A method for enhancing carbon sequestration and paramylum accumulation in euglena gracilis by monoethanolamine

Abstract

The application discloses a method for reinforcing carbon sequestration and byproduct starch accumulation of Leishnmania acutissima by monoethanolamine, which comprises adding MEA in a simplified culture medium, inoculating Leishnmania acutissima, and passing CO2 in the culture process. The application establishes a trinity coordination system of CO2 regulation-MEA reinforcement-simplified culture medium, solves the problems of high-concentration CO2 stress and organic amine toxicity, reduces the culture cost, fills the technical blank of low-cost carbon sequestration and high-value product coordinated production of Leishnmania acutissima, and can be directly applied to carbon emission reduction of industrial flue gas, and simultaneously produces high-value byproduct starch, has environmental protection and economic value, and the simplified culture medium is suitable for large-scale culture and easy to be popularized and applied in industry.

Description

Technical Field

[0001] This invention relates to the field of microalgae cultivation and high-value product development technology, and in particular to a method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa using monoethanolamine. Background Technology

[0002] Currently, global climate change is intensifying, and the amount of carbon dioxide (CO2) in the atmosphere is increasing. CO2 concentrations continue to rise, with global CO2 levels expected to reach a peak in 2023. Emissions reached 37.4 billion tons and are projected to increase to 41.6 billion tons by 2024. The development of efficient CO2 production capacity is crucial. Capture and utilization technologies have become crucial for mitigating climate change. Microalgae, due to their rapid growth rate and high carbon sequestration efficiency (10-50 times that of terrestrial plants), have become a major source of CO2. Preservative high-quality biological materials include β-1,3-glucan from Euglena spp., also known as parastarch. Parastarch is a polysaccharide polymer composed of high-molecular-weight, linear (unbranched) β-1,3-glycosidic bonds, exhibiting high crystallinity in its natural state. Euglena spp. is one of the few algae that can simultaneously produce high-value biological products for human consumption as both food and nutritional supplements. Parastarch possesses various bioactive functions, including enhancing immune function, eliminating toxins, combating radiation damage, promoting cell repair, and lowering blood lipids.

[0003] However, its application has the following technical problems: 1.CO Concentration adaptability limitations: Microalgal carbon fixation efficiency is affected by CO2. Concentration has a significant impact; most microalgae only grow at specific CO2 concentrations. It exhibits highly efficient carbon fixation capabilities within a wide concentration range. For example, *Euglena slenderis* can fix carbon at high concentrations of CO2 of 20% and above. In the environment, cell acidification stress, chloroplast lamellae breakage, cell membrane damage, and other ultrastructural damage can occur, leading to the degradation of photosynthetic pigments (chlorophyll a content is lower than that of 5% CO2). (The group decreased by more than 40%), and its growth and carbon fixation functions were inhibited.

[0004] 2. Differences in the toxicity of chemical absorbents: Biological carbon sequestration alone contains CO. Low transport rate (gas-liquid mass transfer efficiency) Limitations such as insufficient carbon source utilization (30%) necessitate the use of chemical methods to improve efficiency. However, different alcoholic amines exhibit significant differences in toxicity to microalgae: MEA has relatively low toxicity, but concentrations of 0.8 g / L and above still lead to a cell mortality rate exceeding 60% for Euglena filamentosa; DEA and TEA, due to their complex molecular structures (multi-hydroxyl groups, large steric hindrance), inhibit the growth of Euglena filamentosa at a concentration of 0.3 g / L, and CO2... Its absorption efficiency is 30%-40% lower than that of MEA, making it difficult to achieve synergistic effects of "chemical absorption - biological fixation".

[0005] 3. Culture medium cost and compatibility issues: Euglena slenderis can synthesize high-value parastarch (β-1,3-glucan), but the full-component culture media used in existing technologies (such as CM medium and AF-6 medium) contain CaCl₂. 2H O, CaCO The high cost of large-scale cultivation is due to the presence of non-essential and expensive components such as trace elements; furthermore, the relationship between different culture medium components (such as nitrogen and magnesium sources) and CO2 is not clearly defined. —The compatibility of the organic amine synergistic system leads to fluctuations in the efficiency of parastarch accumulation.

[0006] 4. Unclear metabolic mechanisms: Existing studies have not systematically revealed the molecular mechanisms by which organic amines regulate carbon fixation and parastarch accumulation in Euglena slough. In particular, there is a lack of understanding of the regulatory mechanisms of MEA on key pathways of carbon metabolism (such as the Calvin cycle and pentose phosphate pathway) and nitrogen metabolism (such as the ornithine cycle), which limits the precision of technology optimization.

[0007] 5. Insufficient targeting in algal species selection: Existing studies mostly use conventional algal species such as Chlorella and Scenedesmus, focusing on lipid synthesis, without selecting specific algal species for the characteristics of parastarch synthesis. Summary of the Invention

[0008] The purpose of this invention is to address the technical deficiencies in the prior art by providing a method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa using monoethanolamine.

[0009] The technical solution adopted to achieve the purpose of this invention is: A method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa using monoethanolamine (MEA) includes the following steps: adding MEA to a simplified culture medium, then inoculating Euglena filamentosa; continuously purging CO2 gas into the simplified culture medium during cultivation; the simplified culture medium comprises NH4NO3, K2HPO4, MgSO4·7H2O, CaCl2·2H2O, and ferric ammonium citrate, wherein the mass fractions of each component are as follows: NH4NO3: 55.00~60.61 parts; K2HPO4: 12.12~12.50 parts; MgSO4·7H2O: 20.20~22.50 parts; CaCl2·2H2O: 5.05~7.50 parts; Ferric ammonium citrate: 2.02~2.50 parts.

[0010] In the above technical solution, the concentration of MEA in the simplified culture medium is 0.2~1.0 g / L.

[0011] In the above technical solution, the volume fraction of CO2 is 5-20%, the gas supply flow rate is 300-350 mL / min, and the aeration time is 8-8.25 h / d.

[0012] In the above technical solution, the initial cell density of the *Euglena slenderis* is 0.375 × 10⁻⁶. 6 ~0.475×10 6 Cells / mL.

[0013] In the above technical solution, the pH value for culturing the Euglena slenderis is 7.8~9.1.

[0014] In the above technical solution, the culture time of the Euglena filamentosa is 10-11 days, the culture temperature is 24-26°C, the light intensity is 4400-4500 Lux, and the photoperiod is 12-14L:12-14D.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention is the first to systematically compare the effects of three organic amines—MEA, DEA, and TEA—on Euglena filamentosa, determining MEA as the optimal enhancer and establishing a "CO2" enhancement mechanism. The integrated system of "regulation-MEA enhancement-culture medium simplification" solves the problem of high CO2 concentration. This addresses the issues of stress and organic amine toxicity, while also reducing cultivation costs, filling the technological gap in the low-cost carbon fixation and high-value product synergistic production of Euglena filamentosa.

[0016] 2. This invention combines physiological indicators (growth, photosynthesis, enzyme activity), observation of cell ultrastructure, comparison of multiple organic amines, and metabolomics analysis to clarify the superior mechanism of MEA and the inhibitory mechanism of DEA and TEA from the macroscopic to the molecular level, providing multi-dimensional theoretical support for the selection of organic amines in microalgae carbon fixation technology.

[0017] 3. This invention can be directly applied to industrial flue gas (containing 5%-15% CO) This method reduces carbon emissions and simultaneously produces high-value-added by-product starch (used as a raw material for health products and cosmetics), combining environmental and economic value. It also simplifies the culture medium for large-scale cultivation and is easy to promote industrialization.

[0018] 4. The 0.2 g / L MEA of this invention activates ABC transporter proteins, central carbon metabolism (glycolysis, tricarboxylic acid cycle), arginine-proline metabolism, and purine / pyrimidine metabolism pathways by upregulating 77 differential metabolites (such as guanosine, aspartic acid, and gluconic acid), thereby enhancing carbon source absorption and energy supply and promoting parastarch synthesis. DEA and TEA, on the other hand, are difficult for algal cells to utilize due to their complex molecular structures (multi-hydroxyl groups and large steric hindrance), and they also cause cell membrane osmotic pressure imbalance, downregulating photosynthesis-related metabolites (such as chlorophyll synthesis precursors), thereby inhibiting growth and carbon fixation. Attached Figure Description

[0019] Figure 1 The figure shown is a bar chart of Euglena filamentosa cell density from 0 to 10 days according to the present invention.

[0020] Figure 2 The figure shows the relative growth rate of Euglena cells on day 10 of this invention.

[0021] Figure 3 The figure shows the chlorophyll a content under different culture media according to the present invention.

[0022] Figure 4 The figure shows the chlorophyll b content under different culture media according to the present invention.

[0023] Figure 5 The figure shows the carotenoid content under different culture media according to the present invention.

[0024] Figure 6 The figure shows the polysaccharide content under different culture media according to the present invention.

[0025] Figure 7 This is a PLS-DA score chart for different components of the present invention.

[0026] Figure 8 The diagram shown is a volcano diagram of MA vs NC, ME vs NC, and MA vs ME according to the present invention.

[0027] Figure 9 The diagram shown is a classification chart of the metabolites of this invention.

[0028] Figure 10 The diagram shown is a bubble diagram of the MA vss NC KEGG enrichment pathway of the present invention.

[0029] Figure 11 The diagram shown is a bubble diagram of the ME vs NC KEGG enrichment pathway of the present invention.

[0030] Figure 12 The diagram shown is a bubble diagram of the MA vss NC KEGG enrichment pathway of the present invention.

[0031] Figure 13The figure shows the effect of CO2 on the cell density and relative growth rate of Euglena filamentosa according to the present invention.

[0032] Figure 14 The figure shows the effect of CO2 on the biomass of Euglena filamentosa according to the present invention.

[0033] Figure 15 The figure shows the effect of CO2 on the pH of Euglena filamentosa according to the present invention.

[0034] Figure 16 The figure shows the effect of CO2 on the parastarch content and phytouridine diphosphate glucose pyrophosphorylase of Euglena spp. in this invention.

[0035] Figure 17 The figure shows the effect of different concentrations of CO2 on the photosynthetic pigments of Euglena filamentosa.

[0036] Figure 18 The figure shows the effect of CO2 on ribosomal diphosphate carboxylase and plant carbonic anhydrase of Euglena spp. in this invention.

[0037] Figure 19 The figure shows the effect of different concentrations of CO2 on the morphology of Euglena slenderis cells.

[0038] Figure 20 The image shows the ultrastructural changes of Euglena filamentosa under transmission electron microscopy in the control group (air) aeration conditions according to the present invention.

[0039] Figure 21 The image shows the changes in ultrastructure of Euglena filamentosa under 5% CO2 aeration conditions as described in this invention using transmission electron microscopy.

[0040] Figure 22 The image shows the changes in ultrastructure of Euglena filamentosa under 15% CO2 aeration conditions as described in this invention using transmission electron microscopy.

[0041] Figure 23 The image shows the changes in ultrastructure of Euglena filamentosa under 30% CO2 aeration conditions as described in this invention using transmission electron microscopy.

[0042] Figure 24 The figure shows the effect of the organic amines of the present invention on the cell density of Euglena filamentosa.

[0043] Figure 25 The figure shows the effect of the organic amines of the present invention on the polysaccharide content of Euglena filamentosa.

[0044] Figure 26 The figure shows the effect of the organic amines of the present invention on the biomass of Euglena filamentosa.

[0045] Figure 27 The figure shows the effect of different concentrations of MEA of the present invention on the growth and relative growth rate of Euglena filamentosa algal cells.

[0046] Figure 28 The image shows the effect of the MEA of this invention on the biomass of Euglena filamentosa.

[0047] Figure 29 The image shows the effect of the MEA of this invention on the pH of Euglena filamentosa.

[0048] Figure 30 The figure shows the effect of different concentrations of MEA of the present invention on the content of parastarch and glucose pyrophosphorylase in Euglena filamentosa.

[0049] Figure 31 The image shows the effect of the MEA of the present invention on the inorganic carbon concentration (DIC) of Euglena filamentosa.

[0050] Figure 32 The image shows the effect of the MEA of the present invention on the activities of ribosomal diphosphate carboxylase (Rubisco) and plant carbonic anhydrase (CA) in Euglena filamentosa.

[0051] Figure 33 The figure shows the effects of different concentrations of MEA of the present invention on chlorophyll a, chlorophyll b and carotenoids in Euglena filamentosa. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0053] This embodiment uses *Euglena slenderis* as the research object, and uses "CO2" as the basis for the study. A three-stage experiment—concentration screening, monoethanolamine (MEA) concentration optimization, and metabolomics analysis—was conducted to establish a synergistic system of "chemical absorption—biological fixation," clarify the optimal conditions for carbon fixation and parastarch accumulation, and reveal the regulatory mechanism. This was achieved through a 5% CO2... A synergistic system of "optimal carbon fixation concentration + 0.2 g / L MEA (low toxicity and high efficiency) + simplified culture medium (low cost)" is used to directionally activate key enzymes (UDPase) and core metabolic pathways in parastarch synthesis, guiding carbon flow from "basal metabolism" to "parastarch synthesis," achieving simultaneous improvement in carbon fixation efficiency and parastarch accumulation, unlike the lipid synthesis-oriented approach of conventional algae. Urate-1-phosphate glucose pyrophosphorylase (UDPase) is a key enzyme in parastarch synthesis. UDPase catalyzes the conversion of glucose-1-phosphate (G1P) to uridine-1-phosphate glucose (UDP-glucose, UDP-G), an important precursor for polysaccharide synthesis. This indicates that UDPase activity directly affects parastarch synthesis efficiency, thereby influencing algal cell energy storage and their ability to cope with environmental stress. Euglena slenderis can utilize CO2... The conversion of inorganic carbon is used to maintain cell proliferation and metabolic activities, and the supply of carbon sources has an important impact on photosynthesis and the accumulation of stored substances.

[0054] A method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa using monoethanolamine specifically includes the following steps: Step 1: Under conditions of 5% CO2 + 0.2 g / L MEA, the basal AF-6 medium was set as the control group, and the simplified AF-6 medium was set as the experimental group (without Ca²⁺). CO ² (Group 1), No NaNO Group (Group 2), without NaNO NH NO Group (Group 3), no NH NO Group (Group 4), without Mg² Group 5, with 3 replicates per group, and a culture period of 10 days. The basic AF-6 medium consisted of NaNO3, NH4NO3, K2HPO4, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, citric acid, ferric ammonium citrate, and CaCO3.

[0055] Performance tests of Euglena slenderis under different culture medium conditions: The testing process for cell density, relative growth rate, photosynthetic pigments, and parastarch content is the same as in subsequent step 2.

[0056] Reference Figure 1 , Figure 2 With simplified culture media and reagents, the cell density and growth rate of Euglena slenderis are generally inhibited. From Figure 2 The second, third, and fourth groups reveal that nitrogen sources are crucial elements for cell growth; simplifying them leads to a significant decrease in cell growth rate, consistent with the key role of nitrogen sources in cell metabolism. Furthermore, Mg... 2+ The simplification affected intracellular metabolic pathways, thereby impacting the growth and division of *Euglena slenderis* cells. These results indicate that when culturing *Euglena slenderis*, it is essential to ensure that the culture medium contains sufficient nitrogen and magnesium. 2+ .

[0057] refer to Figure 3 , Figure 4 , Figure 5 The second and fourth groups lacked partial nitrogen sources, while the third group lacked all nitrogen sources. This indicates that nitrogen source simplification may have hindered chlorophyll synthesis in *Euglena slenderis*, as chlorophyll synthesis requires nitrogen. Mg... 2+The absence of these elements may also affect the synthesis or stability of photosynthetic pigments, leading to a decrease in their content. These results indicate that nitrogen source and Mg... 2+ The simplification not only affects cell growth, but also the efficiency of cell photosynthesis.

[0058] refer to Figure 6 The polysaccharide production of *Euglena slenderis* also showed significant changes under simplified drug preparation conditions. Nitrogen source and Mg... 2+ The absence of polysaccharides in Euglena slenderis significantly reduces the production of Euglena slenderis.

[0059] Metabolomics analysis of Euglena slenderis under different culture medium conditions: including the following steps: Step a: Three treatment groups were set up: a control group (NC, no MEA added) and experimental groups with 0.2 g / L MEA (ME) and 1.0 g / L MEA (MA). Each group was divided into four replicates. After culturing for 14 days under 5% CO2 aeration, the same volume of Euglena filamentosa was centrifuged, the supernatant was discarded, and the precipitate was immediately transferred to liquid nitrogen and flash-frozen until completely solidified. It was then stored in an ultra-low temperature freezer at -80℃ for subsequent metabolomics analysis.

[0060] Step b: Thaw the frozen Euglena precipitate on ice, extract 20 μL of sample with 120 μL of pre-chilled 50% methanol, vortex for 1 min, and incubate at room temperature for 10 min. Store the extract at -20°C overnight, centrifuge at 4000g for 20 min, and transfer the supernatant to a new 96-well plate. Before LC-MS analysis, store the sample at -80°C. Prepare a mixed quality control sample (QC sample) by taking 10 μL of each extract.

[0061] Chromatographic and mass spectrometric tests were performed on the extracted QC samples: Chromatographic analysis was performed using an UltiMate 3000 UPLC system with an ACQUITY UPLC T3 column (100 mm × 2.1 mm, 1.8 μm) at a column temperature of 40 °C. Mobile phases were A (5 mM ammonium acetate + 5 mM acetic acid) and B (acetonitrile), with gradient elution: 0–0.8 min 2% B, 0.8–2.8 min 2%–70% B, 2.8–5.6 min 70%–90% B, 5.6–6.4 min 90%–100% B, 6.4–8.0 min 100% B, 8.0–8.1 min 100%–2% B, and 8.1–10 min 2% B. Flow rate was 0.3 mL / min.

[0062] Mass spectrometry was performed using a Q-Exactive high-resolution mass spectrometer in positive / negative ion mode, with a primary scan range of m / z 70-1050 (resolution 70000@m / z 200) and a secondary resolution of 17500.

[0063] Reference Figure 7 The PLS-DA scores for all samples are as follows: Figure 12 As shown, PLS-DA is a supervised discriminant analysis method that can reflect the differences between different groups to the greatest extent. This method uses partial least squares regression to establish a relationship model between metabolite abundance and sample category, thereby achieving sample modeling and prediction. The test results show that NC, ME, and MA samples are closely clustered in the instrument, indicating that this study has good reproducibility and the experimental results are reliable.

[0064] Identification of metabolites from Euglena spp. under different culture medium conditions: Peak picking and retention time correction were performed using XCMS software to generate a three-dimensional matrix. The KEGG and HMDB databases were matched (for samples with poor quality). (10 ppm), verified using a laboratory-built secondary mass spectrometry library.

[0065] Differential metabolites among the screened NC, MA, and ME, such as Figure 8 The differential metabolite volcano diagram is shown in the reference diagram. Figure 8 The C, MA, and ME metabolomes showed the most significant differentially regulated metabolites, with 63 differentially regulated metabolites and 63 significantly downregulated metabolites. (Reference) Figure 8 The least differentially expressed metabolome was found among A, MA, and NC, with 60 differentially expressed metabolites significantly upregulated and 33 significantly downregulated. (Reference) Figure 8 In the B, ME, and NC groups, 77 differentially expressed metabolites were significantly upregulated, while 38 differentially expressed metabolites were significantly downregulated.

[0066] In summary, based on the optimization of AF-6 culture medium, the "Ca²-free" culture medium was determined. CO ² But retain the key nitrogen source (NH4+) NO Phosphorus source (K) HPO ), magnesium source (MgSO) 7H A simplified formula of "O)" with 5% CO Under +0.2 g / L MEA conditions, the cell density and parastarch content of Euglena spp. were not significantly different from those in the full-component AF-6 medium. P (0.05), and the cost is reduced by about 20%.

[0067] Metabolic Mechanism Analysis: Referring to step 3, metabolomics analysis was used to reveal the key pathways by which MEA regulates carbon and nitrogen metabolism and parastarch synthesis in Euglena spp. Compared with the inhibitory mechanisms of DEA and TEA, DEA and TEA showed relatively weak promoting effects, and TEA at high concentrations may even inhibit algae growth. Considering biomass, polysaccharide content, and carbon fixation efficiency, the MEA treatment group showed significant advantages and has potential for further research and application. This provides theoretical support for technology optimization. Specifically: The control group (NC, no MEA added) and the experimental groups were set up with three treatment groups: 0.2 g / L MEA (ME) and 1.0 g / L MEA (MA).

[0068] Non-targeted metabolomics analysis revealed the effects of different concentrations of MEA on the physiological metabolism and CO2 fixation mechanisms of Euglena filamentosa. Under 0.2 g / L MEA conditions, the upregulation of fructose, gluconic acid, and xylan activated the TCA cycle and pentose phosphate pathway, promoting photosynthetic carbon fixation and parastarch accumulation. Simultaneously, it enhanced nitrogen metabolism and ammonia detoxification capacity, significantly upregulating amino acids (aspartic acid, arginine, citrulline, and ornithine), mainly concentrated in ABC transporters, lipid metabolism, nucleotide metabolism, arginine and proline metabolism, and central carbon metabolism. 0.2 g / L MEA also promoted cell proliferation, stabilized osmotic pressure, and maintained cell homeostasis by promoting purine and pyrimidine metabolism. MEA enhanced photosynthesis, promoted cell proliferation, and improved metabolic regulation, thereby enhancing the CO2 fixation capacity and polysaccharide accumulation of microalgae.

[0069] MEA demonstrated excellent application effects in this invention, significantly promoting the growth and polysaccharide synthesis of Euglena filamentosa and increasing CO2 levels. The MEA treatment group exhibits superior carbon fixation efficiency. Its excellent biocompatibility and carbon source supply capacity make it a relatively ideal exogenous additive in microalgal carbon fixation systems. In contrast, DEA and TEA have relatively weaker promoting effects, and TEA may even inhibit algae growth at high concentrations. Considering biomass, polysaccharide content, and carbon fixation efficiency, the MEA treatment group demonstrates significant advantages and has the potential for further research and application.

[0070] Screening of differential metabolites of Euglena spp. under different MEA concentrations: setting thresholds P 0.05, |FC|≥1.2, VIP≥1.0, were used to screen for differentially expressed metabolites; among which P Here, FC represents the probability value, FC represents the difference factor, and VIP represents the projective importance of the variable.

[0071] The metabolites identified in this invention are classified and counted based on their chemical classification and attribution information, and the quantity of each metabolite accounts for, for example... Figure 9As shown, there are 10 classes of metabolites, with lipids and lipid molecules accounting for the highest proportion at 52.84%, organic acids and their derivatives accounting for 19.03%, organic heterocyclic compounds accounting for 8.81%, organic oxygen compounds accounting for 7.67%, and nucleosides, nucleotides and analogues accounting for a relatively small proportion at 5.40%.

[0072] Enrichment of Euglena filamentosa pathway under different MEA concentrations: Differential metabolites were mapped to the KEGG pathway for enrichment analysis (taking...). P (Top 20 channels with the smallest value) Differential metabolites from different treatment groups were matched against the KEGG database to obtain information on enriched pathways involved by these metabolites. In this embodiment, enrichment analysis was performed on the annotated results to obtain information on differentially metabolite enriched pathways, such as... Figure 10 , Figure 11 and Figure 12 As shown, take P The top 20 pathways with the lowest values ​​are displayed in a bubble chart. The metabolic pathways that were significantly enriched in all three treatment groups were ABC transporters, glycophospholipid metabolism, nucleotide metabolism, and metabolic pathways. Arginine and proline metabolism, purine metabolism, amino acid biosynthesis, cAMP signaling pathway, and central carbon metabolism were significantly enriched in the control group and the 0.2 g / L MEA treatment group. Arginine and proline metabolism and glycoglycerol metabolism were significantly enriched in the control group and the 1.0 g / L MEA treatment group. In the 0.2 g / L MEA and 1.0 g / L MEA treatment groups, pyrimidine metabolism, carbon metabolism, purine metabolism, amino acid biosynthesis, cAMP signaling pathway, and central carbon metabolism were significantly enriched.

[0073] In summary, 0.2 g / L MEA upregulates 77 differentially metabolites (such as guanosine, aspartic acid, and gluconic acid), activating ABC transporters, central carbon metabolism (glycolysis, tricarboxylic acid cycle), arginine-proline metabolism, and purine / pyrimidine metabolic pathways, thereby enhancing carbon source absorption and energy supply and promoting parastarch synthesis. DEA and TEA, due to their complex molecular structures (multi-hydroxyl groups, large steric hindrance), are difficult for algal cells to utilize and cause cell membrane osmotic pressure imbalance, downregulating photosynthesis-related metabolites (such as chlorophyll synthesis precursors) and inhibiting growth and carbon fixation. Through "CO2..." -MEA synergistic regulation increased parastarch content by 45% compared to the control group, and compared to CO2 alone. The regulation improved by 22%, and the proportion of parastarch in the carbon flow distribution reached 65%, which is significantly different from the characteristic of conventional algae species where the carbon flow flows towards the oil.

[0074] Step 2, CO Concentration screening: using gradient concentrations of CO Culture experiments were conducted to determine the optimal CO2 concentration for the growth, carbon fixation, and parastarch accumulation of Euglena filamentosa. Environmental issues, addressing high CO concentrations Stress issues. *Euglena scabra* (FACHB-848) was provided by the Tianjin Key Laboratory of Aquatic Ecology and Aquaculture. It was cultured using a simplified autoclaved medium. *Euglena scabra* was placed in a light incubator, with the culture temperature maintained at 26°C, light intensity at 4500 Lux, a photoperiod set to 12L:12D, and a culture time of 10 days. Stress was addressed by adjusting the pure CO2. The CO2 concentration (VCO2 / VAir) is controlled by the air flow rate, and the CO2 is introduced into the culture medium. The concentrations were 5%, 10%, 15%, 20%, and 30% for the experimental groups, with air as the control group (denoted as Air). All experimental and control groups used a concentration of 0.375 × 10⁻⁶. 6 The initial cell density was set at 100 slender Euglena cells / mL, and the gas mixture was added to the culture medium at a flow rate of 300 mL / min, and aerated for 8 hours daily under light.

[0075] For different concentrations of CO The performance of the Eugène filamentosa cultured in the following conditions was tested: 1. Samples were taken from the experimental and control groups every 2 days, and the cell density of Euglena fibrousa was measured at 0, 2, 4, 6, 8, and 10 days using the hemocytometer method (Kruse and Patterson, 1973).

[0076] The formula for calculating the relative growth rate of Euglena slenderis is:

[0077] In the formula, For a specific growth rate, For time The cell density of the slender Euglena, The initial time of the experiment The cell density of the slender Euglena.

[0078] Reference Figure 13 In Figure 'a', the effect of different CO2 concentrations on the cell density of Euglena filamentosa is shown. Figure 13 In Figure 'a', the effect of different CO2 concentrations on the relative growth rate of *Euglena spp.* is shown. It can be seen that with increasing CO2 concentration, the cell density and relative growth rate of *Euglena spp.* initially increased and then decreased. With increasing culture time, after 2 days, the cell density of *Euglena spp.* in the 20% CO2 and 30% CO2 treatment groups was significantly lower than that in other treatment groups. Among them, the cell density growth of *Euglena spp.* under 30% CO2 was the slowest, and after 4 days, it was significantly lower than that of other experimental groups and the control group. P 0.05). After 6 days, the cell density of *Euglena scabra* under 10% CO2 and 15% CO2 was significantly lower than that of the control group. After 8 days, the cell density of *Euglena scabra* in the 5% CO2 treatment group was significantly higher than that of other experimental groups and the control group. P 0.05). The relative growth rate of Euglena spp. cells in the control group and the 5% CO2 treatment group was significantly higher than that in other experimental groups, while the relative growth rate of Euglena spp. cells in the 30% CO2 treatment group was the lowest. P 0.05).

[0079] 2. Euglena biomass test: Samples were taken from both the experimental and control groups every two days. 5 mL of Euglena biomass from each group was filtered through a 0.45 μm cellulose membrane, dried at 105 °C to constant weight, and the Euglena biomass was measured. The dry weight of the algae was estimated using a calibration curve relating the OD670 value to the dry weight of Euglena cells in 5 mL of sample. The OD670 value ranged from 0.100 to 1.000. The formula for calculating the Euglena biomass is as follows:

[0080] The effects of different CO2 concentrations on Euglena biomass, such as Figure 14As shown in the figure, the biomass of *Euglena scabra* decreased with increasing CO2 concentration. The biomass of *Euglena scabra* in the 5% CO2 experimental group was significantly higher than that in other experimental groups and the control group, and remained higher than the highest level from day 2 to 10, reaching a maximum of 1.84 g / L at day 10. The biomass of the control group was significantly higher than that in the 10% CO2 experimental group at days 2 and 4, and significantly lower than that in the 10% CO2 experimental group at days 6 and 8. There was no significant difference between the control group and the 15% CO2 experimental group from days 6 to 10. P 0.05). From 2 to 10 days, the 20% CO2 and 30% CO2 experimental groups were significantly lower than other experimental groups and the control group, and the 30% CO2 group was significantly lower than the 20% CO2 experimental group. P 0.05).

[0081] 3. pH Measurement: Every 2 days, a certain volume of the fine Euglena suspension from the experimental group and the control group was filtered through a 0.45 μm membrane filter. The pH value of the filtrate was then measured using a digital pH meter.

[0082] The effect of different CO2 concentrations on the pH of Euglena filamentosa, such as Figure 15 As shown, the pH of the culture medium decreased to varying degrees with increasing CO2 concentration. The 30% CO2 group experienced the largest pH decrease, dropping from 8.2 to 6.7, reaching its lowest value on day 4. On day 8, the pH of each experimental group increased to varying degrees before stabilizing. On day 10, the higher the CO2 concentration, the lower the pH value.

[0083] 4. Photosynthetic pigment test of Euglena spp.: Photosynthetic pigments of Euglena spp. cultured with different concentrations of CO2 were extracted using the ethanol method. The absorbance at 665 nm, 649 nm, and 470 nm was measured, and the contents of chlorophyll a, chlorophyll b, and carotenoids were calculated.

[0084] The photosynthetic pigment content is calculated using the following formula:

[0085] In the formula, , , The values ​​represent the contents of chlorophyll a, chlorophyll b, and carotenoids, respectively. D665, D649, and D470 are the absorbance values ​​of the supernatant at 665 nm, 649 nm, and 470 nm, respectively.

[0086] The effects of different CO2 concentrations on the photosynthetic pigments of Euglena filamentosa, such as Figure 17 As shown. (Refer to...) Figure 17China A Figure 17 China B and Figure 5 In the middle group (C), under 5% CO2 conditions, the contents of chlorophyll a, chlorophyll b, and carotenoids in *Euglena scabra* reached the highest levels. However, under 20% CO2 and 30% CO2 conditions, the contents of photosynthetic pigments were significantly lower than those in the control group (0.04% CO2) and other experimental groups. P 0.05). Reference Figure 17 In group A, compared with the control group, the chlorophyll a content in the 5%, 10%, and 15% CO2 experimental groups was significantly increased at 4 and 6 days of cultivation. P 0.05). At 8 and 10 days, the chlorophyll a content in the 10% and 15% CO2 experimental groups was significantly lower than that in the control group ( P 0.05). Reference Figure 17 In the 15% CO2 treatment group, the chlorophyll b content was significantly lower than that in the control group and the 10% CO2 experimental group at 6 and 10 days. P 0.05), while within 6-10 days, there was no significant difference between the 10% CO2 experimental group and the control group ( P 0.05). Reference Figure 17 In the study, at 8 and 10 days, the carotenoid content in the 15% CO2 treatment group was significantly lower than that in the control group and the 5% and 10% CO2 experimental groups.

[0087] 5. Determination of CO2 biofixation rate of Euglena spp.: The CO2 biofixation rate of Euglena spp. cells was calculated based on changes in Euglena spp. biomass. The specific calculation method is as follows:

[0088] In the formula, CO2 biological fixation rate (unit: g / L / d ). Carbon content in the cell biomass of Euglena slendera (unit, %) w / w ). The molecular weight of CO2 is 44 g / mol. Where is the molecular weight of carbon. It is 12 g / mol. Biomass volume productivity (in units, g / L / d ).

[0089] Among them, biomass volume productivity The calculation formula is:

[0090] In the formula, for Biomass of Euglena spp. (unit, g / L). The initial concentration (g / L) of algal cell biomass. This is the initial time of the experiment.

[0091] The effects of different CO2 concentrations on the CO2 fixation rate of Euglena filamentosa are shown in Table 1. The CO2 fixation rate decreased with time, and the 5% CO2 concentration was significantly higher than that of other experimental groups ( P 0.05). The CO2 fixation rate of the 5% CO2 experimental group was consistently higher than that of the control group, while the CO2 fixation rates of the 20% CO2 and 30% CO2 experimental groups were consistently lower than those of the control group. P 0.05).

[0092] Table 1. Effect of CO2 on CO2 fixation rate of Euglena filamentosa Table 2-2. Comparison of the rate of CO2fixation (g / L / d) with sources of CO2in Euglena gracilis .

[0093] Note: Different letters indicate significant differences between different treatment groups at the same time. P 0.05). Different letters indicate pure significant differences between different treatment groups at the same time ( P 0.05).

[0094] The effects of different CO2 concentrations on ribosomal diphosphate carboxylase and phytocarbonic anhydrase in Euglena filamentosa are as follows: Figure 18 As shown, the control group had the lowest ribosomal diphosphate carboxylase (RuBisCO) activity, significantly lower than other experimental groups, while the 5% CO2 experimental group had the highest RuBisCO enzyme activity. P 0.05). The 5% CO2 experimental group showed the highest carbonic anhydrase (CA) activity, significantly higher than other experimental groups, while the 30% CO2 experimental group showed the lowest CA activity. P 0.05).

[0095] 6. Determination of parastarch content: Using an alkaline extraction method, 50 mL of *Euglena spp.* from each experimental group and control group was centrifuged (5000 rpm, 10 min). The precipitate was frozen overnight at -80℃ and then lyophilized to obtain *Euglena spp.* algal powder. 5 mg of *Euglena spp.* algal powder was defatted with 4 mL of acetone, 1% SDS was added, and the mixture was heated at 85℃ for 30 min. After centrifugation, the precipitate was dried at 50℃ to obtain parastarch. The parastarch was dissolved in 0.7 mol / L NaOH, and the absorbance at 490 nm was measured using the benzene-sulfuric acid method. The content was calculated using a standard curve. The formula for calculating the parastarch content of *Euglena spp.* is as follows:

[0096] The effects of different CO2 concentrations on parastarch accumulation in Euglena filamentosa are as follows: Figure 16 As shown in Figure A, the parastarch content of Euglena filamentosa initially increased and then decreased with increasing CO2 concentration. The parastarch content in the 5% CO2, 10% CO2, and 15% CO2 experimental groups was significantly higher than that in the control group, and in the 20% CO2 and 30% CO2 experimental groups. The 5% CO2 group had the highest parastarch content, increasing by 22% compared to the control group, while the 30% CO2 group had the lowest parastarch content. P 0.05). The effect of different CO2 concentrations on uridine diphosphate glucose pyrophosphorylase (UDPase) in Euglena filamentosa is as follows: Figure 16 As shown in Figure B, the UDPase activity at 5% CO2 was significantly higher than that at 15% CO2, 20% CO2, and 30% CO2. The UDPase activity at 30% CO2 was the lowest, significantly lower than that at the other experimental groups and the control group. P 0.05).

[0097] 7. Cell morphology and ultrastructure tests: Centrifuge 2 mL of Euglena slenderis solution (5000 rpm, 5 min), wash 3 times with PBS, fix with 2.5% glutaraldehyde at 4℃ for 24 h, fix with 1% osmium tetroxide for 1-2 h, dehydrate with graded ethanol, infiltrate with acetone-embedding medium, ultrathin section (70-90 nm), stain with lead citrate and uranium acetate, and observe under a transmission electron microscope.

[0098] The effects of different CO2 concentrations on the cell morphology of Euglena slenderis, such as Figure 19 As shown, with increasing CO2 concentration, the length of *Euglena slenderis* cells gradually decreased while their width increased, and the length-to-width ratio changed from 3.76 to 1.10. The morphology of *Euglena slenderis* cells gradually changed from spindle-shaped and elliptical to round.

[0099] The effects of different CO2 concentrations on the ultrastructure of Euglena filamentosa, such as Figure 20 , Figure 21 , Figure 22 , Figure 23 As shown. The morphology and structure of *Euglena slendera* are not fixed; the outermost cell layer is a serrated cell membrane. The cytoplasm contains multiple chloroplasts, with clearly visible, irregularly shaped individual lamellae. Small ring-shaped parastarch granules surround the chloroplasts; as CO2 concentration increases, the number of parastarch granules increases, while the number of chloroplasts decreases. Under 30% CO2 conditions, *Euglena slendera* cells undergo significant deformation, appearing to be on the verge of rupture. Under 5% and 15% CO2 conditions, the cell morphology gradually becomes rounder. With increasing CO2 concentration, the algal cell motility decreases, swimming slowly, and almost no ruptured algal cells are visible.

[0100] In summary, 5% CO This concentration represents the optimal carbon fixation and growth conditions for Euglena spp. At this concentration, the biomass of Euglena spp. reached 1.84 g / L (a 50% increase compared to the air control group), and CO2... The fixation rate was the highest (reaching 22.32 g / L / d at 10 days), the parastarch content was 22% higher than that of the control group, the content of photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids) was significantly higher than that of other experimental groups, and the cell ultrastructure was intact (chloroplast lamellae were clear and cell membranes were undamaged).

[0101] Step 3, Comparison of multiple types of organic amines: The optimal CO determined in Step 1 At a concentration of 5%, the effects of three common alcoholic amines (MEA, DEA, and TEA) on the growth, carbon fixation, and parastarch accumulation of Euglena filamentosa were compared to screen out the organic amine with the lowest toxicity and the best enhancing effect. Specifically: This embodiment included three treatment groups: *Euglena slenderis* was cultured for 10 days in media containing 0.3 g / L monoethanolamine (MEA treatment group), diethanolamine (DEA treatment group), and triethanolamine (TEA treatment group), respectively. A control group (NC group, no additives) was also included. The 5% CO2 concentration (VCO2 / Vair) was controlled by adjusting the flow rates of pure CO2 and air using a gas mass flow controller. Each treatment group was treated with 0.375 × 10⁻⁶ ppm of CO2. 6 An initial cell density of [number] cells / mL was established. The gas mixture was added to the culture medium at a flow rate of 300 mL / min, and aeration was carried out for 8 hours daily. All treatments were conducted in triplicate.

[0102] Reference Figure 24 The cell density of *Euglena slenderis* in the control group showed a gradual increasing trend, reaching a peak of 0.81 × 10⁻⁶ cells at day 6. 6 The cells / ml value remained close to 0.87 × 10⁻⁶ in the later stages. 6The cell density of *Euglena slenderis* in the MEA-treated group was significantly higher than that in other treatment groups throughout the culture period, with the cell density per ml decreasing. P (0.05). Especially on day 10, the cell density of Euglena slenderis reached 1.25 × 10⁻⁵. 6 The cell density of *Euglena slenderis* in the DEA-treated group was significantly higher than that in other groups. The cell density of *Euglena slenderis* in the DEA-treated group increased slowly in the early stages, reaching a peak of 0.68 × 10⁶ cells / ml at day 4. 6 The cell / ml growth rate then plateaued, reaching 0.92 × 10⁻⁶ cells / ml on day 10. 6 The cell density of *Euglena slenderis* in the TEA-treated group showed a stable growth trend, with a cell density of 0.88 × 10⁶ cells / ml. 6 The cell density of *Euglena slenderis* increased more slowly than that of the control group and the MEA-treated group. The DEA-treated group showed an approximately 44.18% increase in cell density compared to the control group.

[0103] Reference Figure 25 The polysaccharide content determination results of Euglena spp. showed that the MEA treatment group had the highest polysaccharide content (0.73 mg / mL). Throughout the cultivation period, the polysaccharide content of Euglena spp. in the MEA treatment group was significantly higher than that in other treatment groups. P (0.05). After 10 days of cultivation, the polysaccharide content in the MEA-treated group was approximately 75.86% higher than that in the control group. No significant differences were found between the TEA-treated group and the DEA-treated group and the control group.

[0104] Reference Figure 26 During the 6-10 day period, the biomass of Euglena slenderis in the MEA treatment group was significantly higher than that in other treatment groups. P (0.05). Biomass growth in the MEA-treated group accelerated significantly from day 6 to 10, reaching a peak of 0.59 g / L on day 10, compared to 0.35 g / L in the control group. Growth in the DEA-treated and TEA-treated groups slowed after day 6, with biomass reaching 0.32 g / L and 0.33 g / L respectively on day 10, close to or even slightly lower than the control group. The MEA-treated group showed approximately 68.7% higher final biomass than the control group and over 80% higher than the DEA and TEA-treated groups.

[0105] Referring to Table 2, from day 2 to day 4, the carbon fixation rates of the different treatment groups did not differ significantly. P The overall carbon fixation rate remained stable at 0.05 mg / L. From day 6 onwards, the carbon fixation rate in the MEA-treated group increased significantly, reaching 6.14 ± 0.33 mg / L. ¹·d ¹, significantly higher than other treatment groups ( P 0.05). Subsequently, the carbon fixation rate in the MEA-treated group continued to increase, reaching 7.74±0.71 and 7.83±0.34 mg·L on days 8 and 10, respectively. ¹·d ¹, consistently maintained at a high level, significantly higher than other groups ( P (0.05). In contrast, the carbon fixation rate in the control group changed less throughout the culture process, showing a decreasing trend after day 6, and dropping to 3.63±0.35 mg·L on day 10. ¹·d ¹. The carbon fixation rate in the DEA-treated group did not show a significant increase, rising slightly to 4.82 ± 0.06 mg·L on day 6. ¹·d ¹Then it decreased to 3.13 ± 0.40 mg·L on day 10. ¹·d ¹. The TEA-treated group showed similar results to the DEA-treated group, with a slight increase on day 6 (5.81 ± 2.03 mg / L). ¹·d ¹), but on day 10 the level decreased to 3.45 ± 0.13 mg·L. ¹·d ¹.

[0106] Table 2. Effects of organic amines on carbon fixation rate of Euglena filamentosa Note: Different letters indicate significant differences. P 0.05).

[0107] In summary, at a concentration of 0.3 g / L, MEA significantly promoted the growth of Euglena filamentosa compared to DEA and TEA, with the MEA group achieving a cell density of 1.25 × 10⁻⁶ cells / day. CFU / mL (43.7% higher than the control group), CO The fixation rate was 7.83 mg / L / d (2.16 times that of the control group), and the parastarch content was 0.73 mg / mL (an increase of 75.86%); the cell density in the DEA group was 0.92 × 10⁻⁶. CFU / mL (only 5.7% increase), CO The fixation rate was 3.13 mg / L / d (lower than the control group); the cell density in the TEA group was 0.88 × 10⁻⁶. CFU / mL (increased by 1.1%), CO The fixation rate was 3.45 mg / L / d (lower than the control group), indicating that MEA is the most suitable type of organic amine for carbon fixation by Euglena slenderis.

[0108] Step 3, MEA Concentration Optimization: Based on the optimal CO2 concentration (5%) selected in Step 1 and the optimal organic amine (MEA) type selected in Step 2, the concentration of MEA in the culture medium was further optimized to determine a concentration that is non-toxic to Euglena spp. and maximizes carbon fixation and parastarch accumulation, avoiding the toxicity of chemical absorbents and improving carbon source utilization efficiency. Specifically: Euglena filamentosa was cultured at 26°C under a light intensity of 4500 Lux and a photoperiod of 12 L:12 D. Pure CO2 was controlled using a gas mass flow controller. The total flow rate of the gas mixed with air was 300 mL / min. The CO2 concentration in the culture medium was controlled at 5%. The medium was aerated for 8 hours during the light period each day and cultured for 14 days. During the culture, the medium was treated with MEA at concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 g / L, respectively. Each group was repeated 3 times, and a control group of 0 g / L was set up.

[0109] The performance of Euglena filamentosa cultured with different concentrations of MEA was tested, as follows: The specific testing procedures for cell density testing, relative growth rate testing, biomass testing, photosynthetic pigment testing, and parastarch content testing of Euglena spp. are the same as in step 1.

[0110] Effects of different concentrations of MEA on the growth of Euglena filamentosa cells, such as Figure 27 As shown in Figure a, the effect of different concentrations of MEA on the relative growth rate of Euglena filamentosa is as follows: Figure 27 As shown in Figure b, during the entire 14-day culture period, the cell density of *Euglena spp.* in the 0.8 g / L MEA and 1.0 g / L MEA treatment groups showed a trend of first decreasing and then increasing. With increasing time, the cell density of *Euglena spp.* in the 0.8 g / L MEA and 1.0 g / L MEA treatment groups reached their lowest values ​​on days 5 and 6, respectively. The cell density of *Euglena spp.* in the other treatment groups increased with the extension of culture time. Throughout the culture period, the cell density of *Euglena spp.* in the 0.4 g / L MEA and 0.6 g / L MEA treatment groups was consistently lower than that in the control group, while the cell density of *Euglena spp.* in the 0.2 g / L MEA treatment group was significantly higher than that in the control group after day 8. P In the control group (0.05 g / L MEA), the cell density of *Euglena spp.* ceased to increase after 10 days. However, in the 0.2 g / L MEA treatment group, the cell density of *Euglena spp.* continued to increase, reaching its peak at 14 days, with a cell density of 1.84 × 10⁻⁵. 6The relative growth rate of *Euglena scabra* cells decreased with increasing MEA concentration, with the 0.8 g / L and 1.0 g / L MEA treatment groups showing significantly lower rates than other groups. *Euglena scabra* cell density showed a decreasing trend, with the 0.8 g / L and 1.0 g / L MEA treatment groups recovering to normal growth on days 5 and 6, respectively, while the 0.2 g / L MEA treatment group achieved the highest relative growth rate. P 0.05).

[0111] The effects of different concentrations of MEA on the biomass of Euglena filamentosa, such as Figure 28 As shown, the biomass of Euglena spp. decreased with increasing MEA concentration. The 0.2 g / L MEA treatment group showed significantly higher Euglena spp. biomass than other treatment groups from 4 to 14 days, reaching a maximum of 2.47 g / L at 14 days, which was 33% higher than the control group. P 0.05). The biomass content of *Euglena scabra* in the control group was significantly lower than that in the 0.4 g / L MEA treatment group from 8 to 14 days, and the biomass content of *Euglena scabra* in the control group was significantly lower than that in the 0.4 g / L MEA treatment group from 9 to 14 days. P The 0.05 g / L, 0.8 g / L, and 1.0 g / L MEA treatment groups showed significantly lower levels of [missing information] compared to other treatment groups from 2 to 14 days. P 0.05).

[0112] The effect of different concentrations of MEA on pH of Euglena filamentosa, such as Figure 29 As shown, the pH of the culture medium in all treatment groups initially decreased and then increased over time. On day 0, the pH of the medium increased with increasing MEA concentration, rising from 8.2 to 9.1. With the addition of CO2 during the culture process, all treatment groups showed varying degrees of decrease on day 1, reaching a minimum of 7.8 in the 0.8 g / L and 1.0 g / L treatment groups. All treatment groups gradually increased their pH with increasing experimental time, eventually stabilizing at pH 8.

[0113] Figure 30 In Figure A, the effect of different concentrations of MEA on parastarch from Euglena filamentosa is shown. Figure 30 Figure B shows the effect of different concentrations of MEA on glucose pyrophosphorylase in Euglena spp. As can be seen from the figure, the parastarch content in Euglena spp. decreases with increasing MEA concentration, reaching a maximum of 123.57 mg / L in the 0.2 g / L MEA treatment group, which is 45% higher than the control group, and the lowest in the 1.0 g / L MEA treatment group. P 0.05). The UDPase activity of Euglena scabra decreased with increasing MEA concentration. P 0.05). UDPase activity reached its highest levels in the control group and the 0.2 g / L treatment group, with no significant difference between the two groups. UDPase activity was significantly higher than in other treatment groups, and lowest in the 1.0 g / L treatment group. P 0.05).

[0114] pH test of Euglena slenderis cultured with different concentrations of MEA: Samples were taken every 2 days, filtered through a 0.45 μm membrane, and the pH of the filtrate was measured with a digital pH meter.

[0115] Dissolved inorganic carbon (DIC) test of Euglena slough cultured with different concentrations of MEA: Inorganic carbon (DIC) was calculated based on the carbon ionization fraction in the culture medium obtained from total alkalinity (APHA, 1998) and pH value measurements, according to the methods of Brune and Novsak (1981) and Lü Lili et al.

[0116] The effect of different concentrations of MEA on the inorganic carbon (DIC) content in Euglena filamentosa culture medium is as follows: Figure 31 As shown in the figure. After CO2 was introduced into the culture medium, the DIC content in all treatment groups increased significantly. The inorganic carbon content in the culture medium increased with increasing MEA concentration, with the 1.0 g / L treatment group significantly higher than other treatment groups, and the control group significantly lower than other treatment groups. The DIC content in the control group and the 0.2 g / L treatment group was significantly lower than other treatment groups. P 0.05).

[0117] Enzyme activity test of Euglena filamentosa cultured with different concentrations of MEA: Centrifuge 2 mL of algal solution, homogenize the extract in an ice bath, and measure the activities of ribulose-1,5-bisphosphate carboxylase (Rubisco), carbonic anhydrase (CA), and UDPase (kits purchased from China Spectroscopy Biotechnology).

[0118] Reference Figure 32 The UDPase activity of Euglena slenderis decreased with increasing MEA concentration. P 0.05). UDPase activity was highest in the control group and the 0.2 g / L treatment group, with no significant difference between the two groups. It was significantly higher than other treatment groups, and the enzyme activity was lowest in the 1.0 g / L treatment group. P 0.05).

[0119] Figure 33 In the figure, 'a' represents the effect of different concentrations of MEA on the chlorophyll a content of Euglena filamentosa. Figure 33 In Figure b, the effect of different concentrations of MEA on the chlorophyll b content of Euglena filamentosa is shown. Figure 33In Figure c, the effect of different concentrations of MEA on the carotenoid content of Euglena spp. can be seen that the contents of chlorophyll a, chlorophyll b, and carotenoids all decreased to varying degrees with the increase of MEA concentration in the culture medium throughout the entire culture process of Euglena spp. The 0.8 g / L treatment group and the 1.0 g / L treatment group were significantly lower than the other treatment groups. P 0.05). The chlorophyll a content in the 0.2 g / L treatment group was significantly higher than that in the control group, while the chlorophyll a content in the 0.4 g / L and 0.6 g / L treatment groups was significantly lower than that in the control group and the 0.2 g / L treatment group. P 0.05). From 0 to 6 days, the chlorophyll b and carotenoid contents of *Euglena scabra* in the 0.8 g / L and 1.0 g / L treatment groups were significantly lower than those in other treatment groups. P 0.05), at 14 days, the chlorophyll b content in the 0.2 g / L MEA treatment group was significantly higher than that in other treatment groups. From 7 to 14 days, the carotenoid content in the control group and the 0.2 g / L MEA treatment group was significantly higher than that in the 0.4, 0.6, 0.8, and 1.0 g / L MEA treatment groups, with the highest content at 14 days. P 0.05).

[0120] In summary, 0.2 g / L MEA is the optimal concentration for enhancement at 5% CO2 concentration. Under the given conditions, a MEA concentration of 0.2 g / L resulted in a Euglena biomass of 2.47 g / L (a 33% increase compared to the control group), and CO2... The peak fixation rate reached 28.19 g / L / d (an increase of 26%), and the parastarch content reached 123.57 mg / L (an increase of 45%). Moreover, MEA can be gradually degraded during the culture process, avoiding long-term toxicity, while MEA at 0.8 g / L and above causes algal cells to die first and then recover slowly, significantly reducing carbon fixation efficiency.

[0121] Metabolic Mechanism Analysis: Referring to step 3, metabolomics analysis revealed the key pathways by which MEA regulates carbon and nitrogen metabolism and parastarch synthesis in Euglena spp. Compared with the inhibitory mechanisms of DEA and TEA, DEA and TEA showed relatively weak promoting effects, and TEA at high concentrations may even inhibit algae growth. Considering biomass, polysaccharide content, and carbon fixation efficiency, the MEA treatment group showed significant advantages and has potential for further research and application. This provides theoretical support for technology optimization. Specifically: The control group (NC, no MEA added) and the experimental groups were set up with three treatment groups: 0.2 g / L MEA (ME) and 1.0 g / L MEA (MA).

[0122] Non-targeted metabolomics analysis revealed the effects of different concentrations of MEA on the physiological metabolism and CO2 fixation mechanisms of Euglena filamentosa. Under 0.2 g / L MEA conditions, the upregulation of fructose, gluconic acid, and xylan activated the TCA cycle and pentose phosphate pathway, promoting photosynthetic carbon fixation and parastarch accumulation. Simultaneously, it enhanced nitrogen metabolism and ammonia detoxification capacity, significantly upregulating amino acids (aspartic acid, arginine, citrulline, and ornithine), mainly concentrated in ABC transporters, lipid metabolism, nucleotide metabolism, arginine and proline metabolism, and central carbon metabolism. 0.2 g / L MEA also promoted cell proliferation, stabilized osmotic pressure, and maintained cell homeostasis by promoting purine and pyrimidine metabolism. MEA enhanced photosynthesis, promoted cell proliferation, and improved metabolic regulation, thereby enhancing the CO2 fixation capacity and polysaccharide accumulation of microalgae.

[0123] MEA demonstrated excellent application effects in this invention, significantly promoting the growth and polysaccharide synthesis of Euglena filamentosa and increasing CO2 levels. The MEA treatment group exhibits superior carbon fixation efficiency. Its excellent biocompatibility and carbon source supply capacity make it a relatively ideal exogenous additive in microalgal carbon fixation systems. In contrast, DEA and TEA have relatively weaker promoting effects, and TEA may even inhibit algae growth at high concentrations. Considering biomass, polysaccharide content, and carbon fixation efficiency, the MEA treatment group demonstrates significant advantages and has the potential for further research and application.

[0124] Screening of differential metabolites of Euglena filamentosa at different concentrations of organic amines: setting thresholds P 0.05, |FC|≥1.2, VIP≥1.0, were used to screen for differentially expressed metabolites; among which P Here, FC represents the probability value, FC represents the difference factor, and VIP represents the projective importance of the variable.

[0125] The metabolites identified in this invention are classified and counted based on their chemical classification and attribution information, and the quantity of each metabolite accounts for, for example... Figure 30 As shown, there are 10 classes of metabolites, with lipids and lipid molecules accounting for the highest proportion at 52.84%, organic acids and their derivatives accounting for 19.03%, organic heterocyclic compounds accounting for 8.81%, organic oxygen compounds accounting for 7.67%, and nucleosides, nucleotides and analogues accounting for a relatively small proportion at 5.40%.

[0126] Enrichment of different concentrations of organic amines in the Euglena filamentosa pathway: Differential metabolites were mapped to the KEGG pathway for enrichment analysis (taking...). P (Top 20 channels with the smallest value) Differential metabolites from different treatment groups were matched against the KEGG database to obtain information on enriched pathways involved by these metabolites. In this embodiment, enrichment analysis was performed on the annotated results to obtain information on differentially metabolite enriched pathways, such as... Figure 31 , Figure 32 and Figure 33 As shown, take P The top 20 pathways with the lowest values ​​are displayed in a bubble chart. The metabolic pathways that were significantly enriched in all three treatment groups were ABC transporters, glycophospholipid metabolism, nucleotide metabolism, and metabolic pathways. Arginine and proline metabolism, purine metabolism, amino acid biosynthesis, cAMP signaling pathway, and central carbon metabolism were significantly enriched in the control group and the 0.2 g / L MEA treatment group. Arginine and proline metabolism and glycoglycerol metabolism were significantly enriched in the control group and the 1.0 g / L MEA treatment group. In the 0.2 g / L MEA and 1.0 g / L MEA treatment groups, pyrimidine metabolism, carbon metabolism, purine metabolism, amino acid biosynthesis, cAMP signaling pathway, and central carbon metabolism were significantly enriched.

[0127] In summary, 0.2 g / L MEA upregulates 77 differentially metabolites (such as guanosine, aspartic acid, and gluconic acid), activating ABC transporters, central carbon metabolism (glycolysis, tricarboxylic acid cycle), arginine-proline metabolism, and purine / pyrimidine metabolic pathways, thereby enhancing carbon source absorption and energy supply and promoting parastarch synthesis. DEA and TEA, due to their complex molecular structures (multi-hydroxyl groups, large steric hindrance), are difficult for algal cells to utilize and cause cell membrane osmotic pressure imbalance, downregulating photosynthesis-related metabolites (such as chlorophyll synthesis precursors) and inhibiting growth and carbon fixation. Through "CO2..." -MEA synergistic regulation increased parastarch content by 45% compared to the control group, and compared to CO2 alone. The regulation improved by 22%, and the proportion of parastarch in the carbon flow distribution reached 65%, which is significantly different from the characteristic of conventional algae species where the carbon flow flows towards the oil.

[0128] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa using monoethanolamine, characterized in that, Includes the following steps: MEA was added to a simplified culture medium, and then Euglena filamentosa was inoculated. During the culture process, CO2 gas was continuously bubbled into the simplified culture medium, which consisted of NH4NO3, K2HPO4, MgSO4·7H2O, CaCl2·2H2O, and ferric ammonium citrate. The mass fractions of each component were as follows: NH4NO3: 55.00~60.61 parts; K2HPO4: 12.12~12.50 parts; MgSO4·7H2O: 20.20~22.50 parts; CaCl2·2H2O: 5.05~7.50 parts; Ferric ammonium citrate: 2.02~2.50 parts.

2. The method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa as described in claim 1, characterized in that, The concentration of MEA in the simplified culture medium is 0.2~1.0 g / L.

3. The method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa as described in claim 1, characterized in that, The CO2 gas volume fraction is 5-20%, the gas supply flow rate is 300-350 mL / min, and the aeration time is 8-8.25 h / d.

4. The method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa using monoethanolamine as described in claim 1, characterized in that, The initial cell density of the *Euglena slenderis* was 0.375 × 10⁻⁶. 6 ~0.475×10 6 Cells / mL.

5. The method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa using monoethanolamine as described in claim 1, characterized in that, The pH value for culturing the *Euglena slenderis* was 7.8–9.

1.

6. The method for enhancing carbon fixation and parastarch accumulation in Euglena filamentosa as described in claim 1, characterized in that, The culture time of the *Euglena slenderis* was 10-11 days, the culture temperature was 24-26°C, the light intensity was 4400-4500 Lux, and the photoperiod was 12-14 L:12-14 D.

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