0.1% to 0.25% of the volume of the A30 culture medium.

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