Method for improving key enzyme to promote euglena to degrade phenol and enhance carbon sequestration

Abstract

The invention discloses a technology for degrading phenol and sequestering carbon by microalgae, and aims to provide a method for improving key enzyme to promote euglena to degrade phenol and enhance carbon sequestration. Comprising the following steps: performing phenol concentration gradient domestication on euglena cells, and adjusting gene expression quantities of three key enzymes, namely H < + > transport ATP enzyme, pyruvate decarboxylase and isocitrate dehydrogenase in the euglena cells; carrying out enlarged culture growth on the euglena domesticated plant, adding a proper amount of phenol into a culture medium, and continuously introducing gas containing CO2; in the culture process, the carbon sequestration rate of the euglena is calculated by measuring the carbon element content in the biomass of the euglena liquid; and after the culture is finished, measuring the content of euglena polysaccharide in the algae liquid in unit volume, and measuring the content of phenol in the algae liquid to calculate the degradation rate of phenol. According to the method, the euglena cells are domesticated by adopting phenol concentration gradient, the expression quantity of key enzyme genes is increased by several times, the phenol degradation capacity of the euglena cells is enhanced, the carbon sequestration rate of the euglena cells is increased, and the content of polysaccharide in the cells can be increased at the same time.

Description

Technical Field

[0001] The invention relates to microalgae degradation of phenol and carbon fixation technology, and in particular to a method for improving key enzymes to promote euglena to degrade phenol and enhance carbon fixation. Background Art

[0002] Phenol is one of the common organic pollutants emitted by industries such as petroleum refining, plastics, paper and pulp, pharmaceuticals, and coal processing. Phenol easily generates phenoxyl free radicals, which lead to uncoupling of oxidative phosphorylation and inhibit ATP synthesis, thus having harmful and even potentially fatal effects on cell functions.

[0003] At present, there are three main methods for removing phenol: physical, chemical and biological. Compared with physical and chemical methods, phenol can be consumed by microorganisms as a carbon source and thus completely degraded without causing secondary pollution. Therefore, it is urgent to develop sustainable methods for phenol degradation. Microalgae have attracted more and more attention because of their ability to fix carbon dioxide and degrade phenol through photosynthesis. Priyadharshini isolated Chlorella pyrenoidosa from coal chemical plant wastewater, and the removal rate of phenol at 282 mg / L was 92.14% through heterotrophic culture.

[0004] However, most of the existing research on phenol degradation by microalgae is to directly select algae strains from the natural environment for cultivation, but there are few studies on how to improve the phenol degradation ability of existing algae strains, and related research results are difficult to show significant results.

[0005] Therefore, it is necessary to study how to simultaneously improve its phenol degradation ability and growth carbon fixation rate. Summary of the invention

[0006] The main purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for improving key enzymes to promote euglena to degrade phenol and enhance carbon fixation.

[0007] To solve the above technical problems, the solution of the present invention is:

[0008] A method for improving key enzymes to promote euglena to degrade phenol and enhance carbon fixation is provided, comprising the following steps:

[0009] (1) By acclimating the euglena cells to a phenol concentration gradient, the gene expression levels of the three key enzymes, H+ transporting ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase, in the euglena cells were adjusted to 7.0-8.2 times, 4.3-5.5 times, and 3.1-4.2 times of the original expression levels, respectively:

[0010] (2) The euglena domesticated strain was expanded and grown, an appropriate amount of phenol was added to the culture medium, and a gas containing CO2 was continuously introduced; algae liquid samples were collected on the third and fourth days of the culture process, and the carbon fixation rate of the euglena was calculated by measuring the carbon content in the biomass;

[0011] (3) After the culture is completed, the content of euglena polysaccharide per unit volume of algae liquid is measured, and the content of phenol in the algae liquid is measured to calculate the degradation rate of phenol.

[0012] As an improved solution of the present invention, in the step (1), the phenol concentration gradient acclimation of the Euglena cells specifically includes: inoculating the Euglena species into the culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 5-8 mg / L; taking out the algae solution after 5 days of cultivation, transferring it to a new culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 13-16 mg / L; taking out the algae solution after 5 days of cultivation, transferring it to a new culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 22-28 mg / L; taking out the algae solution after 5 days of cultivation, transferring it to a new culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 22-28 mg / L; and subculturing for more than 3 generations under these conditions to finally obtain a Euglena acclimated strain with stable properties and high efficiency in degrading phenol.

[0013] As an improved solution of the present invention, in the step (1), several parallel groups are always set during the domestication process, and the gene expression levels of the three key enzymes in each group of Euglena are measured by transcriptomics after each transfer culture; all transcripts obtained by transcriptome sequencing are compared with the database, and differential gene analysis is performed between the Euglena domesticated strain and the original strain group; and the group with the best gene expression level is selected for the next transfer culture, and finally the gene expression levels of the three key enzymes are adjusted to the above range.

[0014] As an improved solution of the present invention, in step (2), the volume concentration of CO2 in the gas is 3-20%, and the concentration of phenol in the culture medium is 22-28 mg / L.

[0015] As an improved solution of the present invention, standard CM medium is used in the euglena cultivation process, and the temperature conditions during cultivation are controlled to be 20-25°C and the light conditions are 80-100 μmol m -2 s -1 .

[0016] As an improved solution of the present invention, in step (2), the source of phenol is any one of organic wastewater, chemical fume or organic solid waste.

[0017] As an improved solution of the present invention, in step (2), the carbon fixation rate of the euglena is tested by the following method:

[0018] (1) At the same time on the third and fourth days of the culture process, 10 ml of algae liquid sample was taken, and then centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded; the centrifuged euglena was dried at 105°C for 24 hours to constant weight, weighed, and the biomass density DW was calculated;

[0019] (2) Calculate the growth rate v of Euglena according to the following formula, in g / L / d:

[0020] v=(DW2-DW1) / (t2-t1)

[0021] Among them, DW1 is the biomass density at sampling time t1 on the third day, g / L; DW2 is the biomass density at sampling time t2 on the fourth day, g / L;

[0022] (3) Calculate the carbon fixation rate S of Euglena by the following formula, in g / L / d:

[0023] S=v×C×44 / 12

[0024] Wherein, C is the average value of the weight percentage of carbon element in the euglena biomass measured during sampling 2 days before and after step (2).

[0025] As an improved solution of the present invention, in step (3), the polysaccharide content in the euglena is tested by the following method:

[0026] (1) taking 10 ml of algae liquid sample, washing with deionized water and centrifuging to collect euglena cells; adding acetone, vortexing and centrifuging to remove the supernatant; adding sodium dodecyl sulfate (SDS) with a mass percentage concentration of 1% to the precipitate, resuspending and heating at 85°C for 30 minutes, centrifuging to remove the supernatant; drying the precipitate in an oven at 60°C to constant weight to obtain euglena polysaccharide;

[0027] (2) The polysaccharide was dissolved in 0.5 M NaOH solution, and the content of polysaccharide was determined by the phenol-sulfuric acid method based on the glucose measurement standard.

[0028] As an improved solution of the present invention, in step (3), the phenol degradation rate in the euglena liquid is tested by the following method:

[0029] (1) Using the 4-aminoantipyrine method, a standard curve of absorbance versus phenol concentration was drawn;

[0030] (2) Take 10 ml of algae liquid sample, centrifuge at 8000 rpm for 5 minutes, and take the supernatant; add 0.1 ml of NH4Cl buffer, 0.2 ml of 2% 4-aminoantipyrine solution, and 0.2 ml of 8% potassium ferricyanide solution to the supernatant in sequence; after standing at room temperature for 10 minutes, measure the optical density (OD510) at 510 nm on a spectrophotometer;

[0031] (3) Calculate the phenol degradation rate as follows:

[0032] Phenol degradation rate (%) = 1-remaining phenol content in the culture medium after the culture is completed / initial phenol addition amount × 100%.

[0033] As an improved solution of the present invention, the NH4Cl buffer solution is prepared by dissolving 20 g of NH4Cl in 100 mL of ammonia water, and adjusting the pH value to 10.7.

[0034] Description of the invention principle:

[0035] As a type of single-cell eukaryotic organism between animals and plants, euglena is widely distributed in nature and exhibits excellent survival ability. This type of microalgae is known for its strong environmental adaptability and can survive and thrive in extremely harsh environments such as low pH, high salinity, and high-energy ionizing radiation. Euglena cells are not only rich in a variety of nutrients, but also have a very high digestibility and absorption rate of nutrients due to their structural characteristics of having no cell wall.

[0036] Based on the unique biological characteristics of Euglena, the present invention proposes to use phenol concentration gradient to domesticate Euglena cells, adjust the gene expression of H+ transport ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase in Euglena cells to several times that of the original algae strain, and optimize pathways including oxidative phosphorylation, tricarboxylic acid cycle, and glycolysis in this way. These pathways are of great significance in the energy metabolism and material conversion of Euglena, and can jointly maintain the life activities of cells in various ways such as regulating metabolic balance, maintaining cell structure, and affecting cell function.

[0037] The applicant also found that during the phenol concentration gradient domestication process, a small amount of phenol induction can stimulate the repair mechanism of euglena cells. In addition to affecting the above-mentioned key enzymes, genes related to the photosynthesis pathway and genes encoding ATP synthesis in euglena cells will have a certain degree of upregulation of gene expression, thereby promoting the growth of euglena. Moreover, the domesticated euglena cells divide faster, and relatively fewer euglena cells are in the apoptosis stage. Since euglena polysaccharides are storage polysaccharides, they will be synthesized in large quantities during the high-speed growth period of euglena, and a small amount of phenol has a certain degree of promoting effect on the synthesis of euglena polysaccharides.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The present invention uses a phenol concentration gradient to domesticate euglena cells, increases the expression of key enzyme genes by several times, and increases the carbon fixation rate while enhancing the phenol degradation ability of euglena cells. According to experimental verification, the phenol degradation rate of euglena can be increased from 50% to 60% before improvement to 90 to 99%, and the carbon fixation rate can be increased from 0.4 to 0.5 g / L / d before the key enzyme gene is improved to 0.7 to 0.85 g / L / d.

[0040] 2. The present invention can also simultaneously promote the up-regulation of the expression of genes related to the photosynthesis pathway and encoding ATP synthesis, thereby increasing the intracellular polysaccharide content, thereby increasing the polysaccharide content of Euglena from 0.2 to 0.3 g / L before the improvement of the key enzyme gene to 0.4 to 0.5 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a flow chart of the present invention.

[0042] Figure 2 This is a bubble chart of the functional enrichment analysis of the differentially expressed genes between the domesticated and original strains of Euglena on the KEGG pathway. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0044] Part I Implementation of the Invention

[0045] 1. Method for improving key enzymes to promote euglena to degrade phenol and enhance carbon fixation:

[0046] (1) By subjecting naked algae cells to phenol concentration gradient acclimation, the gene expression levels of three key enzymes, namely H+ transporting ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase, in naked algae cells were adjusted to 7.0-8.2 times, 4.3-5.5 times, and 3.1-4.2 times of the original expression levels, respectively. Specifically, the method comprises: inoculating the Euglena species into the culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 5-8 mg / L; taking out the algae solution after 5 days of cultivation, transferring it to a new culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 13-16 mg / L; taking out the algae solution after 5 days of cultivation, transferring it to a new culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 22-28 mg / L; taking out the algae solution after 5 days of cultivation, transferring it to a new culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 22-28 mg / L, and subculturing for more than 3 generations under these conditions to finally obtain an Euglena domesticated strain with stable properties and high efficiency of phenol degradation.

[0047] During the domestication process, several parallel groups are always set up. After each transfer culture, the gene expression levels of the three key enzymes in each group of Euglena are determined by transcriptomics; all transcripts obtained by transcriptome sequencing are compared with the database, and differential gene analysis is performed between the Euglena domesticated strains and the original strains; the group with the best gene expression level is selected for the next transfer culture, and finally the gene expression levels of the three key enzymes are adjusted to the said range.

[0048] The workflow of transcriptomics includes sequencing experiments and machine testing: eukaryotic mRNA sequencing is based on a high-throughput sequencing platform to sequence all mRNAs transcribed from specific tissues or cells of eukaryotic organisms at a certain period. Its operation process includes: extracting total RNA, enriching mRNA with Oligo dT, fragmenting mRNA, reverse synthesizing cDNA, connecting adapters, fragment screening and library enrichment; machine sequencing can be selected from NovaSeq X Plus platform machine sequencing and DNBSEQ-T7 platform machine sequencing. This part of the content belongs to the prior art. The applicant commissioned an external commercial sequencing service agency to perform sequencing. This operation step does not belong to the innovative content of the present invention, so it will not be repeated.

[0049] (2) The domesticated strain of Euglena is expanded and cultured, and an appropriate amount of phenol is added to the culture medium so that the phenol concentration in the culture medium is 22-28 mg / L. The source of phenol is any one of organic wastewater, chemical fume or organic solid waste. Then, a gas containing CO2 is continuously introduced, and the volume concentration of CO2 in the gas is 3-20%. During the culture of Euglena, a standard CM culture medium is used, and the temperature conditions during the culture are controlled to be 20-25°C, and the light conditions are 80-100 μmol m -2 s -1 .

[0050] On the third and fourth days of the culture process, algae liquid samples were collected and the carbon fixation rate of Euglena was calculated by measuring the carbon content in the biomass, as follows:

[0051] (1) At the same time on the third and fourth days, 10 ml of algae liquid sample was taken, and then centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded; the centrifuged euglena was dried at 105°C for 24 hours to constant weight, and the biomass density DW was calculated;

[0052] (2) Calculate the growth rate v of Euglena according to the following formula, in g / L / d:

[0053] v=(DW2-DW1) / (t2-t1)

[0054] Among them, DW1 is the biomass density at sampling time t1 on the third day, g / L; DW2 is the biomass density at sampling time t2 on the fourth day, g / L;

[0055] (3) Calculate the carbon fixation rate S of Euglena by the following formula, in g / L / d:

[0056] S=v×C×44 / 12

[0057] Wherein, C is the average value of the weight percentage of carbon element in the euglena biomass measured during sampling 2 days before and after step (2).

[0058] (3) After the culture is completed, the content of euglena polysaccharide per unit volume of algae liquid is measured, and the content of phenol in the algae liquid is measured to calculate the degradation rate of phenol.

[0059] (3.1) Testing the polysaccharide content in euglena by the following method:

[0060] (1) taking 10 ml of algae liquid sample, washing with deionized water and centrifuging to collect euglena cells; adding acetone, vortexing and centrifuging to remove the supernatant; adding sodium dodecyl sulfate (SDS) with a mass percentage concentration of 1% to the precipitate, resuspending and heating at 85°C for 30 minutes, centrifuging to remove the supernatant; drying the precipitate in an oven at 60°C to constant weight to obtain euglena polysaccharide;

[0061] (b) The euglena polysaccharide was dissolved in 0.5 M NaOH solution, and the euglena polysaccharide content was determined by the phenol-sulfuric acid method based on the glucose measurement standard.

[0062] (3.2) The degradation rate of phenol in the Euglena algae liquid was tested by the following method:

[0063] (a) adopt 4-aminoantipyrine method, draw the standard curve of absorbance to phenol concentration;

[0064] (b) 10 ml of algae liquid sample was taken and centrifuged at 8000 rpm for 5 minutes to obtain the supernatant; 0.1 ml of NH4Cl buffer solution, 0.2 ml of 2% 4-aminoantipyrine solution, and 0.2 ml of 8% potassium ferrocyanide solution were added to the supernatant in sequence; after standing at room temperature for 10 minutes, the optical density (OD510) at 510 nm was measured on a spectrophotometer; the NH4Cl buffer solution was prepared by dissolving 20 g of NH4Cl in 100 ml of ammonia water and adjusting the pH value to 10.7.

[0065] (c) Calculate the phenol degradation rate as follows:

[0066] Phenol degradation rate (%) = 1-remaining phenol content in the culture medium after the culture is completed / initial phenol addition amount × 100%.

[0067] 2. The significance of increasing the gene expression of three key enzymes in Euglena in the present invention:

[0068] exist Figure 2 In the figure, a bubble chart of the functional enrichment analysis of differentially expressed genes between domesticated and original strains of Euglena on the KEGG pathway is shown, and the top 20 KEGG pathways with the highest degree of enrichment are recorded. Among them, the top enriched pathways include oxidative phosphorylation, citric cycle, and glycolysis / gluconeogenesis, etc. These pathways are of great significance in the energy metabolism and material transformation of Euglena, and they jointly maintain the life activities of cells by regulating metabolic balance, maintaining cell structure, and affecting cell function. These pathways play an important role in the energy metabolism and material transformation of organisms, and are interrelated to jointly maintain the life activities of cells.

[0069] (1) The expression of the gene pdc (EC4.1.1.1) encoding pyruvate decarboxylase in the glycolysis pathway was significantly upregulated to a TPM value of 0.33 in the phenol-acclimated strain of Euglena, which was 5.5 times higher than that of the original strain (TPM value of 0.06). Pyruvate decarboxylase indirectly affects the energy metabolism of cells by regulating the metabolic direction of pyruvate.

[0070] (2) The oxidative phosphorylation process of microalgae mainly involves the electron transport chain and the synthesis of ATP. By catalyzing the synthesis of ATP, H+ transporting ATPase provides cells with a large amount of energy, which is used for various cellular activities and biosynthetic processes.

[0071] It was tested that during the phenol concentration gradient domestication process, the expression level of the gene PMA (EC7.1.2.1) encoding the H+ transporting ATPase in the domesticated strain of Euglena (TPM value 2.583) was significantly increased, 8.2 times compared with the original strain (TPM value 0.315). After the addition of phenol, the Euglena cells were subjected to oxidative stress. At this time, the gene expression level of the H+ transporting ATPase increased to help the cells cope with these environmental changes. Therefore, the domesticated Euglena cells showed higher energy conversion efficiency, and by effectively adjusting their energy metabolism pathways and enhancing the synthesis rate of ATP, they were able to adapt to a variety of physiological conditions and metabolic requirements.

[0072] (3) In microalgae, the role of the tricarboxylic acid cycle (TCA cycle) is particularly important. First, microalgae fix CO2 through photosynthesis to produce organic substances such as glucose. After a series of transformations in the cells, these organic substances eventually enter the tricarboxylic acid cycle for oxidative decomposition, providing the microalgae with the energy needed for growth and metabolism. Secondly, when responding to environmental stress (such as nutrient deficiency, phenol stimulation, etc.), microalgae usually adjust their metabolic pathways and rely more on the TCA cycle to provide energy and regulate the growth and reproduction of microalgae.

[0073] It was found that during the phenol concentration gradient domestication process, the expression level of the gene encoding isocitrate dehydrogenase (EC1.1.1.42) in the euglena domesticated strain (TPM value 1.197) was significantly increased, 4.2 times compared with the original strain (TPM value 0.285). When phenol is added, euglena cells face oxidative stress. Isocitrate dehydrogenase is one of the key enzymes in the tricarboxylic acid cycle. It catalyzes the oxidation and decarboxylation of isocitrate to produce α-ketoglutarate, carbon dioxide (CO2) and NADH (or NADPH). Its expression level directly affects the efficiency of the TCA cycle and the energy production of euglena cells. This reaction is an important step in the tricarboxylic acid cycle, providing energy and reducing power for cells.

[0074] Part II Specific implementation examples

[0075] Example 1

[0076] (1) Performing phenol concentration gradient acclimation on the euglena cells, adjusting the gene expression levels of three key enzymes, H+ transporting ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase in the euglena cells to 7.0 times, 4.8 times, and 3.9 times of the original, respectively: inoculating the euglena species with an inoculation amount of 10% into a 400 ml cylindrical bottle (300 ml culture medium), setting up 3 parallel samples, adding 5 mg / L phenol to the culture medium, and culturing for 5 days after 5 days with the same inoculation amount (10%), adding 16 mg / L phenol to the culture medium, and continuing to culture for 5 days; then taking the same inoculation amount (10%) of the euglena from the algae solution at this time and inoculating it into a new culture medium, adding 22 mg / L phenol to the culture medium, and continuing to culture for 5 days; then subculturing for more than 3 generations in a culture medium with a phenol concentration of 22 mg / L to obtain a stable euglena acclimated strain with high efficiency in degrading phenol;

[0077] (2) The domesticated strains and the original strains were expanded and grown, and 24 mg / L phenol was added to the culture medium. During the culture process, CO2 with a volume concentration of 3% was continuously introduced. During the culture period, the temperature of the culture of Euglena was controlled at 20°C and the light condition was 100 μmol m -2 s -1 On the third and fourth days of the culture process, samples of algae solution in the reactor were collected and the carbon fixation rates of the original strain and the domesticated strain were tested to be 0.46 g / L / d and 0.7 g / L / d, respectively.

[0078] (3) After the culture was completed, the algae liquid in the two reactors was collected and the phenol concentration in the original algae liquid was measured to be 12 mg / L, and the phenol degradation rate was 50%; the phenol concentration in the algae liquid of the domesticated algae was 1.5 mg / L, and the phenol degradation rate was 93.8%. After the culture was completed, the polysaccharide contents of the original and domesticated algae per unit volume of the algae liquid were 0.25 g / L and 0.48 g / L, respectively.

[0079] Example 2

[0080] (1) Performing phenol concentration gradient acclimation on the euglena cells, adjusting the gene expression levels of three key enzymes, H+ transporting ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase in the euglena cells to 7.5 times, 4.3 times, and 4.2 times of the original, respectively: inoculating the euglena species with an inoculation amount of 10% into a 400 ml cylindrical bottle (300 ml culture medium), setting up 3 parallel samples, adding 8 mg / L phenol to the culture medium, and culturing for 5 days after 5 days with the same inoculation amount (10%), adding 15 mg / L phenol to the culture medium, and continuing to culture for 5 days; then taking the same inoculation amount (10%) of the euglena from the algae solution at this time and inoculating it into a new culture medium, adding 25 mg / L phenol to the culture medium, and continuing to culture for 5 days; then subculturing for more than 3 generations in a culture medium with a phenol concentration of 25 mg / L to obtain a euglena acclimated strain with stable properties and high efficiency in degrading phenol;

[0081] (2) The domesticated strains and the original strains were expanded and grown, and 22 mg / L phenol was added to the culture medium. During the culture process, CO2 with a volume concentration of 15% was continuously introduced. During the culture period, the temperature of the culture was controlled at 22°C and the light condition was 95 μmol m -2 s -1 On the third and fourth days of the culture process, samples of algae solution in the reactor were collected and the carbon fixation rates of the original strain and the domesticated strain were tested to be 0.4 g / L / d and 0.85 g / L / d, respectively.

[0082] (3) After the culture was completed, the algae liquid in the two reactors was collected and the phenol concentration in the original algae liquid was measured to be 10.3 mg / L, and the phenol degradation rate was 53.2%; the phenol concentration in the algae liquid of the domesticated algae was 2.2 mg / L, and the phenol degradation rate was 90%. After the culture was completed, the polysaccharide contents of the original and domesticated algae liquid per unit volume were 0.2 g / L and 0.4 g / L, respectively.

[0083] Example 3

[0084] (1) Performing phenol concentration gradient acclimation on the euglena cells, adjusting the gene expression levels of three key enzymes, H+ transporting ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase in the euglena cells to 8.2 times, 5.5 times, and 3.1 times of the original, respectively: inoculating the euglena species with an inoculation amount of 10% into a 400 ml cylindrical bottle (300 ml culture medium), setting up 3 parallel samples, adding 6 mg / L phenol to the culture medium, and culturing for 5 days after culturing with the same inoculation amount (10%), adding 13 mg / L phenol to the culture medium, and continuing to culture for 5 days; then taking the same inoculation amount (10%) of the euglena from the algae solution at this time and inoculating it into a new culture medium, adding 28 mg / L phenol to the culture medium, and continuing to culture for 5 days; then subculturing in a culture medium with a phenol concentration of 28 mg / L for more than 3 generations to obtain a euglena acclimated strain with stable characteristics and high efficiency in degrading phenol;

[0085] (2) The domesticated strains and the original strains were expanded and grown, and 28 mg / L phenol was added to the culture medium. CO2 with a volume concentration of 20% was continuously introduced during the culture process. During the culture period, the temperature of the culture of Euglena was controlled at 25°C and the light condition was 80 μmol m -2 s -1 On the third and fourth days of the culture process, samples of algae solution in the reactor were collected and the carbon fixation rates of the original strain and the domesticated strain were tested to be 0.5 g / L / d and 0.84 g / L / d, respectively.

[0086] (3) After the culture was completed, the algae liquid in the two reactors was collected and the phenol concentration in the original algae liquid was measured to be 11.2 mg / L, and the phenol degradation rate was 60%; the phenol concentration in the algae liquid of the domesticated algae was 0.28 mg / L, and the phenol degradation rate was 99%. After the culture was completed, the polysaccharide content of the original and domesticated algae per unit volume of the algae liquid was 0.3 g / L and 0.5 g / L, respectively.

[0087] It can be seen from the data of each embodiment that the carbon fixation rate, phenol degradation rate and polysaccharide content of the Euglena strain obtained by acclimation of the present invention with the phenol concentration gradient are better than those of the unacclimated Euglena strain. In particular, the ability to degrade phenol almost doubles the effect. It can be seen that if the method of the present invention is applied industrially, it will greatly enhance the positive role of Euglena in the application of organic pollutant disposal technology.

[0088] Finally, it should be noted that the above examples are only specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, and many variations are possible. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for improving key enzymes to promote euglena to degrade phenol and enhance carbon fixation, characterized in that: The following steps are involved: (1) By acclimating the euglena cells to a phenol concentration gradient, the gene expression levels of the three key enzymes, H+ transporting ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase, in the euglena cells were adjusted to 7.0-8.2 times, 4.3-5.5 times, and 3.1-4.2 times of the original expression levels, respectively: (2) The euglena domesticated strain was expanded and grown, an appropriate amount of phenol was added to the culture medium, and a gas containing CO2 was continuously introduced; algae liquid samples were collected on the third and fourth days of the culture process, and the carbon fixation rate of the euglena was calculated by measuring the carbon content in the biomass; (3) After the culture is completed, the content of euglena polysaccharide per unit volume of algae liquid is measured, and the content of phenol in the algae liquid is measured to calculate the degradation rate of phenol.

2. The method according to claim 1, characterized in that In the step (1), the phenol concentration gradient acclimation of the euglena cells specifically comprises: inoculating the euglena species into the culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 5-8 mg / L; taking out the algae solution after 5 days of cultivation, transferring it to a new culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 13-16 mg / L; taking out the algae solution after 5 days of cultivation, transferring it to a new culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 22-28 mg / L; taking out the algae solution after 5 days of cultivation, transferring it to a new culture medium at an inoculation amount of 10%, and adding phenol to a concentration of 22-28 mg / L, and subculturing for more than 3 generations under these conditions to finally obtain a euglena acclimated strain with stable properties and high efficiency of phenol degradation.

3. The method according to claim 1, characterized in that In the step (1), several parallel groups are always set during the domestication process, and the gene expression levels of the three key enzymes in each group of Euglena are measured by transcriptomics after each transfer culture; all transcripts obtained by transcriptome sequencing are compared with the database, and differential gene analysis is performed between the Euglena domesticated strain and the original strain group; and the group with the best gene expression level is selected for the next transfer culture, and finally the gene expression levels of the three key enzymes are adjusted to the above range.

4. The method according to claim 1, characterized in that: In the step (2), the volume concentration of CO2 in the gas is 3-20%, and the concentration of phenol in the culture medium is 22-28 mg / L.

5. The method according to claim 1, characterized in that Standard CM medium was used in the culture process of Euglena, and the temperature during culture was controlled at 20-25°C and the light condition was 80-100 μmol m -2 s -1 .

6. The method according to claim 1, characterized in that In the step (2), the source of phenol is any one of organic wastewater, chemical fume or organic solid waste.

7. The method according to claim 1, characterized in that In the step (2), the carbon fixation rate of the euglena is tested by the following method: (1) At the same time on the third and fourth days of the culture process, 10 ml of algae liquid sample was taken, and then centrifuged at 8000 rpm for 5 minutes, and the supernatant was discarded; the centrifuged euglena was dried at 105°C for 24 hours to constant weight, weighed, and the biomass density DW was calculated; (2) Calculate the growth rate v of Euglena according to the following formula, in g / L / d: v=(DW2-DW1) / (t2-t1) Among them, DW1 is the biomass density at sampling time t1 on the third day, g / L; DW2 is the biomass density at sampling time t2 on the fourth day, g / L; (3) Calculate the carbon fixation rate S of Euglena by the following formula, in g / L / d: S=v×C×44 / 12 Wherein, C is the average value of the weight percentage of carbon element in the euglena biomass measured during sampling 2 days before and after step (2).

8. The method according to claim 1, characterized in that In the step (3), the polysaccharide content in the euglena is tested by the following method: (1) taking 10 ml of algae liquid sample, washing with deionized water and centrifuging to collect euglena cells; adding acetone, vortexing and centrifuging to remove the supernatant; adding sodium dodecyl sulfate (SDS) with a mass percentage concentration of 1% to the precipitate, resuspending and heating at 85°C for 30 minutes, centrifuging to remove the supernatant; drying the precipitate in an oven at 60°C to constant weight to obtain euglena polysaccharide; (2) The polysaccharide was dissolved in 0.5 M NaOH solution, and the content of polysaccharide was determined by the phenol-sulfuric acid method based on the glucose measurement standard.

9. The method according to claim 1, characterized in that: In the step (3), the degradation rate of phenol in the euglena liquid is tested by the following method: (1) Using the 4-aminoantipyrine method, a standard curve of absorbance versus phenol concentration was drawn; (2) Take 10 ml of algae liquid sample, centrifuge at 8000 rpm for 5 minutes, and take the supernatant; add 0.1 ml of NH4Cl buffer, 0.2 ml of 2% 4-aminoantipyrine solution, and 0.2 ml of 8% potassium ferricyanide solution to the supernatant in sequence; after standing at room temperature for 10 minutes, measure the optical density (OD510) at 510 nm on a spectrophotometer; (3) Calculate the phenol degradation rate as follows: Phenol degradation rate (%) = 1-remaining phenol content in the culture medium after the culture is completed / initial phenol addition amount × 100%.

10. The method according to claim 9, characterized in that The NH4Cl buffer solution is prepared by dissolving 20 g of NH4Cl in 100 mL of ammonia water, and adjusting the pH value to 10.7.

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