A method for open-culture of oil-producing microalgae

Abstract

This invention relates to a method for open-field cultivation of oil-producing microalgae. The method involves adding microalgae culture medium and microalgae seed solution to an open-field photobioreactor and conducting alternating light and dark cultivation. During the light reaction, CO2 gas is introduced, and during the dark reaction, CO2 and SO2 gases are introduced. After alternating cultivation for a period of time, the cultivation temperature is lowered, and SO2 introduction is stopped during the dark reaction. The cultivation continues until the plateau phase, at which point the microalgae cells are harvested. The microalgae is *Mistylla spp.* Micractinium sp. FSH-BY1, with accession number CGMCC No. 19983. This invention's method, through a specific cultivation approach, solves the problem of contamination by other microorganisms in open microalgae cultivation and reduces cultivation costs.

Description

Technical Field

[0001] This invention belongs to the field of biomass energy technology, specifically relating to a method for open-culture oil-producing microalgae to prevent pollution from harmful organisms. Background Technology

[0002] Microalgae are rich in nutrients such as protein, polysaccharides, and unsaturated fatty acids (e.g., spirulina), and can be used in food, medicine, and energy. They can accumulate large amounts of fatty acids, with some microalgae containing 30%-60% fatty acids by dry weight. Utilizing microalgae cultivation to accumulate oil resources has become one of the most popular research areas for developing renewable resources using solar energy, possessing not only strong market potential but also extraordinary social value.

[0003] Currently, there are two main methods for microalgae cultivation: closed and open. Closed cultivation refers to using closed reactors with different structures, such as airlift, stirred, and tubular reactors. These methods have higher production costs and are used to produce high-value-added products or as seed tanks for open cultivation. Open cultivation refers to using open pool cultivation devices, such as raceway pools and circular shallow pools. These methods are technically simple and require low investment, and have therefore attracted considerable attention from researchers in recent years. However, open cultivation is susceptible to contamination by harmful organisms such as filamentous fungi, rotifers, and protozoa. These harmful organisms grow and reproduce in the algal culture medium. When the number of these harmful organisms reaches a certain density, they affect the growth and reproduction of the cultured algae. Contamination can lead to difficulties in scaling up cultivation, or even complete culture failure. Therefore, effectively preventing contamination by harmful organisms is a key issue for the large-scale cultivation of microalgae.

[0004] In recent years, many researchers have conducted studies on biological control methods for microalgae diseases and pests, and some reports have been published on this topic. Traditional methods include: first, using physical methods such as filtration or acidification to kill and eliminate harmful organisms in algae; second, using chemical methods to control harmful organism contamination in open-field microalgae cultivation; third, using biological control methods to control harmful organism contamination in open-field microalgae cultivation; and fourth, using the addition of plant extracts to control diseases and pests in open-field microalgae cultivation.

[0005] CN103773690A discloses a method for open-culture microalgae. The microalgae culture employs conventional methods and conditions, but at the initial stage of cultivation, plant extracts are directly added to the microalgae culture medium. These plant extracts are one or more selected from *Melia azedarach* extract, *Melia toosendan* extract, and *Cephalotaxus fortunei* extract, added at a concentration of 5-80 mg / L. This method can solve the problem of harmful biological contamination in open-culture microalgae, effectively preventing contamination by miscellaneous bacteria and pests, and has the advantage of not developing drug resistance in subsequent subculturing. However, this method requires the special preparation of plant extracts with specific formulations, increasing the cultivation cost.

[0006] Zheng Chunbo et al. (Zheng Chunbo et al. Control technology of harmful organisms in productive cultivation of flat algae and golden algae[J]. Aquaculture, 2004, 25(3):33) used filtration and acidification to eliminate and kill harmful organisms in algal solution. The filtration method is relatively troublesome, does not clean the solution, and is not suitable for large-scale cultivation. The acidification method requires the addition of acid, which has a certain impact on the growth of microalgae while killing harmful organisms. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for open-culture of oil-producing microalgae. This method, through a specific cultivation approach, solves the problem of contamination by other microorganisms in open-culture microalgae and reduces cultivation costs.

[0008] The method for open-culture of oil-producing microalgae provided by this invention includes the following:

[0009] Microalgae culture medium and microalgae seed solution were added to an open photobioreactor, and cultured under alternating light and dark conditions. CO2 gas was introduced during the light reaction, and CO2 and SO2 gas were introduced during the dark reaction. After alternating culture for a period of time, the culture temperature was lowered, and SO2 introduction was stopped during the dark reaction. The culture was continued until the stationary phase, and then the microalgae cells were harvested. The microalgae were *Mistylla* (a type of algae). Micractinium sp. FSH-BY1, with accession number CGMCC No.19983.

[0010] In this invention, the microalgae ( Micractinium sp. FSH-BY1 was deposited on May 12, 2020, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 19983. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] In this invention, the method for preparing the microalgae seed solution is as follows: microalgae are inoculated into a microalgae culture medium and cultured under the following conditions: pH 7-9, temperature 15-30℃, light cycle 24h, light-dark time ratio 14:10-10:14, and light intensity 2000-20000 Lux, with shaking culture until the logarithmic growth phase is obtained, thus obtaining the microalgae seed solution.

[0012] In this invention, the microalgae culture medium is any one of BG11 medium, SE medium, BBM medium, etc.

[0013] In this invention, the volume ratio of microalgae seed liquid to microalgae culture medium added to the photobioreactor is 1:20 to 1:5.

[0014] In this invention, the light-dark alternating culture has a light-dark cycle of 24 hours, a light-dark time ratio of 14:10 to 10:14, a light intensity of 2000 to 20000 Lux, and a culture temperature of 15 to 30°C.

[0015] In this invention, the CO2-containing gas has a CO2 volume content of 5% to 45%, preferably 5% to 20%, and does not contain SO2, NOx, or other gases.

[0016] In this invention, the CO2- and SO2-containing gas has a CO2 volume content of 5% to 45% and an SO2 volume content of 0.04% to 0.06%. The SO2-containing gas can be flue gas, originating from at least one of the following: S-zorb regeneration tail gas, sulfur recovery unit incineration tail gas, catalytic cracking regeneration tail gas, etc.

[0017] In this invention, after 2-5 days of alternating light and dark culture, the culture temperature is reduced by 5-15°C from the original temperature, and the culture continues until the growth stabilizes.

[0018] In this invention, after cultivation, microalgal cells are harvested by centrifugation, sedimentation, and other methods, and the cell dry weight and lipid content are measured.

[0019] Compared with the prior art, the beneficial effects of the method of the present invention are:

[0020] This invention addresses the industrial properties of microalgae by employing a specific cultivation method to solve the problem of contamination prevention by miscellaneous bacteria in its open cultivation, and it has no toxic effect on the growth of the microalgae themselves, thus ensuring the normal growth of the microalgae.

[0021] The culture method and conditions of this invention facilitate the accumulation of lipids in *Micromistletoe*. Testing showed that the cell dry weight can reach over 6 g / L, and the lipid content can reach over 50% of the cell dry weight.

[0022] The method of this invention also has the advantages of simple operation, readily available raw materials, and low cost, making it suitable for large-scale cultivation and showing good application prospects. Detailed Implementation

[0023] The present invention will be further described in detail below through embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0025] In this invention, the concentrations of CO2 and SO2 in the gas are detected using a flue gas analyzer.

[0026] The microalgae described in this invention ( Micractinium sp. FSH-BY1 is a new algal strain isolated and screened by the inventors and is deposited at the China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC No. 19983; deposit date: May 12, 2020; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0027] The microalgae culture of this invention uses BG11 medium, the formulation of which is shown in Tables 1 and 2.

[0028] Table 1 BG11 culture medium

[0029]

[0030] *Table 2 Composition of A5+Co solution in Table 1

[0031]

[0032] First, BG11 liquid culture medium was prepared according to Tables 1 and 2. The pH of the medium was adjusted to 8.0, and then the microalgae were inoculated into the medium. The culture was carried out in a constant-temperature, light-controlled shaker at 25°C, with a photoperiod of 24 h, a light-dark ratio of 14:10, a light intensity of 5000 Lux, and shaking at 120 rpm until the logarithmic growth phase was reached, thus obtaining the microalgae seed culture.

[0033] Example 1

[0034] (1) Add microalgae (Microalgae) to a 20L open photobioreactor. Micractinium sp. FSH-BY1 seed culture and microalgae culture medium were used. The amount of seed culture added was 800 mL and the amount of microalgae culture medium added was 8 L. The culture was carried out in alternating light and dark conditions with a light-dark cycle of 24 h and a light-dark time ratio of 14:10. The light intensity was 5000 Lux and the culture temperature was 20℃. The CO2 volume content of the gas introduced into the light reaction was 10%, and the CO2 volume content and SO2 volume content of the gas introduced into the dark reaction were 10% and 0.05%, respectively.

[0035] (2) After 3 days of alternating light and dark culture, the dark reaction was introduced with gas similar to the light reaction, i.e., without SO2. The culture temperature was lowered to 10℃. After 5 days of culture, the plant entered the stable growth phase. The culture was then terminated, and the number of contaminating bacteria was found to be below 2.0 × 10⁻⁶. 3 per mL.

[0036] Microalgal cells were harvested by centrifugation, and their dry weight and lipid content were determined. After vacuum freeze-drying to constant weight at -60℃, the dry weight of the algal powder was measured, biomass yield was calculated, and total lipid content was determined using the hexane:ethyl acetate method. The cell dry weight was found to be 6.7 g / L, and the lipid content was 50.89% of the cell dry weight.

[0037] Example 2

[0038] Same as Example 1, except that: the seed culture volume was 800 mL, the microalgae culture medium volume was 4 L, the light-dark time ratio was 10:14, and the light intensity was 10000 Lux; after 4 days of alternating light and dark culture, gas was introduced for photocatalytic reaction, and SO2 was no longer present. After the culture was completed, the number of contaminating bacteria was found to be below 1.5 × 10⁻⁶. 3 The cell count / mL can reach 6.8 g / L, and the lipid content is 52.12% of the cell dry weight.

[0039] Example 3

[0040] Same as Example 1, except that: the amount of seed culture added was 400 mL, the amount of microalgae culture medium added was 8 L, the light-dark time ratio was 12:12, the light intensity was 8000 Lux, and after 5 days of alternating light and dark culture, gas was introduced for photocatalytic reaction, and SO2 was no longer present. After the culture was completed, the number of miscellaneous bacteria was found to be below 2.0 × 10⁻⁶. 3 The cell count is 1 / mL, the cell dry weight can reach 6.4 g / L, and the lipid content is 50.23% of the cell dry weight.

[0041] Example 4

[0042] Similar to Example 1, but with the following difference: the culture temperature was 20°C for the first 3 days of alternating light and dark, and then the temperature for the light reaction was lowered to 15°C, and cultured for another 5 days. After the culture was completed, the number of contaminating bacteria was found to be below 1.9 × 10⁻⁶. 3 The cell count is 1 / mL, the cell dry weight can reach 6.9 g / L, and the lipid content is 50.45% of the cell dry weight.

[0043] Example 5

[0044] Similar to Example 1, but with the following difference: the culture temperature was 30°C for the first 3 days with alternating light and dark conditions; after 3 days, the culture temperature for the light reaction was lowered to 5°C, and culture continued for another 4 days. After the culture was completed, the number of contaminating bacteria was found to be below 2.4 × 10⁻⁶. 3 Cells / mL, cell dry weight can reach 6.2 g / L, and lipid content is 50.89% of cell dry weight.

[0045] Example 6

[0046] Same as Example 1, except that the CO2 volume content was 5% and the SO2 volume content was increased to 0.06%. After cultivation, the number of miscellaneous bacteria was found to be below 1.3 × 10⁻⁶.3 The cell count is 1 / mL, the cell dry weight can reach 6.3 g / L, and the lipid content is 51.23% of the cell dry weight.

[0047] Example 7

[0048] Same as Example 1, except that the CO2 volume content was 25% and the SO2 volume content was reduced to 0.04%. After cultivation, the number of miscellaneous bacteria was found to be below 1.4 × 10⁻⁶. 3 The cell count is 1 / mL, the dry weight of cells can reach 6.1 g / L, and the lipid content is 52.69% of the dry weight of cells.

[0049] Comparative Example 1

[0050] Same as Example 1, except that the microalga used was *Synechocephalus monophylla* SS-B1, disclosed in CN104611228A, with accession number CGMCCNo.7479. After cultivation, the number of contaminating bacteria was found to be below 2.0 × 10⁻⁶. 3 Cells / mL, cell dry weight up to 2.3 g / L, lipid content of 23.41% of cell dry weight.

[0051] Comparative Example 2

[0052] Same as Example 1, except that the same CO2-containing gas was introduced into both the light and dark reactions, while SO2 was not added to the dark reaction. After cultivation, the number of contaminating bacteria was measured to be 3.3 × 10⁻⁶. 5 The cell count was 5.9 g / L, and the lipid content was 39.92% of the cell dry weight.

[0053] Comparative Example 3

[0054] Same as Example 1, except that the same CO2 and SO2 gases were introduced into both the light and dark reactions. After cultivation, the number of contaminating bacteria was measured to be 1.1 × 10⁻⁶. 3 The cell count / mL can reach 5.7 g / L, and the lipid content is 40.29% of the cell dry weight.

[0055] Comparative Example 4

[0056] Same as Example 1, except that the incubation temperature for both the light and dark reactions was consistently 10°C. After incubation, the number of contaminating bacteria was measured to be 1.1 × 10⁻⁶. 3 The cell count / mL can reach 4.5 g / L, and the lipid content is 38.91% of the cell dry weight.

[0057] Comparative Example 5

[0058] Same as Example 1, except that the incubation temperature for both the light and dark reactions was consistently 20°C. After incubation, the number of contaminating bacteria was measured to be 3.5 × 10⁻⁶. 5The cell count / mL can reach 5.4 g / L, and the lipid content is 40.27% of the cell dry weight.

Claims

1. A method for open culture of oleaginous microalgae, characterized in that The application relates to a method for culturing microalgae, which comprises the following steps: adding a microalgae culture medium and a microalgae seed solution into an open photobioreactor, carrying out light-dark alternate culture, passing in CO2-containing gas during light reaction, and passing in CO2 and SO2-containing gas during dark reaction, wherein the volume content of CO2 is 5%-45%, the volume content of SO2 is 0.04%-0.06%, the culture temperature is 15-30 DEG C, the culture temperature is reduced by 5-15 DEG C on the original basis after alternate culture for a period of time, SO2 is stopped from being passed in during dark reaction, the microalgae cells are harvested after the culture reaches the stable period, and the microalgae is Micractinium chthonoplastics FSH-BY1 with a preservation number of CGMCC No. 19983. Micractinium sp. ​ 2. The method of claim 1, wherein: The microalgae culture medium is any one of BG11 medium, SE medium and BBM medium.

3. The method according to claim 1 or 2, characterized in that: The preparation method of the microalgae seed liquid is as follows: inoculating the microalgae into the microalgae culture medium, and culturing under the conditions of pH 7-9, temperature 15-30 DEG C, light cycle 24 h, light and dark time ratio 14:10-10:14, and light intensity 2000-20000 Lux, to obtain the microalgae seed liquid in the logarithmic growth phase.

4. The method according to claim 1 or 2 or 3, characterized in that: The volume ratio of the microalgae seed liquid to the microalgae culture medium in the photobioreactor is 1:20-1:

5.

5. The method of claim 1, wherein: The light and dark cycle is 24 h, the light and dark time ratio is 14:10-10:14, and the light intensity is 2000-20000 Lux.

6. The method of claim 1, wherein: The CO2-containing gas contains 5%-45% CO2 by volume, and does not contain SO2 and NOx gas.

7. The method of claim 6, wherein: The CO2-containing gas contains 5%-20% CO2 by volume.

8. The method of claim 1, wherein: The SO2-containing gas is derived from at least one of S-zorb regeneration tail gas, sulfur recovery device incineration tail gas and catalytic cracking regeneration tail gas.

9. The method of claim 1, wherein: The light and dark cycle is 24 h, the light and dark time ratio is 14:10-10:14, and the light intensity is 2000-20000 Lux.

10. The method of claim 1, wherein: After the culture is completed, the microalgae cells are harvested by centrifugation and sedimentation. After the culture is completed, the microalgae cells are harvested by centrifugation and sedimentation.

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