Method for regulating the production of biomass of special environment microalgae and application thereof

Abstract

The application provides a precise regulation method for rapid growth of high-fat acid microalgae biomass in Xinjiang special environment. Through red light / blue light alternate irradiation, dynamic nitrogen supply and salinity-low temperature combined stress, the daily average growth rate of microalgae biomass is greater than or equal to 120 mg / L, and the total fat content is greater than or equal to 35%. The method breaks through the contradiction between growth and lipid accumulation in traditional culture, and the light energy utilization rate is increased by 25%, which is suitable for large-scale development of desert microalgae resources and biodiesel production.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and microalgae cultivation technology, specifically involving a precise regulation method for the rapid biomass growth of microalgae with high fatty acid content (such as Dunaliella salina, Chlorella vulgaris, and Microcystis aeruginosa isolated from the Gurbantünggüt Desert) under special environments such as high salinity, drought and strong light in Xinjiang. By optimizing the light environment, nutrient supply and metabolic regulation strategies, the growth rate and fatty acid accumulation efficiency of microalgae are significantly improved, which is suitable for the large-scale production of biodiesel feedstock. Background Technology

[0002] Microalgae are microorganisms containing chlorophyll a and capable of photosynthesis, including eukaryotic microalgae and prokaryotic cyanobacteria. Microalgae cultivated or produced in large quantities using biotechnology belong to four phyla: Chlorophyta, Cyanophyta, Chrysophyta, and Rhodophyta. Chlorophyta includes Dunaliella salina and Chlorella vulgaris. Dunaliella salina is extremely salt-tolerant, surviving in near-freshwater to saturated saline (salinity > 5 mol / L NaCl) environments, and is commonly found in high-salinity environments such as salt pans and salt lakes. Chlorella vulgaris is a highly adaptable green algae, capable of operating in a wide salinity range (freshwater to brackish water), temperatures of 10-36℃ (optimal 24-28℃), and light intensities of approximately 150 μmol·m⁻¹. -2 ·s -1 They reproduce rapidly in their environment.

[0003] Microalgae have a wide range of applications in many fields. High-oil species such as Dunaliella salina, Chlorella, and Gurbantünggüt algae can even be used to produce biodiesel. Microalgal cells contain valuable nutrients and chemical raw materials, including proteins, lipids, algal polysaccharides, β-carotene, and various inorganic elements (such as Cu, Fe, Se, Mn, Zn, etc.). For example, Chlorella has a protein content as high as 50%-57%, is rich in fats, carbohydrates, and vitamins (A, C, E), and has a chlorophyll content of 3000-7000 mg / 100g. Dunaliella salina contains high concentrations of β-carotene (13% dry weight), α-carotene, and chlorophyll, with α-carotene having an antioxidant capacity more than 10 times that of β-carotene; it is also rich in glycerol (50%), protein (40%), vitamins (such as vitamins A and E), minerals (more than 70 kinds), and polysaccharides, with elemental ratios highly similar to those in human body fluids.

[0004] However, the following problems exist in the production of microalgal biomass in the existing technology: (1) Low growth rate and low yield: Under the traditional culture method (such as BG11 medium + constant light), the biomass growth rate of Xinjiang high-lipid microalgae (Microcystis aeruginosa) is slow, with an average daily growth of only 50-60 mg / L and a culture cycle of 15-20 days. Under standard culture conditions, the total lipid content reaches 33.2%, but the biomass is only 580 mg / L, and the overall lipid production efficiency does not meet the industrial requirements. (2) Insufficient fatty acid accumulation: Although conventional stress methods (such as nitrogen restriction) can increase lipid content, they lead to a 30%-40% decrease in biomass, and the overall lipid production has no obvious advantage. Under the traditional stress culture (such as phosphorus restriction), the microalgal biomass decreases to 400 mg / L, the total lipid content increases to 40%, but the total lipid production is still lower than the level before optimization. (3) Poor environmental adaptability: Xinjiang has a large diurnal temperature range (daily average temperature range > 15℃) and strong ultraviolet radiation. Existing photobioreactors are difficult to dynamically adjust the light intensity and spectrum, resulting in photoinhibition or low light energy utilization. Summary of the Invention

[0005] The purpose of this invention is to address the problem of slow microalgae growth in existing technologies by providing a method for regulating and improving microalgae biomass.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for regulating microalgal biomass includes ambient light regulation: alternating red and blue light irradiation is used, with a photocycle consisting of a 12-16 h light period and an 8-12 h dark period.

[0007] The optical cycle is typically 8-12, with 10 being the preferred number.

[0008] Preferably, the red light wavelength is 615-660 nm and the blue light wavelength is 445-480 nm. During the 12-16h illumination period, red light and blue light are alternately irradiated, with the cumulative red light irradiation time in each light cycle being 8-12 h and the cumulative blue light irradiation time being 4-8 h.

[0009] Preferably, the red light wavelength is 615-660 nm, and the blue light wavelength is 445-480 nm. The cumulative irradiation with red light is 8-12 hours, and the cumulative irradiation with blue light is 4-8 hours.

[0010] Preferably, the light wavelength is 640 nm, and the blue light wavelength is 465 nm. Preferably, the red light is irradiated for 7 hours, and the blue light is irradiated for 5 hours.

[0011] In a preferred embodiment, during a 12-hour light period, red light is applied for a cumulative total of 9 hours, and blue light for a cumulative total of 3 hours, with an alternation pattern of 3 hours of red light followed by 1 hour of blue light. The light cycle is 12 hours of light / 12 hours of darkness, simulating the natural light cycle in spring and autumn in regions between 40° and 45° north latitude, thus improving light adaptation efficiency.

[0012] Preferably, the intensity of the irradiated light is 200-400 μmol•m. -2 •s -1 Including but not limited to 200, 250, 300, 350, and 400 μmol•m -2 •s -1 .

[0013] Preferably, the intensity of the irradiated light is 200~300 μmol•m. -2 •s -1 More preferably, 300 μmol•m -2 •s -1 .

[0014] Furthermore, the microalgal biomass regulation method of the present invention also includes a dynamic nitrogen supply strategy: an initial nitrogen concentration of 1.2-1.8 g / L, which is decreased daily by 0.2-0.4 g / L during the middle and late stages of cultivation. The initial cultivation period is 1-3 days, and the middle and late stages are 4-7 days.

[0015] Furthermore, in the microalgal biomass regulation method of the present invention, the stress treatment includes: using the initial salinity as a reference, periodically fluctuating within a range of ±5%-10% daily, with a fluctuation frequency of 1-2 times per hour.

[0016] Preferably, the stress treatment further includes daily low-temperature shock at 13-17°C for 3-5 hours. More preferably, daily low-temperature shock culture at 15°C for 4 hours.

[0017] Furthermore, the present invention provides a microalgae cultivation method, characterized in that it includes inoculating fatty acid-producing microalgae into a culture medium and then carrying out controlled cultivation through the above-described method.

[0018] This invention improves the growth rate and yield of microalgae, while simultaneously addressing insufficient fatty acid accumulation. The biomass achieved using this method reaches 1250 mg / L (daily average growth rate of 125 mg / L), with a total lipid content of 37.5%. Furthermore, this method shortens the cultivation period. Attached Figure Description

[0019] Figure 1 Cell morphology of *Microcystis globulus* was observed using an optical microscope. Figure 2 PCR amplification pattern of Micrococcus pluvialis. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure.

[0021] Example 1 (Microcystis globulus) 1. Obtaining Microcystis aeruginosa (1) Source and separation: The algae were isolated from the saline-alkali waters of Ebinur Lake in Xinjiang (44°53'N, 82°53'E). The isolation was performed using conventional microalgae isolation methods (Pan Huifei, Cultivation Process of Micrococcus microcarpa in Alkali Lake Water and Algal Cell Response to Alkali Lake Water Environment, Master's Thesis, 2013).

[0022] (2) Identification Pure algal strains need to be identified by combining morphological and molecular biological identification to ensure that they are *Microcystis globulus*.

[0023] Morphological identification: The purified algal strain was inoculated into liquid culture medium and cultured until the logarithmic growth phase. A small amount of culture medium was taken to prepare a slide, and cell morphology was observed under an optical microscope. Figure 1 As shown. Microcystis cells are spherical or nearly spherical, without flagella, and are single-celled or aggregated with a few cells. The chloroplasts are cup-shaped or disc-shaped, and are yellowish-green to dark green in color, with a diameter of 2-5 μm.

[0024] Molecular biological identification: DNA Extraction: Collect algal cells in the logarithmic growth phase and extract genomic DNA using a kit; PCR amplification: Select universal primers for microalgae classification and identification, such as 18S rRNA gene primers and ITS sequence gene primers, for PCR amplification. The amplification pattern is shown below. Figure 2 As shown.

[0025] Sequencing and alignment: The PCR products were sequenced, and the sequencing results were submitted to databases such as NCBI for BLAST comparison. If the sequence similarity with known *Microcystis* species was ≥98%, it could be identified as belonging to the genus *Microcystis*. Combined with phylogenetic tree construction, the species classification was further confirmed as *Microcystis*. Nannochloropsis ).

[0026] 2. Detailed cultivation process (1) Inoculation and culture medium Basic culture medium: Modified f / 2 formula, containing: NaNO31.5 g, NaH2PO4·2H2O 0.03g, Na2SiO3·9H2O 0.02 g; 1 mL of trace element stock solution contains: 2.86 g / L H3BO4, 1.81 g / L MnCl2·4H2O, 0.222 g / L ZnSO4, 0.39 g / L Na2MoO4, 0.079 g / L CuSO4·5H2O, and 0.025 g / L CoCl2·6H2O; 1 mL of vitamin stock solution contains: Vitamin B12: 0.001 g / L, Biotin: 0.001 g / L, Thiamine Hydrochloride: 0.2 g / L, and Distilled Water: 1000 mL. Note the following when preparing this solution: Vitamin B12 and Biotin must first be prepared as crystal solutions (B12 with 11% water, Biotin with 4% water), then dissolved in distilled water. After sterilization by filtration, dispense into individual containers and store in a refrigerator.

[0027] Salinity baseline value 5% (w / v, adjusted with NaCl).

[0028] Inoculation procedure: Collect algal cells by centrifugation at 4000 rpm for 5 min, and resuspend in sterile culture medium to OD. 680 =0.1, loaded into a 5L flat-plate photobioreactor (working volume 3 L).

[0029] (2) Light control system Light source configuration: The reactor has a built-in LED array (red light 620±5 nm, blue light 450±5 nm). The photoperiod schedule is shown in Table 1: Table 1 Red and blue light alternation configuration during illumination period

[0030] Each day's light period is followed by a dark period, and the light cycle is repeated the next day. The total culture period is 10 days, and OD is measured daily. 680 value.

[0031] (3) Nutritional supply strategy High nitrogen period (D1-D3): Maintain NaNO3 at 1.5 g / L and pH at 7.8±0.2 (adjusted by automatically introducing 1% CO2).

[0032] Nitrogen reduction period (D4-D10): 10% of the culture medium is drawn off at 08:00 every day and an equal volume of nitrogen-free culture medium is added.

[0033] Measured nitrogen concentration: Based on the cultivation process records, the measured daily nitrogen concentrations during the nitrogen reduction period are shown in Table 2 below: Table 2. Measured daily nitrogen concentrations during the nitrogen reduction period.

[0034] The measured values ​​were consistent with the calculation results of the dilution model, verifying that the nitrogen concentration could be gradually reduced by diluting by 10% daily, which meets the process requirements of "gradual stress-induced lipid accumulation" in microalgae culture.

[0035] (4) Coercion Management Salinity fluctuations: Add NaCl to 10% at 08:00 daily (0.83% increase / h); dilute back to 5% at 14:00 (0.83% decrease / h); monitor in real time using an online conductivity meter.

[0036] (5) Low-temperature shock: The circulating water bath system should be activated daily. During the day, the temperature is 25℃-28℃. At night, the temperature drops to 15℃ at a rate of 2.5℃ / h and is maintained for 4 hours. Then, the temperature is gradually increased to 25℃-28℃ at a rate of 2.5℃ / h.

[0037] Stir at a constant speed (150 rpm) throughout the process.

[0038] 3. Test Results (1) Biomass (1250±35 mg / L): determined by daily OD 680 Monitoring and plotting growth curves, R ² =0.993 indicates that the model fit is extremely high and the data is reliable.

[0039] (2) Total lipid production was 468.8 mg / L / d, and total lipid content was (37.5 ± 1.2%): The sulfuric acid-vanillin method was used for chemical determination, and repeated experiments verified the stability of lipid accumulation.

[0040] (3) The specific value of SOD activity was 12.8 U / (g·min), which was obtained by Western blot quantitative analysis.

[0041] (4) The lipid production rate per unit of light energy is 0.0181 mg·mL -1 (g fatty acid / mol photons), with an energy conversion efficiency of 0.0595%.

[0042] All experimental data above are based on at least three independent replicates, and the results are expressed as mean ± standard deviation to ensure data reliability.

[0043] Example 2 (Comparison with traditional methods)

[0044] Except for the conditions set for the control group and extreme group described below, everything else was the same as in Example 1, and the experiment was conducted concurrently with that in Example 1. Details are shown in Table 3 below: Table 3 Condition parameters for each experimental group

[0045] The key experimental results are shown in Table 4 below.

[0046] The table analysis shows that the experimental results of Example 1 are better than those of the control group and the extreme group.

[0047] Biomass increased from 580 mg / L in the control group to 1250 mg / L, a biomass increase rate of 115%; total lipid production increased from 192.6 mg / L / d in the control group to 468.8 mg / L / d, a total lipid production increase rate of 143%; EPA content increased from 5.2% to 8.5%, a content increase rate of 63.5%; and the culture period was shortened from 14 days to 10 days, a shortening rate of 28.6%.

[0048] Example 3 (Verification of extreme salinity adaptation of Microcystis aeruginosa)

[0049] In this example, the extreme salinity adaptability verification experiment, except for the gradient salinity test, follows the same culture conditions as in Example 1, specifically including: Stress management: During the salinity gradient test, the salinity was adjusted to the target value (3%, 5%, 8%, 10%, 13%, 15%, 18%, 23%) at 08:00 every day, as shown in Table 5, and monitored in real time by an online conductivity meter.

[0050] Table 5. Concentrations for Salinity Gradient Test

[0051] Biomass was determined using spectrophotometry (OD 680 nm), with three replicates and the average value calculated. The standard deviation was then determined. Measurements were taken at the 240th hour (10 days) of culture.

[0052] The results of the gradient salinity test are shown in Table 6 below.

[0053] Table 6 Gradient Salinity Test Results

[0054] The aforementioned differences are actually due to the unique regulatory effect of salinity stress on microalgal metabolism, as shown by the experimental results. Microalgae exhibit higher biomass at salinity levels of 5–18%. This result further confirms that moderate salinity stress can trigger compensatory growth mechanisms in microalgae, while excessive stress (such as above 18%) inhibits growth. Therefore, 13% salinity serves as a "stress threshold," achieving a balance between peak biomass and stress tolerance.

[0055] Example 4 (Optimization Experiment of Light Quality Combination)

[0056] In this example, the light quality combination optimization experiment, except for the seven-color LED orthogonal experiment and dynamic spectral control, the other cultivation conditions are basically the same as in Example 1, specifically including: Light control system: Light source configuration: The reactor has a built-in LED array that supports independent control of red light (620±5 nm), blue light (450±5 nm), and far-red light (730±5 nm).

[0057] Light intensity control: Precise adjustment of the light intensity ratio is achieved through PWM dimming technology (e.g., a 4:3:1 red:blue:far-red light intensity ratio). Red light: 160–200 μmol·m -2 ·s -1 (4 parts per batch); Blue light: 120–150 μmol·m -2 ·s -1 (3 parts per batch); Far-red light: 40–50 μmol·m -2 ·s -1 (1 serving).

[0058] Light quality regulation is a key method to improve microalgal EPA production. Different light quality combinations significantly alter EPA content by affecting photosynthetic efficiency and lipid synthesis pathways. Specific results are shown in Table 7.

[0059] Table 7 Experimental Results

[0060] According to the experimental results, the optimal combination is red (620nm) + blue (450nm).

[0061] The red-blue light combination (4:3) not only increased photosynthetic efficiency by 27%, but also achieved an EPA content of up to 18.9%, shortened the culture period to 10 days, and produced a total lipid yield of 468.8 mg / L / d, which was significantly better than other ratios.

[0062] Example 5 (50L scale-up production verification) 50L of microalgae

[0063] The culture was carried out based on the culture method of Example 1, and the following engineering parameters were modified:

[0064] Reduce the stirring speed to 100 rpm (to prevent shear damage); Airlift mixing is used instead of magnetic stirring; Load it into a 50 L photobioreactor.

[0065] Semi-continuous mode (15% harvest per day): can operate stably for 45 days.

[0066] result: (1) Biomass (1250±35 mg / L): determined by daily OD 680 Monitoring and plotting growth curves, R ² =0.993 indicates that the model fit is extremely high and the data is reliable.

[0067] (2) Total lipid content (37.5±1.2%): The total lipid content was determined by the sulfuric acid-vanillin method. Repeated experiments verified the stability of lipid accumulation.

[0068] (3) The specific value of SOD activity was 12±2 U / (g·min), which was obtained by Western blot quantitative analysis.

Claims

1. A method for regulating microalgal biomass, characterized in that, This includes ambient light regulation: alternating red and blue light is used, with a photocycle consisting of a 12-16 hour light period and an 8-12 hour dark period.

2. The method as described in claim 1, characterized in that, The red light wavelength is 615-660 nm, and the blue light wavelength is 445-480 nm. During the 12-16 h illumination period, red light and blue light are alternately irradiated, with the cumulative irradiation time of red light being 8-12 h and the cumulative irradiation time of blue light being 4-8 h.

3. The method as described in claim 2, characterized in that, The red light wavelength is 640 nm, the blue light wavelength is 465 nm, and the red light is cumulatively irradiated for 7 hours and the blue light is cumulatively irradiated for 5 hours.

4. The method as described in claim 1, characterized in that, The intensity of the irradiated light is 200-400 μmol•m. -2 •s -1 .

5. The method as described in claim 4, characterized in that, The intensity of the irradiated light was 300 μmol•m. -2 •s -1 .

6. The method as described in claim 1, characterized in that, It also includes a dynamic nitrogen supply strategy: an initial nitrogen concentration of 1.2-1.8 g / L, which is reduced by 0.05-0.4 g / L daily during the middle and late stages of cultivation.

7. The method as described in claim 1, characterized in that, It also includes stress treatment: the daily culture medium salinity is based on the baseline salinity and fluctuates periodically within the range of 5%-10%.

8. The method as described in claim 7, characterized in that, The stress treatment also includes daily low-temperature shocks of 13-17°C for 3-5 hours.

9. A method for cultivating microalgae, characterized in that, This includes inoculating fatty acid-producing microalgae into a culture medium and then regulating their cultivation using the method described in any one of claims 1 to 8.

Images