Method for directionally improving tolerance of haematococcus pluvialis acetate

Abstract

The invention relates to a method for directionally improving the tolerance of haematococcus pluvialis acetate, aiming at the problem that haematococcus pluvialis is sensitive to high-concentration acetate, the method comprises the following steps: gradually improving the tolerance of algae species through a staged adaptive evolution strategy, gradually adapting WT to 2.5 g / L sodium acetate through short-period-multi-cycle domestication, and obtaining a domesticated algae species HpAC25; on the basis of HpAC25, domesticating the HpAC25 to 5g / L to obtain HpAC50; further, on the basis of HpAC50, domesticating to obtain 7.5 g / L of sodium acetate, so as to obtain HpAC75. In the domestication process, suspended green motile cells are enriched through centrifugation, circulation passage is carried out every 3 days, the cell density is monitored, and the accumulative culture period reaches 189 days. Experiments show that the domesticated algae species HpAC50 and HpAC75 are cultured for 10 days under 5g / L sodium acetate and 7.5 g / L sodium acetate, and the survival rates of the domesticated algae species HpAC50 and HpAC75 reach 81.22% and 43.51% respectively and are increased by 422% and 460% compared with WT. The method is simple and convenient to operate, the tolerance of haematococcus pluvialis to acetate is remarkably enhanced, and a technical basis is provided for efficient production of astaxanthin and large-scale culture.

Description

Technical Field

[0001] The present invention relates to a method for directionally improving the acetate tolerance of Haematococcus pluvialis. Background Art

[0002] Haematococcus pluvialis is an important economic microalgae due to its high concentration of high-value natural astaxanthin. Haematococcus pluvialis can grow both autotrophically and heterotrophically using organic carbon sources. Acetate is a commonly used organic carbon source in Haematococcus pluvialis aquaculture, promoting both its growth and the accumulation of oil and astaxanthin under stress conditions. However, Haematococcus pluvialis has a low tolerance to acetate. High concentrations (>2.5 g / L) inhibit photosynthesis, causing cells to enter dormancy and accompanied by extensive cell damage and death. Targeted improvements in Haematococcus pluvialis's acetate tolerance would greatly promote the development of both the algae aquaculture industry and the natural astaxanthin industry. Currently, techniques for breeding salt-tolerant microalgae include mutagenesis, genetic engineering, and directed adaptive evolution. However, limited technical expertise exists in Haematococcus pluvialis. A search revealed one study that employed mutagenesis followed by targeted screening for algal strains tolerant to high concentrations of inorganic salts. (Yang, HE, Yu, BS, Sim, SJ2023. Enhanced astaxanthin production of Haematococcus pluvialis strains induced salt and high light resistance with gamma irradiation. Bioresource technology, 372, 128651.) Under stress conditions, the green, motile cells in the supernatant of Haematococcus pluvialis rapidly transform into dormant, immobile cells that settle to the bottom of the culture flask. Although dormant cells have higher stress resistance, their reduced metabolic activity slows their adaptive evolution, while active, motile cells have the potential for faster adaptive evolution. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a directed evolution method for acetate-resistant Haematococcus pluvialis, wherein green motile cells in the supernatant are collected, and short-cycle multi-cycle acclimation and passage are performed. After long-term acclimation and culture, acclimated Haematococcus pluvialis strains resistant to high-concentration acetate are obtained.

[0004] To solve the above technical problems, the present invention provides a method for targetedly improving the acetate tolerance of Haematococcus pluvialis, comprising the following steps:

[0005] (1) Basic culture: The modified BG11 medium was used as the basic culture medium to culture Haematococcus pluvialis WT. The specific components of the modified BG11 medium were: NaNO3 0.25 g / L, K2HPO4·3H2O 0.045 g / L, KH2PO4 0.175 g / L, MgSO4·7H2O0.1257 g / L, CaCl2·2H2O 0.0266 g / L, ferric citrate 0.0076 g / L, A5 mother liquor 1 mL / L, NaAc·3H2O 0.68 g / L, 100 mg / L ampicillin. The components of the A5 mother liquor were H3BO4 0.42 g / L, MnCl2·4H2O 1.42 g / L, NaMoO4 0.83 g / L, CuSO4·7H2O 1.57 g / L, CoSO4 0.1 g / L, culture system is a 250 mL conical flask with a liquid volume of 200 mL, culture temperature is 23 ° C, light intensity is 1500-2000 lux, light: dark = 12h:12h;

[0006] (2) WT adaptation to 2.5 g / L sodium acetate: The initial concentration of sodium acetate added to Haematococcus pluvialis during its green growth phase was controlled within 1 g / L. To obtain a stable acetate-tolerant algae strain, the sodium acetate concentration was first increased slightly from 1 g / L to 2.5 g / L. A high-density algal cell inoculation of 4 × 10 5 cells / mL for long-term culture, and the cell density was determined by hemocytometer every 3 days. All algal cells in the culture system were collected by centrifugation at 3500 rpm for 10 minutes and transferred to fresh 2.5 g / L sodium acetate acclimation medium: modified BG11 medium with a sodium acetate concentration of 2.5 g / L. The culture was repeated every 3 days. After 21 cycles, or 63 days of long-term culture, the surviving algal cells in the culture system were collected, plated and streaked for preservation, and the purified algal strain was named HpAC25. To prevent the degeneration of HpAC25 traits, it was cultured under 2.5 g / L sodium acetate for a long time.

[0007] (3) Adaptive evolution of HpAC25 to 5 g / L sodium acetate: HpAC25 was cultured in 2.5 g / L sodium acetate acclimation medium until the logarithmic growth phase. An appropriate amount of the upper algal liquid was collected and centrifuged at 3500 rpm for 10 min to collect the green motile cells. The cells were then inoculated into 5 g / L sodium acetate acclimation medium at an initial density of approximately 1.3 × 10 5cells / mL, samples were taken every 3 days to calculate the cell density, and an appropriate amount of the upper layer of algae liquid was taken and centrifuged to collect the green motile cells. The cells were then transferred to fresh 5g / L sodium acetate acclimation medium. This cycle was repeated for 3 days. After 24 cycles, or 72 days of long-term culture, the suspended green motile cells in the culture system were collected and plated for preservation. The purified algae strain was named HpAC50. To prevent the degradation of HpAC50 traits, it was stored in 5g / L sodium acetate culture conditions for a long time.

[0008] (4) Adaptive evolution of HpAC50 to 7.5 g / L sodium acetate: HpAC50 was cultured in 5 g / L sodium acetate acclimation medium until the logarithmic growth phase. An appropriate amount of the upper algal liquid was collected and centrifuged at 3500 rpm for 10 min to collect the green motile cells. The cells were then inoculated into 7.5 g / L sodium acetate acclimation medium at an initial density of approximately 1 × 10 5 cells / mL, samples were taken every 3 days to calculate the cell density, and an appropriate amount of the upper and middle layer algae liquid was taken and centrifuged to collect the green motile cells, which were then transferred to fresh 7.5 g / L sodium acetate acclimation medium. This cycle was repeated, with 3 days as one cycle. After 18 cycles, or 54 days of long-term culture, the suspended green motile cells in the culture system were collected, plated and streaked for preservation, and the purified algae strain was named HpAC75. To prevent the degeneration of HpAC75 traits, it was stored long-term in 7.5 g / L sodium acetate culture conditions.

[0009] Advantages of the present invention: To address the shortcomings of poor acetate tolerance of Haematococcus pluvialis, the present invention specifically targets suspended green motile cells with greater adaptive evolutionary potential. Through a long-term, short-cycle, multi-cycle acclimation method, the acetate culture concentration is gradually increased. The resulting acclimated algae strains, after 10 days of culturing under high sodium acetate concentrations (5 g / L and 7.5 g / L), have cell survival rates of 81.22% and 43.51%, respectively, compared to the survival rates of the initial algae strain WT (15.54% and 7.77%). The acclimated algae strains of Haematococcus pluvialis obtained based on this method have significantly improved acetate tolerance. This technical solution is simple and easy to implement, and has reference significance for the targeted acclimation of Haematococcus pluvialis and related species. It significantly improves the acetate tolerance of Haematococcus pluvialis, laying the foundation for improving the aquaculture level of Haematococcus pluvialis and astaxanthin production capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the algal solution and cell morphology of WT cultured under different acetate gradients for 6 days.

[0011] Figure 2 This is a graph showing changes in cell density and specific growth rate of WT after acclimation to 2.5 g / L sodium acetate.

[0012] Figure 3This is a graph showing changes in cell density and specific growth rate of HpAC25 after acclimation to 5 g / L sodium acetate.

[0013] Figure 4 This is a graph showing the changes in cell density and specific growth rate of HpAC50 after acclimation to 7.5 g / L sodium acetate.

[0014] Figure 5 This is the live-dead staining image of the acclimated algae and WT on the 10th day of culture under acetate stress.

[0015] Figure 6 This is a statistical chart of the survival rates of acclimated algae and WT on the 10th day of culture under acetate stress. DETAILED DESCRIPTION

[0016] A method for targetedly improving the acetate tolerance of Haematococcus pluvialis comprises the following steps:

[0017] (1) Basic culture conditions: The modified BG11 medium was used as the basic culture medium to culture Haematococcus pluvialis. The specific components were NaNO3 0.25 g / L, K2HPO4·3H2O 0.045 g / L, KH2PO4 0.175 g / L, MgSO4·7H2O 0.1257 g / L, CaCl2·2H2O 0.0266 g / L, ferric citrate 0.0076 g / L, A5 mother solution 1 mL / L, NaAc·3H2O 0.68 g / L, and 100 mg / L ampicillin. The A5 mother solution components were H3BO4 0.42 g / L, MnCl2·4H2O 1.42 g / L, NaMoO4 0.83 g / L, CuSO4·7H2O 1.57 g / L, CoSO4 0.1 g / L, culture system is a 250 mL conical flask with a liquid volume of 200 mL, culture temperature is 23 ° C, light intensity is 1500-2000 lux, light: dark = 12h:12h;

[0018] (2) Microalgae cell density counting: After mixing the liquid in the above culture medium, take 10 μL of algae liquid and count the cell density using a hemocytometer under an inverted fluorescence microscope. Take samples and count them 6 times to get the average value. Calculate the specific growth rate: The specific growth rate calculation formula is:

[0019] μ(d -1 )=(lnX2-lnX1) / (t2-t1)

[0020] Where X2 is the cell density on culture day t2, and X1 is the cell density on culture day t1;

[0021] (3) WT sodium acetate tolerance test: Set the sodium acetate concentration gradient at 0, 0.5, 1, 1.5, 2.5, and 5 g / L, culture the initial algae strain WT of Haematococcus pluvialis, count the changes in cell density, and use a microscope to take pictures of the algal cell morphology at a 40x field of view. Figure 1 As shown, it shows that sodium acetate above 2.5g / L will cause the WT algae liquid to sink to the bottom, the algae liquid to bleach, and a large number of cells to die. Based on this, it can be judged that sodium acetate above 2.5g / L has a significant growth inhibition on Haematococcus pluvialis WT;

[0022] (4) WT adaptation to 2.5 g / L sodium acetate: The concentration of sodium acetate added during the green growth phase of Haematococcus pluvialis was between 0 and 1 g / L. To obtain a stable acetate-tolerant algae strain, the sodium acetate concentration was first increased slightly from 1 g / L to 2.5 g / L. A high-density algal cell inoculation of 4 × 10 5 Cells / mL were cultured for a long time. The cell density was detected by hemocytometer every 3 days. All algal cells in the culture system were collected by centrifugation at 3500 rpm for 10 min and added to fresh 2.5 g / L sodium acetate acclimation medium: modified BG11 medium with a sodium acetate concentration of 2.5 g / L. The culture was repeated every 3 days. After 21 cycles, i.e. 63 days of long-term culture, the changes in cell density and specific growth rate during the acclimation culture process are shown in the attached figure. Figure 2 As shown, the surviving algal cells in the culture system were collected and plated for preservation, and the purified algal species was named HpAC25. To prevent the degradation of HpAC25 traits, it was stored in 2.5 g / L sodium acetate culture conditions for a long time.

[0023] (5) Adaptive evolution of HpAC25 to 5 g / L sodium acetate: HpAC25 was cultured in 2.5 g / L sodium acetate acclimation medium until the logarithmic growth phase. An appropriate amount of the upper algal liquid was collected by centrifugation at 3500 rpm for 10 min to collect the green motile cells, which were then inoculated into 5 g / L sodium acetate acclimation medium at an initial density of approximately 1.3 × 10 5 cells / mL, sample the cell density every 3 days, and take an appropriate amount of the upper layer of algae liquid, centrifuge to collect the green swimming cells, and transfer them to fresh 5g / L sodium acetate acclimation medium. This cycle is repeated, with 3 days as one cycle. After 24 cycles, that is, 72 days of long-term culture, the cell density changes during the acclimation culture process are as follows Figure 3 As shown, the green motile cells suspended in the culture system were collected and plated for preservation, and the purified algae species was named HpAC50. To prevent the degradation of HpAC50 traits, it was stored in 5g / L sodium acetate culture conditions for a long time;

[0024] (6) Adaptive evolution of HpAC50 to 7.5 g / L sodium acetate: HpAC50 was cultured in 5 g / L sodium acetate acclimation medium until the logarithmic growth phase. An appropriate amount of the upper algal liquid was collected and centrifuged at 3500 rpm for 10 min to collect the green motile cells. The cells were then inoculated into 7.5 g / L sodium acetate acclimation medium at an initial density of approximately 1 × 10 5 cells / mL, sampled every 3 days to count the cell density, and at the same time, took an appropriate amount of the upper layer of algae liquid, centrifuged to collect the green swimming cells, and inoculated them into fresh 7.5g / L sodium acetate acclimation medium. This cycle was repeated, with 3 days as one cycle. After 18 cycles, that is, 54 days of long-term culture, the cell density changes during the acclimation culture process were as follows Figure 4 As shown, the green motile cells suspended in the culture system were collected and plated for preservation, and the purified algae species was named HpAC75. To prevent the degradation of the characteristics of HpAC75, it was stored in 7.5 g / L sodium acetate culture conditions for a long time;

[0025] (7) Observation and statistics of cell survival rate under sodium acetate stress: The algal cells of the domesticated algae species and WT were cultured and stained for live and dead at the concentrations of 2.5 g / L, 5 g / L, and 7.5 g / L sodium acetate, respectively. Fluorescein diacetate (FDA) was used to stain the living cells, which showed green fluorescence, and propidium iodide (PI) was used to stain the dead cells, which showed red fluorescence. Figure 5 As shown. A 5 mg / mL FDA master stock solution was prepared with dimethyl sulfoxide (DMSO). The master stock solution was then diluted 100-fold to prepare a working stock solution of FDA. The solution was stored on ice in the dark. 40 μL of the FDA working stock solution was added to 1 mL of the reaction sample to make the final concentration of FDA in the reaction mixture 2 μg / mL. A 3 mg / mL PI master stock solution was prepared with ultrapure water and stored at 4°C. The master stock solution was diluted 10-fold with ultrapure water to prepare a fresh working stock solution for each use. 10 μL of the PI working stock solution was added to 1 mL of the reaction sample to make the final concentration of PI in the reaction sample 3 μg / mL. The reaction mixture was incubated on ice in the dark for 30 minutes. Finally, the algal cells were detected for live and dead cells using an inverted fluorescence microscope. The number of live and dead cells at random viewing angles was counted using Image J software. The counts were repeated 9 times and the average was calculated using the following formula:

[0026] Cell viability (%) = number of living cells (number of living cells + number of dead cells) × 100%.

[0027] Under the corresponding acetate stress, the survival rate of the domesticated algae species increased significantly, especially HpAC50 and HpAC75, the survival rate increased by 422% and 460%, respectively. Figure 6 shown.

Claims

1. A method for targetedly improving the acetate tolerance of Haematococcus pluvialis, characterized in that: The following steps are involved: (1) Basic culture: The modified BG11 medium was used as the basic culture medium to culture Haematococcus pluvialis WT. The specific components of the modified BG11 medium were: NaNO3 0.25 g / L, K2HPO4·3H2O 0.045 g / L, KH2PO4 0.175 g / L, MgSO4·7H2O0.1257 g / L, CaCl2·2H2O 0.0266 g / L, ferric citrate 0.0076 g / L, A5 mother liquor 1 mL / L, NaAc·3H2O 0.68 g / L, 100 mg / L ampicillin. The components of the A5 mother liquor were H3BO4 0.42 g / L, MnCl2·4H2O 1.42 g / L, NaMoO4 0.83 g / L, CuSO4·7H2O 1.57 g / L, CoSO4 0.1 g / L, culture system is a 250 mL conical flask with a liquid volume of 200 mL, culture temperature is 23 ° C, light intensity is 1500-2000 lux, light: dark = 12h:12h; (2) WT adaptation to 2.5 g / L sodium acetate: The initial concentration of sodium acetate added to Haematococcus pluvialis during its green growth phase was controlled within 1 g / L. To obtain a stable acetate-tolerant algae strain, the sodium acetate concentration was first increased slightly from 1 g / L to 2.5 g / L. A high-density algal cell inoculation of 4 × 10 5 cells / mL for long-term culture, and the cell density was determined by hemocytometer every 3 days. All algal cells in the culture system were collected by centrifugation at 3500 rpm for 10 minutes and transferred to fresh 2.5 g / L sodium acetate acclimation medium: modified BG11 medium with a sodium acetate concentration of 2.5 g / L. The culture was repeated every 3 days. After 21 cycles, or 63 days of long-term culture, the surviving algal cells in the culture system were collected, plated and streaked for preservation, and the purified algal strain was named HpAC25. To prevent the degeneration of HpAC25 traits, it was cultured under 2.5 g / L sodium acetate for a long time. (3) Adaptive evolution of HpAC25 to 5 g / L sodium acetate: HpAC25 was cultured in 2.5 g / L sodium acetate acclimation medium until the logarithmic growth phase. An appropriate amount of the upper algal liquid was collected and centrifuged at 3500 rpm for 10 min to collect the green motile cells. The cells were then inoculated into 5 g / L sodium acetate acclimation medium at an initial density of approximately 1.3 × 10 5 cells / mL, samples were taken every 3 days to calculate the cell density, and an appropriate amount of the upper layer of algae liquid was taken and centrifuged to collect the green motile cells. The cells were then transferred to fresh 5g / L sodium acetate acclimation medium. This cycle was repeated for 3 days. After 24 cycles, or 72 days of long-term culture, the suspended green motile cells in the culture system were collected and plated for preservation. The purified algae strain was named HpAC50. To prevent the degradation of HpAC50 traits, it was stored in 5g / L sodium acetate culture conditions for a long time. (4) Adaptive evolution of HpAC50 to 7.5 g / L sodium acetate: HpAC50 was cultured in 5 g / L sodium acetate acclimation medium until the logarithmic growth phase. An appropriate amount of the upper algal liquid was collected and centrifuged at 3500 rpm for 10 min to collect the green motile cells. The cells were then inoculated into 7.5 g / L sodium acetate acclimation medium at an initial density of approximately 1 × 10 5 cells / mL, samples were taken every 3 days to calculate the cell density, and an appropriate amount of the upper and middle layer algae liquid was taken and centrifuged to collect the green motile cells, which were then transferred to fresh 7.5 g / L sodium acetate acclimation medium. This cycle was repeated, with 3 days as one cycle. After 18 cycles, or 54 days of long-term culture, the suspended green motile cells in the culture system were collected, plated and streaked for preservation, and the purified algae strain was named HpAC75. To prevent the degeneration of HpAC75 traits, it was stored long-term in 7.5 g / L sodium acetate culture conditions.

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