Method for improving carbon fixation by regulating photosynthetic disintegration metabolism function distribution of microalgae

Abstract

The present application relates to microalgae biology and carbon emission reduction technology, aiming to provide a method for improving carbon fixation amount by environmental regulation of microalgae photosynthetic splitting and metabolic function distribution. The method comprises: inoculating microalgae cells in a basic medium, and pre-culturing under the condition of CO2-containing flue gas being introduced; detecting the photosynthetic pigment content and gene expression characteristics of the microalgae cell population, and selecting samples that meet the requirements of chlorophyll and carotenoid concentration ratio, photosynthetic index, splitting index and metabolic index at the same time; and transferring the microalgae cell population screened to a photobioreactor for engineering scale-up culture to realize carbon fixation. The present application establishes a scientific function state screening method by detecting the chlorophyll and carotenoid concentration ratio and the expression level of three types of function genes, which can accurately identify the microalgae population with the best photosynthetic-splitting-metabolic function distribution in the pre-culture stage; without genetic engineering or chemical additives, it can be realized only by simple environmental regulation, which has significant economic benefits.

Description

Technical Field

[0001] This invention belongs to the fields of microalgae biotechnology and carbon emission reduction technology, specifically involving a method to increase carbon sequestration by regulating the allocation of photosynthetic, fission, and metabolic functions of microalgae through environmental regulation. Background Technology

[0002] Microalgae, as photosynthetic autotrophic microorganisms, possess advantages such as high photosynthetic efficiency, short growth cycle, and strong CO2 fixation capacity, making them ideal carriers for biofuel production and CO2 fixation. Utilizing microalgae to convert and utilize flue gas from coal-fired power plants (containing 10%–15% CO2) has enormous potential; however, its actual efficiency remains constrained by fundamental issues such as unclear mechanisms of division of labor and cooperation within microalgal communities and undefined responses to environmental factors.

[0003] Previous research has proposed many effective strategies to promote CO2 conversion into microalgal biomass, such as using nutrient-rich wastewater to increase microalgal growth rates, exploring the effects of auxins on microalgal biomass dry weight and fatty acid accumulation, and studying the synergistic effects of nutrients and flash frequency on *Microcystis aeruginosa*. However, most studies have focused on macroscopic experiments, often neglecting in-depth exploration of underlying scientific mechanisms. Although a few studies have used genomics, transcriptomics, and proteomics to explore internal mechanisms, these studies mainly focus on the overall characteristics of the entire cell population under specific conditions and have not yet proposed optimized strategies for microalgal carbon fixation from the perspective of photosynthetic-division-metabolic functional allocation.

[0004] Therefore, proposing a method to increase carbon fixation by regulating the allocation of photosynthetic, fission, and metabolic functions of microalgae through environmental regulation is of great significance for improving the economic efficiency of microalgae in industrial production. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for improving carbon fixation by regulating the allocation of photosynthetic division and metabolic functions of microalgae through environmental regulation.

[0006] To solve the technical problem, the solution of the present invention is:

[0007] A method for improving carbon fixation by regulating the allocation of photosynthetic fission metabolic functions in microalgae through environmental regulation includes the following steps:

[0008] (1) Inoculate microalgae cells into a basic culture medium and pre-culture for 2-4 days under conditions of CO2 flue gas introduction;

[0009] (2) Detect the photosynthetic pigment content and gene expression characteristics of the microalgal cell population after pre-culture, and select microalgal population samples that meet the following requirements: chlorophyll to carotenoid concentration ratio of 4 to 4.6, photosynthetic index of 0.35 to 0.4, cell division index of 0.45 to 0.49, and metabolic index of 0.15 to 0.16;

[0010] (3) The selected microalgal cell population is transferred into a photoreactor for engineering-scale culture to achieve carbon fixation.

[0011] As a preferred embodiment of the present invention, the microalgae is one or more of Haematococcus pluvialis, Chlorella proteoglycans, or Micrococcus microcarpa.

[0012] As a preferred embodiment of the present invention, the initial microalgal cells used for pre-culture have a dry weight of 0.2~0.4 g / L.

[0013] As a preferred embodiment of the present invention, the basal culture medium refers to BG-11 basal culture medium, which is prepared as follows: Add the following components in the following amounts to 1L of water: 1mg disodium ethylenediaminetetraacetate, 6mg ferric ammonium citrate, 6mg citric acid monohydrate, 36mg calcium chloride dihydrate, 75mg magnesium sulfate heptahydrate, 45mg dipotassium hydrogen phosphate trihydrate, and 20mg sodium carbonate; after mixing, sterilize at 121°C for 15 minutes; then add 1.5g sodium nitrate, mix well, and sterilize again at 121°C for 15 minutes, and then cool for later use.

[0014] As a preferred embodiment of the present invention, the flue gas introduced during the pre-cultivation and expansion cultivation is obtained by removing dust and desulfurizing the exhaust gas from a coal-fired boiler, and the flue gas contains 10% to 20% CO2 by volume.

[0015] As a preferred embodiment of the present invention, the chlorophyll to carotenoid concentration ratio is determined by spectrophotometry.

[0016] As a preferred embodiment of the present invention, the methods for measuring the photosynthetic index, cell division index, and metabolic index are as follows:

[0017] (1) The expression levels of three types of functional genes in microalgal cell populations—photosynthetic functional genes, division functional genes, and metabolic functional genes—were determined by real-time quantitative PCR.

[0018] (2) Calculate the average expression levels of the three types of genes respectively;

[0019] (3) Normalize the average expression levels of the three types of genes to convert them into proportional values, which gives the photosynthetic index, cell division index and metabolic index of the sample. The sum of the three is 1.

[0020] As a preferred embodiment of the present invention, the photosynthetic functional genes are ribulose-1,5-bisphosphate carboxylase gene, cytochrome b / f complex gene, and ATP-dependent Clp protease gene; the mitotic functional genes are S-adenosyl-L-methionine-dependent methyltransferase gene, protein CCDC61 gene containing a coiled-coil domain, and ribosomal protein S3 gene; the metabolic functional genes are proline dipeptidase gene, E3 ubiquitin protein ligase HRD1 gene, and 3'5'-cyclic nucleotide phosphodiesterase gene.

[0021] As a preferred embodiment of the present invention, during the engineering scale-up culture, the light intensity is controlled at 5000~10000 Lux, the temperature at 20~28℃, and the culture cycle is 6 days; the algal solution in the photoreactor is collected every day to test the growth rate and carbon fixation rate of the microalgae.

[0022] As a preferred embodiment of the present invention, the growth rate and carbon fixation rate of microalgae are determined according to the following method:

[0023] (1) Collect algal powder and test the carbon content T (%) in microalgal biomass using the Dumall combustion total organic carbon (TOC) method of an elemental analyzer.

[0024] (2) Collect algal liquid samples daily, centrifuge and rinse multiple times, dry to constant weight, weigh and calculate biomass density DW;

[0025] (3) The calculation method for the microalgal growth rate u is as follows:

[0026] u (g / L / day)=(DW2-DW1) / (t2-t1)

[0027] Fixed flue gas CO2 rate V CO2 The calculation method is as follows:

[0028] V CO2 =u×T×44 / 12

[0029] Where DW2 is the biomass density (g / L) at day t2, and DW1 is the biomass density (g / L) at day t1.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention establishes a scientific method for screening the functional status of microalgal populations by detecting the concentration ratio of chlorophyll to carotenoids and the expression levels of three types of functional genes. This method can accurately identify microalgal populations with optimal photosynthetic-division-metabolic functional allocation during the pre-culture stage.

[0031] 2. The method of the present invention does not require genetic engineering or chemical additives, and can be achieved through simple environmental regulation, which is a zero-cost CO2 fixation optimization strategy.

[0032] 3. Compared with traditional cultivation techniques, this invention has significant economic benefits. Under identical conditions, the average carbon fixation rate of unselected primitive microalgae is 0.19–0.24 g / L / d. After pre-culture and screening, the average carbon fixation rate is 0.4–0.5 g / L / d, representing an increase of 108%–119%. Attached Figure Description

[0033] Figure 1 This is a flowchart of the method described in this invention. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0035] Part One: Implementation Scheme of the Invention

[0036] 1. The method for improving carbon fixation by regulating the allocation of photosynthetic fission metabolic functions in microalgae according to the present invention includes the following steps: (1) Pre-culture stage:

[0037] Microalgae cells were inoculated into a basal culture medium and pre-cultured for 2-4 days under conditions of CO2 flue gas.

[0038] As an optional example, the microalgae is one or more of Haematococcus pluvialis, Chlorella proteoglycans, or Micrococcus microcarpa. The dry weight of the initial microalgal cells used for pre-culture is 0.2–0.4 g / L.

[0039] The basal culture medium refers to BG-11 basal culture medium, which is prepared as follows: Add the following components to 1L of water in the following amounts: 1mg disodium ethylenediaminetetraacetate, 6mg ferric ammonium citrate, 6mg citric acid monohydrate, 36mg calcium chloride dihydrate, 75mg magnesium sulfate heptahydrate, 45mg dipotassium hydrogen phosphate trihydrate, and 20mg sodium carbonate; mix well and sterilize at 121℃ for 15 minutes; then add 1.5g sodium nitrate, mix well and sterilize again at 121℃ for 15 minutes, and cool before use.

[0040] During the pre-cultivation and scale-up cultivation, the flue gas introduced is obtained by removing dust and desulfurizing the exhaust gas from the coal-fired boiler. The flue gas contains 10% to 20% CO2 by volume.

[0041] (2) Functional indicator detection and screening:

[0042] The photosynthetic pigment content and gene expression characteristics of microalgal cell populations after pre-culture were detected. Microalgal population samples that simultaneously met the following requirements were selected: chlorophyll to carotenoid concentration ratio of 4-4.6, photosynthetic index of 0.35-0.4, cell division index of 0.45-0.49, and metabolic index of 0.15-0.16.

[0043] The chlorophyll to carotenoid concentration ratio was determined spectrophotometrically. An example is shown below:

[0044] Centrifuge 1 mL of algal solution to remove the supernatant, then add deionized water to wash and collect the algal sludge. Then add 3 mL of anhydrous methanol, mix evenly with the algal sludge, and treat in the dark for 15 min before centrifugation. Collect the supernatant and use a spectrophotometer to measure the absorbance at wavelengths of 665 nm, 652 nm and 480 nm, and record them as A665, A652 and A480 respectively.

[0045] [Chlorophyll a] μg / mL = 16.29 × A 665 – 8.54 × A 652

[0046] [Chlorophyll b] μg / mL = 27.44×A 652 -12.17×A 665

[0047] [Carotenoids] μg / mL = 4 × A 480

[0048] Chlorophyll to carotenoid concentration ratio = (chlorophyll a + chlorophyll b) / carotenoid

[0049] The methods for measuring photosynthetic, fibrillation, and metabolic indicators are as follows:

[0050] (2.1) The expression levels of three types of functional genes in microalgal cell populations—photosynthetic functional genes, division functional genes, and metabolic functional genes—were determined by real-time quantitative PCR.

[0051] The photosynthetic functional genes are ribulose-1,5-bisphosphate carboxylase gene, cytochrome b / f complex gene, and ATP-dependent Clp protease gene; the mitotic functional genes are S-adenosyl-L-methionine-dependent methyltransferase gene, CCDC61 gene containing a coiled-coil domain, and ribosomal protein S3 gene; the metabolic functional genes are proline dipeptidase gene, E3 ubiquitin protein ligase HRD1 gene, and 3'5'-cyclic nucleotide phosphodiesterase gene. The expression level determination of these functional genes is a well-known technique and will not be elaborated further in this invention.

[0052] (2.2) Calculate the average expression levels of the three types of genes respectively;

[0053] (2.3) Normalize the average expression levels of the three types of genes to convert them into proportional values, which gives the photosynthetic index, cell division index and metabolic index of the sample. The sum of the three is 1.

[0054] (3) Engineering-scale cultivation:

[0055] The selected microalgal cell populations were transferred into a photoreactor for engineered large-scale culture to achieve carbon fixation.

[0056] During the large-scale engineering cultivation, the light intensity was controlled at 5000~10000 Lux, the temperature at 20~28℃, and the cultivation cycle was 6 days. The algal solution in the photoreactor was collected daily to test the growth rate and carbon fixation rate of the microalgae.

[0057] Specifically, the growth rate and carbon fixation rate of microalgae were determined using the following methods:

[0058] (1) Collect algal powder and test the carbon content T (%) in microalgal biomass using the Dumall combustion total organic carbon (TOC) method of an elemental analyzer.

[0059] (2) Collect algal liquid samples daily, centrifuge and rinse multiple times, dry to constant weight, weigh and calculate biomass density DW;

[0060] (3) The calculation method for the microalgal growth rate u is as follows:

[0061] u (g / L / day)=(DW2-DW1) / (t2-t1)

[0062] Fixed flue gas CO2 rate V CO2 The calculation method is as follows:

[0063] V CO2 =u×T×44 / 12

[0064] Where DW2 is the biomass density (g / L) at day t2, and DW1 is the biomass density (g / L) at day t1.

[0065] 2. The significance of increasing the expression levels of nine key genes in microalgae in this invention:

[0066] In microalgae, changes in the expression levels of these nine genes indicate physiological states at three levels: photosynthesis, cell division, and metabolism.

[0067] (1) In terms of photosynthesis: upregulation of the ribulose-1,5-bisphosphate carboxylase (Rubisco) gene indicates enhanced carbon assimilation (rapid growth or low CO2 stress), while downregulation suggests photoinhibition or nitrogen deficiency; high expression of the cytochrome b / f complex gene reflects active electron transport, while low expression indicates electron chain obstruction or photodamage; upregulation of the ATP-dependent Clp protease gene indicates the need to repair photosystem II protein under photooxidative stress, while downregulation leads to damage accumulation.

[0068] (2) In terms of division: high expression of S-adenosine-L-methionine-dependent methyltransferase gene promotes cell cycle and epigenetic regulation, while low expression causes cell cycle arrest; upregulation of the coiled-coil domain protein CCDC61 gene indicates mitosis or flagellar regeneration, while downregulation leads to abnormal division; high expression of ribosomal protein S3 gene reflects vigorous protein synthesis and rapid proliferation, while low expression indicates quiescence or stress tolerance.

[0069] (3) In terms of metabolism: Upregulation of the proline dipeptidase gene responds to hyperosmolarity, heavy metals, or nitrogen starvation by releasing proline to protect cells, while downregulation conserves resources under non-stress conditions; high expression of the E3 ubiquitin ligase HRD1 gene indicates endoplasmic reticulum stress and clearance of misfolded proteins, while low expression may lead to the accumulation of toxic proteins; high expression of the 3',5'-cyclic nucleotide phosphodiesterase gene reduces cAMP levels and shuts down signaling, while low expression prolongs cAMP signaling to adapt to sudden environmental changes. These gene expression profiles can be used to diagnose the physiological state of microalgae, optimize culture conditions, and guide genetic modification.

[0070] Part Two: Specific Implementation Examples

[0071] Example 1

[0072] (1) Pre-culture stage

[0073] Using Haematococcus pluvialis as the experimental algae species, microalgae with an initial biomass dry weight of 0.3 g / L were placed in an Erlenmeyer flask and cultured in flue gas containing 15% CO2.

[0074] (2) Functional indicator detection and screening

[0075] After 3 days of cultivation, the photosynthetic pigment content of the microalgal cell population was measured, and the chlorophyll to carotenoid concentration ratio was found to be 4.3. The expression levels of three functional genes were determined using real-time quantitative PCR. After normalization, the photosynthetic index was 0.38, the cell division index was 0.465, and the metabolic index was 0.155, which met the screening criteria.

[0076] (3) Engineering-scale cultivation

[0077] The selected microalgal population was transferred to a column-type photobioreactor, where the CO2 concentration was controlled at 15%, the light intensity at 8000 Lux, and the temperature at 25℃, and cultured for 6 days. The average carbon fixation rate was measured to be 0.46 g / L / d.

[0078] Example 2

[0079] (1) Pre-culture stage

[0080] Using Chlorella pyrenoidosa as the experimental algae species, microalgae with an initial biomass dry weight of 0.2 g / L were placed in conical flasks and cultured in flue gas containing 10% CO2.

[0081] (2) Functional indicator detection and screening

[0082] After 2 days of cultivation, the photosynthetic pigment content of the microalgal cell population was measured, and the chlorophyll to carotenoid concentration ratio was found to be 4. The functional indicators were: photosynthetic index 0.35, cell division index 0.49, and metabolic index 0.16, which met the screening criteria.

[0083] (3) Engineering-scale cultivation

[0084] The selected microalgal population was transferred to a photoreactor, where the CO2 concentration was controlled at 10%, the light intensity at 5000 Lux, and the temperature at 20℃, and cultured for 6 days. The average carbon fixation rate was measured to be 0.5 g / L / d.

[0085] Example 3

[0086] (1) Pre-culture stage

[0087] Using *Nannochloropsis gaditana* as the experimental algae species, microalgae with an initial biomass dry weight of 0.4 g / L were placed in conical flasks and cultured in flue gas containing 20% ​​CO2.

[0088] (2) Functional indicator detection and screening

[0089] After 4 days of cultivation, the photosynthetic pigment content of the microalgal cell population was measured, and the chlorophyll to carotenoid concentration ratio was found to be 4.6. The functional indicators were: photosynthetic index 0.4, cell division index 0.45, and metabolic index 0.15, which met the screening criteria.

[0090] (3) Engineering-scale cultivation

[0091] The selected microalgal population was transferred to a photoreactor, where the CO2 concentration was controlled at 20%, the light intensity at 10,000 Lux, and the temperature at 28℃, and cultured for 6 days. The average carbon fixation rate was measured to be 0.4 g / L / d.

[0092] Comparative Example 1

[0093] Under the exact same conditions as in Example 1, unpre-cultured and unscreened primitive Haematococcus pluvialis were cultured, with an average carbon fixation rate of 0.21 g / L / d.

[0094] Comparative Example 2

[0095] Under the exact same conditions as in Example 2, unpre-cultured and unscreened Chlorella nucleatum were cultured, with an average carbon fixation rate of 0.24 g / L / d.

[0096] Comparative Example 3

[0097] Under the exact same conditions as in Example 3, unpre-cultured and unscreened primitive Micrococcus was cultured, with an average carbon fixation rate of 0.19 g / L / d.

[0098] As can be seen from the experimental data of the various embodiments and comparative examples, under exactly the same culture conditions, the present invention can effectively improve the carbon fixation efficiency of microalgae. Compared with their respective comparative examples, the carbon fixation efficiency of the embodiments of the present invention is increased by 119%, 108%, and 111%, respectively, thus making it highly economical for industrial-scale microalgae carbon fixation production.

[0099] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of this invention should be considered within the scope of protection of this invention.

Claims

1. A method for improving carbon fixation by regulating the allocation of photosynthetic fission metabolic functions in microalgae through environmental regulation, characterized in that... Includes the following steps: (1) Inoculate microalgae cells into a basic culture medium and pre-culture for 2-4 days under conditions of CO2 flue gas introduction; (2) Detect the photosynthetic pigment content and gene expression characteristics of the microalgal cell population after pre-culture, and select microalgal population samples that meet the following requirements: chlorophyll to carotenoid concentration ratio of 4 to 4.6, photosynthetic index of 0.35 to 0.4, cell division index of 0.45 to 0.49, and metabolic index of 0.15 to 0.16; (3) The selected microalgal cell population is transferred into a photoreactor for engineering-scale culture to achieve carbon fixation.

2. The method according to claim 1, wherein the microalgae is one or more of Haematococcus pluvialis, Chlorella proteoglycans, or Micrococcus microcarpa.

3. The method according to claim 1, characterized in that, The initial microalgal cells used for pre-culture have a dry weight of 0.2~0.4 g / L.

4. The method according to claim 1, characterized in that, The basal culture medium refers to BG-11 basal culture medium, which is prepared as follows: Add the following components to 1L of water in the following amounts: 2mg disodium ethylenediaminetetraacetate, 6mg ferric ammonium citrate, 6mg citric acid monohydrate, 36mg calcium chloride dihydrate, 75mg magnesium sulfate heptahydrate, 45mg dipotassium hydrogen phosphate trihydrate, and 20mg sodium carbonate; mix well and sterilize at 121℃ for 15 minutes; then add 1.5g sodium nitrate, mix well and sterilize again at 121℃ for 15 minutes, and cool before use.

5. The method according to claim 1, characterized in that, The flue gas introduced during the pre-cultivation and expansion cultivation is obtained by removing dust and desulfurizing the exhaust gas from a coal-fired boiler, and the flue gas contains 10% to 20% CO2 by volume.

6. The method according to claim 1, characterized in that, The chlorophyll to carotenoid concentration ratio was determined by spectrophotometry.

7. The method according to claim 1, characterized in that, The methods for measuring the photosynthetic, fibrillation, and metabolic indicators are as follows: (1) The expression levels of three types of functional genes in microalgal cell populations—photosynthetic functional genes, division functional genes, and metabolic functional genes—were determined by real-time quantitative PCR. (2) Calculate the average expression levels of the three types of genes respectively; (3) Normalize the average expression levels of the three types of genes to convert them into proportional values, which gives the photosynthetic index, cell division index and metabolic index of the sample. The sum of the three is 1.

8. The method according to claim 5, characterized in that, The photosynthetic functional genes are ribulose-1,5-bisphosphate carboxylase gene, cytochrome b / f complex gene, and ATP-dependent Clp protease gene; the mitotic functional genes are S-adenosyl-L-methionine-dependent methyltransferase gene, CCDC61 protein gene containing coiled-coil domain, and ribosomal protein S3 gene; the metabolic functional genes are proline dipeptidase gene, E3 ubiquitin protein ligase HRD1 gene, and 3'5'-cyclic nucleotide phosphodiesterase gene.

9. The method according to claim 1, characterized in that, During the large-scale engineering cultivation, the light intensity was controlled at 5000~10000 Lux, the temperature at 20~28℃, and the cultivation cycle was 6 days. The algal solution in the photoreactor was collected daily to test the growth rate and carbon fixation rate of the microalgae.

10. The method according to claim 1, characterized in that, The growth rate and carbon fixation rate of microalgae were determined using the following methods: (1) Collect algal powder and test the carbon content T (%) in microalgal biomass using the Dumall combustion total organic carbon method with an elemental analyzer. (2) Collect algal liquid samples daily, centrifuge and rinse multiple times, dry to constant weight, weigh and calculate biomass density DW; (3) The calculation method for the microalgal growth rate u is as follows: u (g / L / day)=(DW2-DW1) / (t2-t1) Fixed flue gas CO2 rate V CO2 The calculation method is as follows: In CO2 =u×T×44 / 12 Where DW2 is the biomass density (g / L) at day t2, and DW1 is the biomass density (g / L) at day t1.

Images