A method for promoting the degradation of phenol by euglena by improving key enzymes to enhance carbon sequestration

Abstract

The microalgae phenol degradation and carbon fixation technology aims to provide a method for improving key enzymes to promote euglena to degrade phenol and enhance carbon fixation. The method comprises the following steps: adjusting the gene expression amount of three key enzymes, H+ transport ATPase, pyruvate decarboxylase and isocitrate dehydrogenase in euglena cells by performing phenol concentration gradient domestication on the euglena cells; performing large-scale culture growth on the euglena domesticated strains, adding appropriate phenol in the culture medium, and continuously feeding the gas containing CO2; calculating the carbon fixation rate of the euglena by measuring the carbon element content in the biomass of the algal liquid during the culture process; after the culture is completed, the euglena polysaccharide content in the unit volume of the algal liquid is measured, and the phenol content in the algal liquid is measured to calculate the phenol degradation rate. The euglena cells are domesticated by using the phenol concentration gradient, the gene expression amount of the key enzymes is improved by several times, the phenol degradation capacity of the euglena cells is enhanced, the carbon fixation rate is improved, and the polysaccharide content in the cells is also improved.

Description

Technical Field

[0001] This invention relates to microalgae degradation of phenol and carbon fixation technology, and particularly to a method for improving key enzymes to promote phenol degradation by Euglena and enhance carbon fixation. Background Technology

[0002] Phenol is a common organic pollutant emitted by industries such as petroleum refining, plastics, papermaking and pulping, pharmaceuticals, and coal processing. Phenol readily generates phenoxy radicals, leading to oxidative phosphorylation uncoupling and inhibiting ATP synthesis, thereby having harmful or even potentially fatal effects on cell function.

[0003] Currently, there are three main methods for phenol removal: physical, chemical, and biological. Compared to physical and chemical methods, phenol can be completely degraded by microorganisms as a carbon source, without causing secondary pollution. Therefore, there is an urgent need to develop sustainable phenol degradation methods. Microalgae have attracted increasing attention due to their ability to fix carbon dioxide and simultaneously degrade phenol through photosynthesis. Priyadharshini isolated *Chlorella pyrenoidosa* from coal chemical plant wastewater and achieved a 92.14% removal rate of phenol at 282 mg / L through heterotrophic culture.

[0004] However, most existing research on microalgae degradation of phenol involves directly screening algal strains from the natural environment for cultivation, but there is little research on how to improve the phenol degradation capacity of existing algal strains, and the relevant research results are not very significant.

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

[0006] The main objective of this invention is to overcome the shortcomings of the prior art and provide a method for improving key enzymes to promote the degradation of phenol by Euglena and enhance carbon fixation.

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

[0008] A method for improving a key enzyme to promote phenol degradation and enhance carbon fixation in Euglena is provided, comprising the following steps:

[0009] (1) By subjecting Euglena cells to a phenol concentration gradient acclimatization, the gene expression levels of three key enzymes in Euglena cells—H+ transport ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase—were adjusted to increase their original expression levels to 7.0–8.2 times, 4.3–5.5 times, and 3.1–4.2 times, respectively.

[0010] (2) The domesticated strain of Euglena was cultured and grown on a large scale. An appropriate amount of phenol was added to the culture medium and CO2 gas was continuously introduced. On the third and fourth days of the culture process, algal liquid samples were collected and the carbon fixation rate of Euglena was calculated by measuring the carbon content in the biomass.

[0011] (3) After the culture is completed, the content of Euglena polysaccharide in a unit volume of algal solution is measured, and the content of phenol in the algal solution is measured to calculate the degradation rate of phenol.

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

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

[0014] As an improvement 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 improvement to the present invention, standard CM culture medium is used during the Euglena cultivation process, and the temperature conditions during cultivation are controlled at 20–25°C, and the light conditions are controlled at 80–100 μmol m2. -2 s -1 .

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

[0017] As an improvement to the present invention, in step (2), the carbon fixation rate of Euglena is tested in the following manner:

[0018] (1) At the same time on the third and fourth days of the cultivation process, take 10 ml of algal solution sample, then centrifuge at 8000 rpm for 5 minutes and discard the supernatant; dry the centrifuged Euglena at 105℃ for 24 hours until constant weight, weigh and calculate the biomass density DW;

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

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

[0021] Wherein, 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 using the following formula, in g / L / d:

[0023] S = v × C × 44 / 12

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

[0025] As an improvement to the present invention, in step (3), the polysaccharide content in Euglena is tested in the following manner:

[0026] (1) Take 10 ml of algal solution sample, wash with deionized water and centrifuge to collect Euglena cells; add acetone, vortex and centrifuge to remove supernatant; add 1% sodium dodecyl sulfate (SDS) to the precipitate, resuspend and heat at 85℃ for 30 minutes, centrifuge to remove supernatant; dry the precipitate in an oven at 60℃ to constant weight to obtain Euglena polysaccharide;

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

[0028] As an improvement to the present invention, in step (3), the phenol degradation rate in the Euglena algae solution is tested in the following manner:

[0029] (1) The standard curve of absorbance versus phenol concentration was plotted using the 4-aminoantipyrine method;

[0030] (2) Take 10 mL of algal solution 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 cultivation / Initial phenol addition amount × 100%.

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

[0034] Description of the invention principle:

[0035] Euglena, a type of single-celled eukaryotic organism intermediate between animals and plants, are widely distributed in nature and exhibit remarkable survival abilities. These microalgae are renowned for their exceptional environmental adaptability, surviving and thriving in extreme environments such as low pH, high salinity, and high-energy ionizing radiation. Euglena cells are not only rich in various nutrients, but their cell wall-less structure also results in an extremely high rate of nutrient digestibility and absorption.

[0036] Based on the unique biological characteristics of Euglena, this invention proposes using a phenol concentration gradient to domesticate Euglena cells, adjusting the gene expression levels of H+ transport ATPase (PMA), pyruvate decarboxylase (PDC), and isocitrate dehydrogenase in Euglena cells to several times that of the original algal strain. This optimizes pathways including oxidative phosphorylation, the tricarboxylic acid cycle, and glycolysis. These pathways are crucial for energy metabolism and material transformation in Euglena, jointly maintaining cellular life activities through various means such as regulating metabolic homeostasis, maintaining cell structure, and influencing cell function.

[0037] The applicant also discovered that during the phenol concentration gradient acclimation process, a small amount of phenol induced the repair mechanism of Euglena cells. Besides affecting the aforementioned key enzymes, the expression levels of genes related to the photosynthetic pathway and genes encoding ATP synthesis within Euglena cells were upregulated to some extent, thereby promoting Euglena growth. Furthermore, acclimated Euglena cells divided more rapidly, with relatively fewer cells in the apoptotic phase. Since Euglena polysaccharides are storage polysaccharides, they are synthesized in large quantities during the rapid growth phase of Euglena, and a small amount of phenol played a certain role in promoting the synthesis of Euglena polysaccharides.

[0038] Compared with the prior art, the beneficial effects of the present invention are:

[0039] 1. This invention employs a phenol concentration gradient to acclimatize Euglena cells, increasing the expression level of key enzyme genes several times over. This enhances the phenol degradation capacity of Euglena cells while simultaneously improving their carbon fixation rate. Experimental verification shows that the phenol degradation rate of Euglena can be increased from 50%–60% before the modification to 90–99%, and the carbon fixation rate can be increased from 0.4–0.5 g / L / d before the key enzyme gene modification to 0.7–0.85 g / L / d.

[0040] 2. This invention can also simultaneously promote the upregulation of the expression of genes related to the photosynthetic pathway and encoding ATP synthesis, increase the intracellular polysaccharide content, and increase the polysaccharide content of Euglena from 0.2-0.3 g / L before the modification of the key enzyme gene to 0.4-0.5 g / L. Attached Figure Description

[0041] Figure 1 This is a flowchart of the present invention.

[0042] Figure 2 A bubble diagram showing the functional enrichment of differentially expressed genes in the KEGG pathway between domesticated and original Euglena strains. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0044] Part One: Implementation Scheme of the Invention

[0045] 1. Methods to improve key enzymes to promote phenol degradation in Euglena and enhance carbon fixation:

[0046] (1) By subjecting Euglena cells to phenol concentration gradient acclimatization, the gene expression levels of three key enzymes in Euglena cells, namely H+ transport ATPase (PMA), pyruvate decarboxylase (pdc) and isocitrate dehydrogenase, were adjusted to increase them to 7.0–8.2 times, 4.3–5.5 times, and 3.1–4.2 times the original expression levels, respectively. Specifically, the process involves: inoculating the Euglena species into the culture medium at a 10% inoculum, adding phenol to a concentration of 5–8 mg / L; after culturing for 5 days, removing the algal solution and transferring it to a new culture medium at a 10% inoculum, adding phenol to a concentration of 13–16 mg / L; after culturing for 5 days, removing the algal solution and transferring it to a new culture medium at a 10% inoculum, adding phenol to a concentration of 22–28 mg / L; after culturing for 5 days, removing the algal solution and transferring it to a new culture medium at a 10% inoculum, adding phenol to a concentration of 22–28 mg / L; and then subculturing under these conditions for at least 3 generations to finally obtain a stable Euglena strain that efficiently degrades phenol.

[0047] Several parallel groups were always set up during the domestication process. After each transfer culture, the gene expression levels of three key enzymes in each group of Euglena were measured by transcriptomics. All transcripts obtained from transcriptome sequencing were compared with the database to perform differential gene analysis between the domesticated Euglena strain and the original strain. The group with the best gene expression level was selected for the next transfer culture, and finally the gene expression levels of the three key enzymes were adjusted to the specified range.

[0048] The transcriptomics workflow includes sequencing experiments and sequencing: eukaryotic mRNA sequencing, based on a high-throughput sequencing platform, sequences all mRNAs transcribed from specific eukaryotic tissues or cells at a certain stage. The process includes: total RNA extraction, Oligo dT enrichment of mRNA, mRNA fragmentation, reverse DNA synthesis, adapter ligation, fragment selection, and library enrichment. Sequencing can be performed on the NovaSeq X Plus platform or the DNBSEQ-T7 platform. This part is prior art; the applicant commissioned an external commercial sequencing service provider to perform the sequencing, and this step is not part of the innovation of this invention, therefore it will not be described further.

[0049] (2) Expand the growth of the Euglena domesticated strain by adding an appropriate amount of phenol to the culture medium to achieve a phenol concentration of 22–28 mg / L. The phenol source can be any one of organic wastewater, chemical flue gas, or organic solid waste. Then, continuously introduce CO2 gas with a volume concentration of 3–20%. Use standard CM medium during Euglena cultivation, controlling the temperature at 20–25℃ and the light intensity at 80–100 μmol / L. -2 s -1 .

[0050] Algal culture samples were collected on the third and fourth days of the cultivation process. The carbon fixation rate of Euglena was calculated by measuring the carbon content in the biomass, as detailed below:

[0051] (1) At the same time on the third and fourth days, take 10 ml of algal liquid sample, then centrifuge at 8000 rpm for 5 minutes and discard the supernatant; dry the centrifuged Euglena at 105℃ for 24 hours until constant weight, weigh and calculate the biomass density DW;

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

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

[0054] Wherein, 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 using the following formula, in g / L / d:

[0056] S = v × C × 44 / 12

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

[0058] (3) After the culture is completed, the content of Euglena polysaccharide in a unit volume of algal solution is measured, and the content of phenol in the algal solution is measured to calculate the degradation rate of phenol.

[0059] (3.1) The polysaccharide content in Euglena was tested using the following method:

[0060] (1) Take 10 ml of algal solution sample, wash with deionized water and centrifuge to collect Euglena cells; add acetone, vortex and centrifuge to remove supernatant; add 1% sodium dodecyl sulfate (SDS) to the precipitate, resuspend and heat at 85℃ for 30 minutes, centrifuge to remove supernatant; dry the precipitate in an oven at 60℃ to constant weight to obtain Euglena polysaccharide;

[0061] (b) The content of Euglena polysaccharide was determined by dissolving it in 0.5M NaOH solution and using the phenol-sulfuric acid method based on the glucose measurement standard.

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

[0063] (a) A standard curve of absorbance versus phenol concentration was plotted using the 4-aminoantipyrine method;

[0064] (b) Take 10 mL of algal solution sample, centrifuge at 8000 rpm for 5 minutes, and collect 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; the NH4Cl buffer is prepared by dissolving 20 g of NH4Cl in 100 mL of ammonia water and adjusting the pH 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 cultivation / Initial phenol addition amount × 100%.

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

[0068] exist Figure 2 The image shows a bubble chart illustrating the functional enrichment of differentially expressed genes in KEGG pathways between domesticated and original Euglena strains. The chart lists the top 20 KEGG pathways with the highest enrichment levels. Among these, the most enriched pathways include oxidative phosphorylation, the citric cycle, and glycolysis / gluconeogenesis. These pathways are crucial in the energy metabolism and material transformation of Euglena, working together to maintain cellular life activities through various mechanisms such as regulating metabolic homeostasis, maintaining cell structure, and influencing cell function. These pathways play important roles in the energy metabolism and material transformation of organisms, and are interconnected to jointly maintain cellular life activities.

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

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

[0071] During the phenol concentration gradient acclimation process, the expression level of the gene PMA (EC7.1.2.1) encoding the H+ transport ATPase in the acclimated Euglena strain (TPM value 2.583) was significantly increased, representing an 8.2-fold increase compared to the original strain (TPM value 0.315). After the addition of phenol, Euglena cells experienced oxidative stress, at which point the increased expression level of the H+ transport ATPase gene helped the cells cope with these environmental changes. Therefore, the acclimated Euglena cells exhibited higher energy conversion efficiency, effectively adjusting their energy metabolism pathways and enhancing the rate of ATP synthesis, thereby adapting to various physiological conditions and metabolic requirements.

[0072] (3) The tricarboxylic acid cycle (TCA cycle) plays a particularly important role in microalgae. First, microalgae fix CO2 through photosynthesis to produce organic matter such as glucose. After a series of transformations within the cell, these organic substances eventually enter the TCA cycle for oxidative decomposition, providing the energy required for the growth and metabolism of microalgae. Second, when coping with environmental stresses (such as nutrient deficiency, phenol stimulation, etc.), microalgae usually adjust their metabolic pathways, relying more on the TCA cycle to provide energy and regulate their growth and reproduction.

[0073] During the phenol concentration gradient acclimation process, the expression level of the gene encoding isocitrate dehydrogenase (EC1.1.1.42) in the acclimated Euglena strain (TPM value 1.197) was significantly increased, representing a 4.2-fold increase compared to the original strain (TPM value 0.285). When phenol is added, Euglena cells face oxidative stress. Isocitrate dehydrogenase is a key enzyme in the tricarboxylic acid cycle (TCA cycle), catalyzing the oxidative 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 TCA cycle, providing energy and reducing power for the cell.

[0074] Part Two: Specific Implementation Examples

[0075] Example 1

[0076] (1) Euglena cells were acclimatized with phenol concentration gradients to adjust the gene expression levels of three key enzymes in Euglena cells: H+ transport ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase, increasing them to 7.0, 4.8, and 3.9 times the original levels, respectively. Euglena cells were inoculated at a 10% inoculum into 400 ml cylindrical flasks (300 ml of culture medium), with three parallel samples. 5 mg / L phenol was added to the culture medium, and the cells were cultured for 5 days. Then, the cells were transferred with the same inoculum (10%), and 16 mg / L phenol was added to the culture medium, followed by another 5 days of culture. Then, Euglena cells with the same inoculum (10%) were inoculated into a new culture medium, and 22 mg / L phenol was added to the culture medium, followed by another 5 days of culture. Finally, the cells were passaged for more than 3 generations in a culture medium with a phenol concentration of 22 mg / L to obtain a stable Euglena strain that efficiently degrades phenol.

[0077] (2) Both the domesticated and undomesticated Euglena strains were cultured and expanded, with 24 mg / L phenol added to the culture medium. During cultivation, 3% CO2 was continuously introduced. The temperature was controlled at 20℃ and the light intensity at 100 μmol / L. -2 s -1 On the third and fourth days of the cultivation process, algal liquid samples were collected from the reactor, 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 cultivation, the algal solutions of Euglena were collected from both reactors. The concentration of phenol in the algal solution of the original Euglena strain was 12 mg / L, indicating a phenol degradation rate of 50%. The concentration of phenol in the algal solution of the domesticated Euglena strain was 1.5 mg / L, indicating a phenol degradation rate of 93.8%. The polysaccharide content of the original and domesticated Euglena strains per unit volume of algal solution after cultivation was 0.25 g / L and 0.48 g / L, respectively.

[0079] Example 2

[0080] (1) Euglena cells were acclimatized with phenol concentration gradients to adjust the gene expression levels of three key enzymes in Euglena cells: H+ transport ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase, increasing them to 7.5, 4.3, and 4.2 times the original levels, respectively. Euglena cells were inoculated at a 10% inoculum into 400 ml cylindrical flasks (300 ml of culture medium), with three parallel samples. 8 mg / L phenol was added to the culture medium, and the cells were cultured for 5 days. Then, the cells were transferred with the same inoculum (10%), and 15 mg / L phenol was added to the culture medium, followed by another 5 days of culture. Then, Euglena cells with the same inoculum (10%) were inoculated into a new culture medium, and 25 mg / L phenol was added to the culture medium, followed by another 5 days of culture. Finally, the cells were passaged for more than 3 generations in a culture medium with a phenol concentration of 25 mg / L to obtain a stable Euglena strain that efficiently degrades phenol.

[0081] (2) Both the domesticated and undomesticated Euglena strains were cultured and expanded, with 22 mg / L phenol added to the culture medium. During cultivation, 15% CO2 was continuously introduced. The temperature was controlled at 22℃ and the light intensity at 95 μmol / L. -2 s -1 On the third and fourth days of the cultivation process, algal liquid samples were collected from the reactor, 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 cultivation, the algal solutions of Euglena were collected from both reactors. The concentration of phenol in the algal solution of the original Euglena strain was 10.3 mg / L, indicating a phenol degradation rate of 53.2%. The concentration of phenol in the algal solution of the domesticated Euglena strain was 2.2 mg / L, indicating a phenol degradation rate of 90%. The polysaccharide content of the original and domesticated Euglena strains per unit volume of algal solution after cultivation was 0.2 g / L and 0.4 g / L, respectively.

[0083] Example 3

[0084] (1) Euglena cells were acclimatized with phenol concentration gradients to adjust the gene expression levels of three key enzymes in Euglena cells: H+ transport ATPase (PMA), pyruvate decarboxylase (pdc), and isocitrate dehydrogenase, increasing them to 8.2, 5.5, and 3.1 times the original levels, respectively. Euglena cells were inoculated at a 10% inoculum into 400 ml cylindrical flasks (300 ml of culture medium), with three parallel samples. 6 mg / L phenol was added to the culture medium, and the cells were cultured for 5 days. Then, Euglena cells were transferred at the same inoculum level (10%), and 13 mg / L phenol was added to the culture medium, followed by another 5 days of culture. Then, Euglena cells with the same inoculum level (10%) were inoculated into a new culture medium, and 28 mg / L phenol was added to the culture medium, followed by another 5 days of culture. Finally, the cells were passaged for more than 3 generations in a culture medium with a phenol concentration of 28 mg / L to obtain a stable Euglena strain that efficiently degrades phenol.

[0085] (2) Both the domesticated and undomesticated Euglena strains were cultured and expanded, with 28 mg / L phenol added to the culture medium. During cultivation, CO2 at a volume concentration of 20% was continuously introduced. The temperature was controlled at 25℃ and the light intensity at 80 μmol / L. -2 s -1 On the third and fourth days of the cultivation process, algal liquid samples were collected from the reactor, 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 cultivation, the algal solutions of Euglena were collected from both reactors. The concentration of phenol in the algal solution of the original Euglena strain was 11.2 mg / L, indicating a phenol degradation rate of 60%. The concentration of phenol in the algal solution of the domesticated Euglena strain was 0.28 mg / L, indicating a phenol degradation rate of 99%. The polysaccharide content of the original and domesticated Euglena strains per unit volume of algal solution after cultivation was 0.3 g / L and 0.5 g / L, respectively.

[0087] As can be seen from the data of the various embodiments, the Euglena strain obtained by acclimation with a phenol concentration gradient according to the present invention exhibits superior carbon fixation rate, phenol degradation rate, and polysaccharide content compared to the unacclimated Euglena strain. In particular, its phenol degradation capacity almost doubles. Therefore, the industrial application of the method of the present invention will greatly enhance the positive role of Euglena in the treatment of organic pollutants.

[0088] 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 sequestration by promoting degradation of phenol by Euglena by key enzymes, characterized in that, The method comprises the following steps: (1) By gradient acclimation of phenol concentration to euglena cells, the H + The gene expression of the three key enzymes, PMA, pyruvate decarboxylase, and isocitrate dehydrogenase, is increased to 7.0-8.2 times, 4.3-5.5 times, and 3.1-4.2 times of the original expression, respectively: The phenol concentration gradient acclimation specifically comprises: inoculating the Euglena sp. into the culture medium at an inoculation amount of 10%, adding phenol to make the concentration of phenol 5-8 mg / L; after 5 days of culture, taking out the algal liquid, inoculating into a new culture medium at an inoculation amount of 10%, and adding phenol to make the concentration of phenol 13-16 mg / L; after 5 days of culture, taking out the algal liquid, inoculating into a new culture medium at an inoculation amount of 10%, and adding phenol to make the concentration of phenol 22-28 mg / L; after 5 days of culture, taking out the algal liquid, inoculating into a new culture medium at an inoculation amount of 10%, and adding phenol to make the concentration of phenol 22-28 mg / L; under the above conditions, subculturing for more than 3 generations to obtain a phenol-degrading Euglena acclimation strain with stable properties; During the acclimation process, a plurality of parallel groups are always set, and the gene expression amounts of the three key enzymes in the Euglena in each group are determined by transcriptomics after each subculture; all the transcripts obtained by transcriptome sequencing are compared with a database to analyze the difference genes between the Euglena acclimation strain and the original strain; the group with the optimal gene expression amount is selected for the next subculture, and finally the gene expression amounts of the three key enzymes are adjusted to the range; (2) The Euglena acclimation strain is subcultured to grow, and an appropriate amount of phenol is added to the culture medium, and CO2-containing gas is continuously introduced; on the third day and the fourth day of the culture, algal liquid samples are collected, and the carbon fixation rate of the Euglena is calculated by determining the carbon element content in the biomass; In the culture process of euglena, the standard CM medium is used, the phenol concentration in the medium is 22-28 mg / L, the CO2 volume concentration in the gas is controlled to be 3-20%, the temperature condition during the culture is 20-25℃, and the light condition is 80-100 μmol m -2 s -1 ; (3) After the culture is completed, the Euglena polysaccharide content in the unit volume of algal liquid is determined, and the phenol content in the algal liquid is determined to calculate the phenol degradation rate.

2. The method of claim 1, wherein, In the step (2), the source of the phenol is any one of organic wastewater, chemical gas or organic solid waste.

3. The method of claim 1, wherein, In the step (2), the carbon fixation rate of the Euglena is tested by the following methods: (1) At the same time on the third day and the fourth day of the culture, 10 milliliters of algal liquid samples are taken, and then centrifuged at 8000 revolutions per minute for 5 minutes, and the supernatant is discarded; the Euglena obtained by centrifugation is dried at 105°C for 24 hours to constant weight, and the biomass density DW is calculated; (2) The growth rate v of the Euglena is calculated according to the following formula, with the unit of g / L / d: v = (DW2-DW1) / (t2-t1) Wherein, DW1 is the biomass density at the third day t1, g / L; DW2 is the biomass density at the fourth day t2, g / L; (3) The carbon fixation rate S of the Euglena is calculated according to the following formula, with the unit of g / L / d: S = v x C x 44 / 12 Wherein, C is the average value of the carbon element weight percentage in the biomass of the Euglena measured in the step (2) on the two sampling days.

4. The method of claim 1, wherein, In the step (3), the polysaccharide content in the Euglena is tested by the following methods: (1) 10 milliliters of algal liquid samples are taken, and the Euglena cells are collected after washing with deionized water and centrifugation; after vortexing after adding acetone, the supernatant is removed by centrifugation; 1% sodium dodecyl sulfate is added to the precipitate, resuspended and heated at 85°C for 30 minutes, and the supernatant is removed by centrifugation; the precipitate is dried to constant weight in a 60°C oven to obtain the Euglena polysaccharide; (2) The paramylon is dissolved in 0.5 M NaOH solution, and the content of the paramylon is measured by the phenol-sulfuric acid method based on a glucose measurement standard.

5. The method of claim 1, wherein, In the step (3), the phenol degradation rate in the paramylon algae solution is tested by the following method: (1) The standard curve of absorbance vs. phenol concentration is drawn by using the 4-aminoantipyrine method; (2) 10 mL of the algae solution sample is centrifuged at 8000 rpm for 5 minutes by using a centrifuge, and the supernatant is taken; 0.1 mL of NH4Cl buffer, 0.2 mL of 4-aminoantipyrine solution with a mass percentage of 2%, and 0.2 mL of potassium ferricyanide solution with a mass percentage of 8% are sequentially added into the supernatant; after being placed at room temperature for 10 minutes, the optical density at 510 nm is measured on a spectrophotometer; (3) The phenol degradation rate is calculated as follows: Phenol degradation rate = 1 - residual phenol content in the culture medium after the culture ends / initial phenol addition amount × 100%.

6. The method of claim 5, wherein, The NH4Cl buffer 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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