Microalga with high lipid content and culture application thereof

Abstract

The present application relates to a strain of microalgae (Nannochloropsis gaditana) Micractinium sp. ) FSH-BY1, which has been preserved in the China General Microbiological Culture Collection Center on May 12, 2020, with a preservation number of CGMCC No. 19983. The present application also provides a culture method and application of the microalgae (Nannochloropsis gaditana) Micractinium sp. ) FSH-BY1. The microalgae provided by the present application can utilize carbon dioxide for rapid growth of light energy, obtain biomass rich in oil, and have the properties of tolerating SO2 and low temperature.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and bioenergy, and particularly relates to a kind of oil-rich microchaetae and its culture application. BACKGROUND

[0002] Since the 21st century, the development of economy and energy consumption are closely related, and energy security is a strategic problem for each country, so finding a clean, economic, efficient and renewable new energy has become an urgent problem to be solved, among which biological energy has been an important reserve technology direction, and each country maintains interest in it. Among many biological energy, microalgae has the advantages of high photosynthetic efficiency, strong environmental adaptability, high comprehensive utilization value, and does not compete with grain for land and people for grain. The fatty acids obtained from microalgae can be converted into fatty acid methyl ester, i.e. biodiesel, so the oil-rich microalgae is often considered as an ideal raw material for the third generation of biological fuel.

[0003] Microalgae cells accumulate oil, starch, protein, pigment and other components during growth, and the content of starch and glycogen in some algae accounts for 30% of the dry weight of cells. Some algae can have an oil content of up to 80% under specific culture conditions. The solar energy conversion efficiency of microalgae can reach 3.5%, which is a potential resource for producing pharmaceuticals and fine chemicals. How to effectively screen new algae species with high oil content, strong environmental adaptability, large-scale cultivation and low cost has become the focus of researchers in each country.

[0004] CN108660079A discloses a kind of microchaetae (Nannochloropsis sp.) 74C and its culture application. The microchaetae can produce high amount of triacylglycerol, can absorb carbon dioxide during culture, can be used for carbon sequestration, and has the characteristics of high heat value, low ash content and low sulfur content, and can be used as a raw material for producing biodiesel and edible oil. However, the culture temperature of the microchaetae 74C disclosed in the patent is 15-60℃, preferably 20-40℃, and it does not have low temperature resistance. Micractinium sp.

[0005] Temperature has a great influence on the growth and reproduction of microalgae. The optimal survival temperature of microalgae is usually 15-35℃, and low temperature will cause slow growth or even stop growth of microalgae. In addition, in cold weather in winter, microalgae need to consume more energy to grow. Therefore, it is necessary to select microalgae with low temperature resistance or multi-function to make it more suitable for industrial application. SUMMARY

[0006] In view of the shortcomings of the prior art, the present application provides an oil-rich microchaetae and its culture application. The microchaetae provided by the present application can utilize carbon dioxide for rapid growth of light energy, obtain oil-rich biomass, and has the properties of SO2 tolerance and low temperature resistance.​

[0007] The application provides a microalgae (Nannochloropsis) rich in oil and fat. Micractinium sp. The microalgae (Nannochloropsis) FSH-BY1 is preserved in the China General Microbiological Culture Collection Center on May 12, 2020, and has a preservation number of CGMCC No. 19983.

[0008] The application provides a microalgae (Nannochloropsis) rich in oil and fat. Micractinium sp. The 18S rDNA gene sequencing analysis result of the microalgae (Nannochloropsis) FSH-BY1 provided by the application is shown in a sequence table. Micractinium sp. According to sequence alignment, the 18S rDNA data of the microalgae (Nannochloropsis) FSH-BY1 is different from the published microalgae.

[0009] The application provides a microalgae (Nannochloropsis) rich in oil and fat. Micractinium sp. The microalgae (Nannochloropsis) FSH-BY1 provided by the application is green under a microscope, a single cell is spherical, the diameter is about 4-8 μm, and four cells are usually gathered into a square or tetrahedron, and sometimes eight cells are arranged into a spherical shape.

[0010] The application provides a microalgae (Nannochloropsis) rich in oil and fat. Micractinium sp. The microalgae (Nannochloropsis) FSH-BY1 provided by the application can tolerate a CO2 concentration of 40 v%, can tolerate a SO2 concentration of 0.04 v%, and can tolerate a low temperature of 5 ℃.

[0011] The application further provides a culture method of the microalgae (Nannochloropsis) FSH-BY1. Micractinium sp. In the culture method, the freshwater culture is any one of BG11 culture medium, SE culture medium or D1 culture medium.

[0012] In the culture method, the CO2-containing gas also contains SO2, and the SO2 concentration is not higher than 0.04 v%.

[0013] The application provides an application of the microalgae (Nannochloropsis) FSH-BY1 in fixing CO2.

[0014] The application provides an application of the microalgae (Nannochloropsis) FSH-BY1 in fixing CO2. Micractinium sp. The strain can grow by photoautotrophy using CO2, has a high CO2 fixing efficiency, and can tolerate a CO2 concentration of 40 v%, preferably 5 v%-30 v%.

[0015] The microalgae (Nannochloropsis) FSH-BY1 of the present application can be used for producing microalgae oil. Micractinium sp. The microalgae (Nannochloropsis) FSH-BY1 of the present application can be used for purifying waste gas or flue gas containing CO2 and SO2.The concentration of CO2 in the waste gas or flue gas is ≤30v%, and the concentration of SO2 is ≤0.04v%.

[0016] The microalgae (Nannochloropsis) FSH-BY1 of the present application can be used for purifying waste gas or flue gas containing CO2 and SO2.The concentration of CO2 in the waste gas or flue gas is ≤30v%, and the concentration of SO2 is ≤0.04v%. Micractinium sp. Compared with the prior art, the present application has the following beneficial effects:

[0017] (1) The microalgae (Nannochloropsis) FSH-BY1 selected by the present application can utilize CO2 to grow efficiently under autotrophic conditions, has high carbon fixation efficiency, and can alleviate the CO2 greenhouse effect problem caused by the current industrial society.

[0018] Micractinium sp. (2) The algal strain also has the function of tolerating SO2. When CO2 and SO2 exist simultaneously in the waste gas, the inhibition of SO2 on the photosynthesis of microalgae can be avoided when the microalgae grow using the waste gas, and the normal growth of the microalgae can be maintained.

[0019] (3) The algal strain also has the function of tolerating low temperature. At 5-15℃, the normal growth rate can still be maintained, and the cultivation cost can be significantly reduced in winter.

[0020] (4) After cultivation, the dry weight of the algal cells can reach more than 6g / L, and the total lipid content of the cells accounts for more than 45% of the dry weight of the cells, which is suitable for producing biodiesel.

[0021]

[0022] Biological material preservation instructions

[0023] The microalgae (Nannochloropsis) FSH-BY1 provided by the present application is preserved in the General Microbiological Center of the China Microorganism Strain Preservation Management Committee; the preservation number is CGMCC No. 19983; the preservation date is May 12, 2020; and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology. Micractinium sp. DETAILED DESCRIPTION The microalgae and its cultivation application of the present application will be further described in detail below in combination with specific embodiments. The embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0024]

[0025] ​The experimental methods in the following examples are all conventional methods in the art, unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores, unless otherwise specified. In the present application, v% is the volume fraction.

[0026] Example 1 Isolation and acclimation screening of microalgae FSH-BY1

[0027] (1) Obtaining of the starting algae strain: 150 mL of water sample was taken from Hunhe River in Fushun City, Liaoning Province in October 2015, the water sample was filtered with gauze to remove impurities, and 50 mL of the filtered water sample was inoculated into 200 mL of BG11 medium for enrichment culture. The light intensity for the culture was 5000 Lux, the temperature was 15°C, the light cycle was 24 h, and the light / dark time ratio was 14:10. After about half a month of culture, the culture medium was green. The water sample of the enrichment culture was diluted to 10 -5 times under sterile conditions and spread on a BG11 solid plate for culture. The light intensity for the culture was 5000 Lux, the temperature was 15°C, and after about 10 days of culture, green single algae colonies appeared on the plate. The single algae colonies were picked and cultured in a shake flask, the culture temperature was 15°C, and the light intensity was 5000 Lux. After 8 days of culture, microscopic observation was performed to determine whether it was a pure algae strain. If it was not a pure strain, the above steps were repeated until a pure culture algae strain was obtained. After repeated culture, a pure algae strain numbered FSH-B1 was obtained.

[0028] (2) CO2 acclimation culture: the pure algae strain cultured in the shake flask in step (1) was inoculated into a microalgae aeration culture device for acclimation culture. The light intensity was 5000 Lux, the temperature was 10°C, and the CO2 content in the gas was increased from 5v% to 40v% in steps of 5v%. The culture was ended after 8 days of culture, and the acclimation culture was repeated for 3 times.

[0029] (3) The algae liquid acclimated and cultured in step (2) was cultured to obtain a pure algae strain by the way of plate streaking, and the culture steps were the same as in (1).

[0030] (4) SO2 acclimation culture: the algae liquid of the logarithmic growth phase of the strain in step (3) was passed through a mixed gas with a CO2 content of 20v%. The light intensity was 5000 Lux, the temperature was 10°C, and the SO2 gas was injected into the mixed gas to culture the algae strain for SO2 tolerance. The SO2 content in the mixed gas was increased from 0.01% to 0.04% in steps, and the content was increased by 0.01% every two days during the culture. After the culture was ended, the acclimation culture was repeated for 3 times, and the SO2 tolerant algae liquid was harvested.

[0031] (5) The algae liquid domesticated in step (4) is cultured by the way of plate streaking to obtain pure algae species, and the culture step is the same as (1). After the culture, the larger algae are selected for shake flask culture to obtain the target algae strain, which is named Microalga FSH-BY1.

[0032] Example 2 Identification of the algae strain

[0033] The DNA of the obtained FSH-BY1 microalgae is extracted by the CTAB method, and the 18S rDNA gene is cloned. The obtained three positive clones are sent to Shanghai Shenguo Company for sequencing. The 18S rDNA gene sequencing analysis result is shown in the sequence table. The 18S rDNA sequence is registered in the Genbank database for Blast comparison. The result shows that it has the largest similarity with Micractinium sp. , and the BLASTn value is 2935, and the Max index value is 99.63%, so it can be determined that FSH-BY1 is Microalga Micractinium sp. Micractinium ).

[0034] Example 3 Culture application of Microalga FSH-BY1

[0035] The algae liquid of the logarithmic growth period of Microalga FSH-BY1 is inoculated in the BG11 culture medium for culture. The formula of the BG11 culture medium is shown in Table 1 and Table 2. The culture is carried out in a photobioreactor. The OD 690 of the culture liquid after inoculation is 0.25. The flue gas with a CO2 content of 40v% and a SO2 content of 0.04% is introduced from the bottom of the reactor. The light intensity during the culture is 8000Lux, the culture temperature is 15℃, the pH value is controlled at 7-8, the light cycle is 24h, the light and dark time ratio is 14:10, the culture time is 8 days, and the culture is ended. The algae liquid is collected by centrifugation, and the algae powder is measured after vacuum freeze-drying under the condition of-60℃ until the constant weight. The biomass is calculated, and the total lipid content is measured by the n-hexane: ethyl acetate method. The biomass yield of FSH-BY1 algae strain is 6.32g / L, and the total lipid content of the cells accounts for 45.12% of the dry weight of the cells.

[0036] Table 1 BG11 culture medium

[0037]

[0038] Table 2 Composition of A5+Co solution in Table 1

[0039]

[0040] Example 4 Comparison of culture effects of FSH-BY1 and FSH-B1

[0041] The algal liquid of FSH-BY1 and FSH-B1 in the logarithmic growth phase was inoculated in BG11 medium for culture, the culture was carried out in a photobioreactor, the OD 690 was 0.25. The light intensity during the culture was 8000 Lux, the culture was carried out under different CO2 and SO2 concentrations, the culture temperature was 25°C, the pH value was controlled at 7-8, the light cycle was 24 h, the light / dark time ratio was 14:10, the culture time was 8 days, after the culture was completed, the algal cells were collected by centrifugation, and the algal powder dry weight was measured after vacuum freeze-drying under the condition of-60°C until the constant weight, the biomass was calculated, and the total lipid content of the cells was measured by the n-hexane: ethyl acetate method. The results are shown in Table 3.

[0042] Table 3 Comparison of culture results of FSH-BY1 and FSH-B1

[0043]

[0044] As can be seen from Table 3, the microchaete FSH-BY1 screened in the present application has better tolerance to CO2 and SO2 than the initial algal strain FSH-B1, and has good stability of biomass and total lipid content.

[0045] Example 5 Comparison of culture temperatures of FSH-BY1 and FSH-B1

[0046] The algal liquid of FSH-BY1 and FSH-B1 in the logarithmic growth phase was inoculated in BG11 medium for culture, the culture was carried out in a photobioreactor, the OD 690 was 0.25. The CO2-containing gas with a content of 10v% was introduced from the bottom of the reactor. The light intensity during the culture was 8000 Lux, the pH value was controlled at 7-8, the light cycle was 24 h, the light / dark time ratio was 14:10, and the culture time was 7 days. After the culture was completed, the algal cells were collected by centrifugation, and the algal powder dry weight was measured after vacuum freeze-drying under the condition of-60°C until the constant weight, the biomass was calculated, and the total lipid content of the cells was measured by the n-hexane: ethyl acetate method. The results are shown in Table 4.

[0047] Table 4 Comparison of culture results of FSH-BY1 and FSH-B1

[0048]

[0049] As can be seen from Table 4, the FSH-BY1 of the present application has better low-temperature tolerance than the initial algal strain FSH-B1, but both of them are not resistant to high temperature.

[0050] Comparative Example 1

[0051] The same as Example 3, except that the Scenedesmus HCS-02 (CN107177505A) was used instead of FSH-BY1. sp.), and the preservation number is CGMCC No. 10766, and the culture was ended after 8 days. It was detected that the biomass yield of the HCS-02 strain was 2.8 g / L, and the total lipid content of the cells accounted for 26.78% of the dry weight of the cells. Since the microalgae cannot tolerate SO2, the culture effect is poor.

[0052] Comparative Example 2

[0053] The same as Example 3, except that the Chlorella vulgaris MH-04 disclosed in CN106467897A was used Scenedesmus sp. Desmodesmus sp. ), and the preservation number is CGMCC No. 10765, and the culture was ended after 8 days. It was detected that the biomass yield of the MH-04 strain was 1.59 g / L, and the total lipid content of the cells accounted for 20.7% of the dry weight of the cells. Since the microalgae cannot tolerate SO2, the culture effect is poor. SEQUENCE LISTING <110> China Petroleum & Chemical Corporation Dalian Research Institute of Petroleum & Chemical Industry of China Petroleum & Chemical Corporation <120> A strain of microalgae rich in oil and its culture application <130> NEW PATENT APPLICATION <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1607 <212> DNA <213> Micractinium sp. <400> 1 tagtcatatg cttgtctcaa agattaagcc atgcatgtct aagtataaac tgctttatac 60 tgtgaaattg cgaatggctc attaaatcag ttatagttta tttgatggta cctactactc 120 ggatacccgt agtaaatatg gagctaatac gtgcgtaaat cccgacttct ggaagggacg 180 tatttattag ataaaaggcc gaccgggggt tgcccgactc gcggtgaatc atgataactt 240 cacgaatcgc atggccttgt gccggcgatg tttcattcaa atttctgccc tatcaacttt 300 cgatggtagg atagaggcct accatggtgg taacgggtga cggaggatta gggttcgatt 360 ccggagaggg agcctgagaa acggctacca catccaagga aggcagcagg cgcgcaaatt 420 acccaatcct gacacaggga ggtagtgaca ataaataaca atactgggcc ttttcaggtc 480 tggtaattgg aatgagtaca atctaaaccc cttaacgagg atcaattgga gggcaagtct 540 ggtgccagca gccgcggtaa ttccagctcc aatagcgtat atttaagttg ctgcagttaa 600 aaagctcgta gttggatttc gggtggggcc tgccggtccg ccgtttcggt gtgcactggc 660 agggcccacc ttgttgccgg ggacgggctc ctgggcttca ctgtccggga ctcggagtcg 720 gcgctgttac tttgagtaaa ttagagtgtt caaagcaggc ctacgctctg aatacattag 780 catggaataa cacgatagga ctctggccta tcctgttggt ctgtaggacc ggagtaatga 840 ttaagaggga cagtcggggg cattcgtatt tcattgtcag aggtgaaatt cttggattta 900 tgaaagacga actactgcga aagcatttgc caaggatgtt ttcattaatc aagaacgaaa 960 gttgggggct cgaagacgat tagataccgt cctagtctca accataaacg atgccgacta 1020 gggatcggcg gatgtttctt cgatgactcc gccggcacct tatgagaaat caaagttttt 1080 gggttccggg gggagtatgg tcgcaaggct gaaacttaaa ggaattgacg gaagggcacc 1140 accaggcgtg gagcctgcgg cttaatttga ctcaacacgg gaaaacttac caggtccaga 1200 catagtgagg attgacagat tgagagctct ttcttgattc tatgggtggt ggtgcatggc 1260 cgttcttagt tggtgggttg ccttgtcagg ttgattccgg taacgaacga gacctcagcc 1320 tgctaaatag tcacggttgg ctcgccagcc ggcggacttc ttagagggac tattggcgac 1380 tagccaatgg aagcatgagg caataacagg tctgtgatgc ccttagatgt tctgggccgc 1440 acgcgcgcta cactgatgca ttcaacgagc ttagccttgg ccgagaggcc cgggtaatct 1500 ttgaaactgc atcgtgatgg ggatagatta ttgcaattat taatcttcaa cgaggaatgc 1560 ctagtaagcg caagtcatca gcttgcgttg attacgtccc tgccctt 1607

Claims

1. A strain of Botryococcus braunii enriched in lipids, characterized in that: The algal strain is Micractinium chthonoplastics (Chod.) Kome Micractinium sp. ) FSH-BY1, which was preserved in China General Microbiological Culture Collection Center on May 12, 2020, and the preservation number is CGMCC No. 19983.

2. The Botryococcus braunii according to claim 1, characterized in that: Microchaete sp. (Fig. 1) Micractinium sp. The sequencing results of the 18S rDNA gene of FSH-BY1 are shown in the sequence listing.

3. The Botryococcus braunii according to claim 1, characterized in that: Microchaete sp. Micractinium sp. Microchaete sp. (Fig. 1) FSH-BY1 The algal cells are green under microscope, and the single cell is spherical with a diameter of about 4-8 μm. Usually, four cells are aggregated into a square or tetrahedron, and sometimes eight cells are arranged into a sphere.

4. The microalga of claim 1, wherein: Microchaete sp. Micractinium sp. FSH-BY1 can tolerate CO2 concentration up to 40 v%, SO2 concentration up to 0.04 v%, and low temperature down to 5℃.

5. A method of culturing Botryococcus braunii (B. braunii) according to claim 1, wherein the B. braunii is a micro-algae. Micractinium sp. ) A method of culturing FSH-BY1, characterized by: In the photobioreactor, the fresh water medium is used for culture, and the gas with 1.0v%-40% CO2 content is used for culture.

6. The culturing method according to claim 5, characterized by: The gas with 5v%-30v% CO2 content is used for culture.

7. The culturing method according to claim 5, characterized by: The culture condition is: light intensity 1500-20000Lux, pH value 6-9, temperature 5-35℃, light cycle 24h, light and dark time ratio 14:

10.

8. The culturing method according to claim 7, characterized by: The temperature is 10-30℃.

9. The culturing method according to claim 5, characterized by: The fresh water culture is any one of BG11 medium, SE medium or D1 medium.

10. The culturing method of claim 5, wherein: The gas with CO2 and SO2 is used for culture, and the SO2 concentration is not higher than 0.04v%.

11. The microalga (B. braunii) of claim 1, wherein the microalga is capable of producing FSH-BY1. Micractinium sp. ) Use of FSH-BY1 in the fixation of CO2.

12. The microalgae (B. braunii) of claim 1, Micractinium sp. ) Use of FSH-BY1 in the production of microalgal lipids.

13. The microalga Botryococcus braunii (1 Micractinium sp. ) FSH-BY1 for use in the purification of exhaust or flue gases containing CO2 and SO2.

14. The use according to claim 13, characterized in that: The CO2 concentration in the waste gas or flue gas is ≤30v%, and the SO2 concentration is ≤0.04v%.

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