Method integrating microalgae lipid accumulation promotion, algae cell flocculation and lipid extraction

Abstract

The invention discloses a method integrating microalgae lipid accumulation promotion, algae cell flocculation and lipid extraction, and belongs to the field of bioengineering and energy. An AIE group is used as a main body structure, positive charges and hydrophobic chains are modified through quaternization, so that AIE molecules can be combined with algae cells through electrostatic interaction and hydrophobic interaction, and lipid accumulation of the algae cells is promoted by improving the light energy utilization rate of the algae cells. Furthermore, integration from lipid synthesis to extraction is realized based on the positive charge and active oxygen generation performance of molecules.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and energy technology, and specifically relates to a method that integrates the promotion of microalgal lipid accumulation, algal cell flocculation and lipid extraction. Background Technology

[0002] Photosynthesis is the best option for alleviating the energy crisis and achieving sustainable food production. Algae, as photosynthetic organisms, have become an important raw material for biodiesel due to their high lipid content. However, algal photosynthetic efficiency is low, only about 1%, which greatly limits their application in the biodiesel field. Therefore, improving their photosynthetic efficiency is crucial. Currently, fluorescent materials, as artificial antenna molecules, are used to improve the light energy utilization rate of photosynthetic organisms. For example, patent (application number: 202311835264.4) has demonstrated that a class of AIE compounds has a superior promoting effect on microalgal photosynthesis.

[0003] Photosynthetic microalgae, with their high lipid content and rapid growth and reproduction, have become a major raw material for third-generation biofuels. However, efficient lipid extraction from algae requires cell destruction. Common industrial methods, such as mechanical and ultrasonic treatments, are energy-intensive and suffer from high energy consumption in practical applications. Given the excellent performance of AIE molecules in generating reactive oxygen species (ROS) under light, developing a green technology for photoinduced ROS destruction of algal cells to improve lipid extraction efficiency will contribute to the sustainable development of algae as a raw material for biodiesel production. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0005] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0006] Therefore, the object of the present invention is to overcome the shortcomings of the prior art and provide a compound.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a compound, the structural formula of which is:

[0008] ;

[0009] Wherein, G is selected from straight-chain saturated alkanes with 4 to 16 carbon atoms;

[0010] X - Selected from anions with one charge; R is independently selected from hydrogen, hydroxyl, amino or alkyl.

[0011] As a preferred embodiment of the compound described in this invention, the structure of the compound is as follows:

[0012] .

[0013] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a compound in a method that integrates the promotion of microalgal lipid accumulation, algal cell flocculation, and lipid extraction.

[0014] As a preferred embodiment of the application described in this invention, the method includes adding the compound to a microalgae culture system for cultivation to promote microalgae growth and lipid accumulation.

[0015] As a preferred embodiment of the application described in this invention, the method includes adding the compound to a microalgae culture system to promote microalgae flocculation.

[0016] As a preferred embodiment of the application described in this invention, the method includes adding the compound to a microalgae culture system and irradiating it with light to promote the generation of ROS by the compound, which disrupts the microalgae structure and improves lipid extraction efficiency.

[0017] In a preferred embodiment of the application described in this invention, the concentration of the compound added is 0.2-5.0 µM.

[0018] In a preferred embodiment of the application described in this invention, the concentration of the compound added is greater than 5 µM, and the flocculation time is greater than 1 hour.

[0019] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method that integrates the promotion of microalgal lipid accumulation, algal cell flocculation, and lipid extraction, comprising,

[0020] The compound was added to the microalgae culture system and cultured to promote microalgae growth and lipid accumulation.

[0021] The addition of the aforementioned compound promotes microalgae flocculation;

[0022] Applying SLight illumination generates ROS that disrupt the microalgal structure, improving lipid extraction efficiency and achieving a combined effect of promoting microalgal lipid accumulation, algal cell flocculation, and lipid extraction.

[0023] Beneficial effects of this invention:

[0024] This invention provides a method that integrates the promotion of microalgal lipid accumulation, algal cell flocculation, and lipid extraction. By utilizing the TPyD molecule, which combines weak toxicity and strong ROS properties, it is suitable for promoting the growth, flocculation, and lipid extraction of Chlorella in an integrated process. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0026] Figure 1 This is the synthetic route for the TPyD molecule in this invention, as well as the TPy-C8 and TPy-C10 molecules in the comparative examples.

[0027] Figure 2 For the TPyD molecule in this invention 1 H NMR spectrum (600 MHz, Chloroform-d).

[0028] Figure 3 For the TPyD molecule in this invention 13 C10 NMR spectrum (600 MHz, Chloroform-d).

[0029] Figure 4 This is the MALDI-TOF high-resolution mass spectrum of the TPy-D molecule in this invention.

[0030] Figure 5 In this invention, DCFH-DA (ROS probe) is prepared in a BG11 solvent containing 1.0 µM TPyD molecules under light irradiation (PLight: 1200 µmol·m⁻¹). -2 ·s -1 SLight: 1000 W·m -2 The fluorescence intensity varies with time. The excitation wavelength is 488 nm.

[0031] Figure 6 The lipid content of *Chlorella proteoglycans* before and after interaction with 1.0 μM TPyD molecules, after 12 days of cultivation in an artificial climate chamber. Cultivation conditions: light / dark = 12 h / 12 ​​h, light intensity 27.6 µmol·m⁻¹. -2 ·s -1 .

[0032] Figure 7 The protein-nucleated Chlorella (C. pyre, OD) in this invention 680 The flow cytometry results of 2.0 μM TPyD molecules before and after interaction with 1.0 µM TPyD molecules, after 30 min of illumination under PLight and SLight conditions, and after 5 min of iodide pyridine staining.

[0033] Figure 8The protein-nucleated Chlorella (C. pyre, OD) in this invention 680 = 2.0) before and after the interaction of 1.0 µM TPyD molecules, under Slight conditions and after 30 min of illumination, and in Chlorella pyreus (C. pyre, OD 680 = 2.0) The lipid content extracted after being treated with methanol and ultrasound for 30 min.

[0034] Figure 9 The protein-nucleated Chlorella (C. pyre, OD) in this invention 680 = 2.0) after being reacted with different concentrations of TPyD molecules (0, 2, 5, 10 µM), the following images show the flocculation of Chlorella proteoglycans at different time points (1, 2, 3, 5 h): (a) flocculation photographs and (b) flocculation results.

[0035] Figure 10 The lipid content is calculated as follows: before and after the Chlorella proteoglycans reacted with 1.0 μM TPyD molecules, after being cultured in an artificial climate chamber for 12 days, and after reacting with 10 μM TPyD molecules and flocculating for 5 h, the lipids were extracted under SLight conditions after 5 min of light irradiation.

[0036] Figure 11 The fluorescence intensity (PLight: 1200 µmol·m⁻¹) of DCFH-DA (ROS probe) in this invention in BG11 solvent containing 1.0 µM TPy-C8, TPyD, and TPy-C12 molecules after 120 s of illumination is measured. -2 ·s -1 SLight: 1000 W·m -2 The excitation wavelength is 488 nm.

[0037] Figure 12 The protein-nucleated Chlorella (C. pyre, OD) in this invention 680 = 0.3) OD of TPy-C8, TPyD and TPy-C12 molecules at a concentration of 1.0 μM co-cultured in an artificial climate chamber for 12 days 680 nm The line graph shows the changes over time, reflecting the biotoxicity of the three molecules during the growth period of Chlorella.

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0039] Explanation of names in the embodiments of this invention:

[0040] TPEPy: 4-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)styryl)pyridine, purchased from Jilin Zhongke Science & Technology Co., Ltd.

[0041] BrC 10 H 21 : 1-Bromodecane; BrC8H 17 : 1-Bromooctane; BCr 12 H 25 : 1-Bromododecane;

[0042] DMF: N,N-dimethylformamide, is a common commercially available product.

[0043] Example 1

[0044] (1) Preparation of TPyD and the comparative molecules TPy-C8 and TPy-C12:

[0045] like Figure 1 As shown, using compound TPEPy (100 mg) as a raw material, it was reacted with BrC... 10 H 21 (110 µL), BRC8H 17 (105µL), BrC 12 H 25 The reaction was carried out with 120 µL of DMF as solvent under nitrogen protection at 80 °C for 24 h.

[0046] After the reaction was completed, the sample was purified by column chromatography with eluent (dichloromethane / methanol), and dried by rotary evaporation to obtain red solid powder TPyD, as well as two comparative molecules TPy-C8 and TPy-C12.

[0047] The structural formula of the TPyD molecule is:

[0048] .

[0049] (2) Confirmation of the molecular structure of TPyD:

[0050] Each of the TPyD molecules 1 H NMR spectrum ( Figure 2 ), 13 C NMR spectrum ( Figure 3 ) and high-resolution mass spectra ( Figure 4 The structure of the TPyD molecule can be confirmed by this.

[0051] (3) Characterization of ROS generation performance of TPyD molecules:

[0052] The fluorescence intensity of the DCFH-DA probe as a function of illumination time was used to reflect the ROS production of 1.0 μM TPyD molecules under photosynthetic light intensity (PLight) and strong light (SLight) irradiation. The specific procedure was as follows: A 1.0 μM TPyD molecular solution was prepared in 2 mL of BG11 solvent. After self-assembly in the dark for 30 min, 50 µM of the DCFH-DA probe was added. The solution was irradiated with photosynthetic light intensity (PLight) and strong light (SLight) for 0, 30, 60, 90, and 120 s, respectively. The fluorescence emission spectrum of the solution was measured under excitation light at 488 nm. The highest emission peak was plotted as a broken-line graph (PLight: 1200 µmol·m⁻¹). -2 ·s -1 SLight: 1000 W·m -2 ).

[0053] like Figure 5 As shown, TPyD molecules exhibit a weaker ROS generation capacity under PLight conditions, while their ROS generation capacity significantly increases under SLight conditions.

[0054] Example 2

[0055] Characterization of the promoting effect of TPyD molecules on lipid content in Chlorella vulgaris:

[0056] Using methyl tert-butyl ether as a lipid extractant and methanol as an organic reagent to disrupt the cell structure of *Chlorella proteoglycans*, the lipid content of *Chlorella proteoglycans* before and after interaction with TPyD (1.0 μM) molecules and after 12 days of cultivation in an artificial climate chamber was determined. The specific procedures were as follows:

[0057] 25 mL of initial concentration OD 680 = 0.3% Chlorella proteoglycans were added with 1.0 μM TPyD molecules, and an equal amount of DMSO was added to the blank group. After culturing in an artificial climate chamber for 12 days, 2 mL of methanol and 5 mL of methyl tert-butyl ether were added. After standing for 30 min, the organic phase was collected. This process was repeated 3 times. The organic phase was dried and weighed.

[0058] The results are as follows Figure 6 As shown, the lipid content in Chlorella proteoglycans co-cultured with TPyD molecules was significantly higher than that in Chlorella itself, indicating that TPyD molecules have the ability to promote lipid accumulation in Chlorella proteoglycans.

[0059] Example 3

[0060] Characterization of TPyD molecules disrupting the cell membrane structure of Chlorella protein nuclei under strong light:

[0061] Take 2 mL of Chlorella proteoglycans in the logarithmic phase (OD) 680 = 2.0), add 1.0 μM TPyD molecules, add an equal amount of DMSO to the blank group, apply SLight and PLight light for 30 min respectively, add 5 μg / mL pyridine iodide for staining, and then perform flow cytometry analysis;

[0062] like Figure 7 As shown, Chlorella proteoglycans treated with TPyD (1.0 μM) molecules showed a staining rate of nearly 90% after 30 min of SLight treatment. This indicates that the ROS generated by TPyD molecules after strong light irradiation can efficiently destroy the cell structure of Chlorella proteoglycans.

[0063] Example 4

[0064] Characterization of lipid content extracted under conditions of strong light, methanol, and ultrasound using TPyD molecules:

[0065] Using methyl tert-butyl ether as the lipid extraction solvent, 20 mL of Chlorella proteoglycans (OD200) was extracted. 680 = 2.0), add 1.0 μM TPyD molecules, add an equal amount of DMSO to the blank group, then add 5 mL of methyl tert-butyl ether, and apply strong light (1000 W·m) respectively. -2 Irradiation, methanol (1.5 mL), and sonication (80 W, 40 kHz) for 30 min were performed sequentially. The organic phase was collected, dried, and weighed. The results are as follows: Figure 8 As shown, the lipid content extracted by the TPyD molecule binding strong light irradiation method is comparable to that extracted by methanol and ultrasonic membrane rupture methods used in industry.

[0066] Example 5

[0067] Characterization of the flocculation effect of TPyD molecules on Chlorella proteoglycans: 5 mL of Chlorella in the logarithmic growth phase (OD) was taken. 680 =2.0), and different concentrations of TPyD molecules (0, 2.0, 5.0, 10.0 μM) were added sequentially. The control group was added with the same volume of DMSO, ensuring a final volume of 5 mL. After thorough mixing, the mixture was placed in the dark and allowed to stand. The OD value of the Chlorella supernatant at 680 nm was measured before the molecules were added and recorded as A0. The OD value of the supernatant was measured at 1 h, 2 h, 3 h, and 5 h and recorded as A0. t , through (A0-A t The flocculation efficiency of TPyD molecules on Chlorella at different times is calculated using 100% / A0.

[0068] like Figure 9As shown, different concentrations of TPyD molecules had different flocculation effects on Chlorella proteoglycans at different time periods. Among them, 10 μM TPyD molecules had a flocculation effect of about 90% on Chlorella proteoglycans within 5 h.

[0069] Example 6

[0070] Characterization of lipid content after TPyD promotes lipid accumulation, flocculation, and membrane rupture in an integrated manner:

[0071] Take 25 mL OD 680 =0.3% Chlorella proteoglycans were added with 1.0 μM TPyD molecules, while the control group was added with an equal amount of DMSO. After culturing in an artificial climate chamber for 12 days, 10 μM TPyD molecules were added for flocculation for 5 h, followed by the addition of 5 mL of methyl tert-butyl ether and application of SLight illumination for 5 min. The organic phase was then collected, dried, and weighed. The results are as follows. Figure 10 As shown, this strategy of combining TPyD and light to optimize the entire lipid production process yielded a lipid concentration of 290 mg / L, which is significantly higher than that of Chlorella proteoglycans itself and the lipid extraction results after no light treatment.

[0072] Comparative Example 1

[0073] (1) Comparison of ROS generation capabilities of TPy-C8, TPyD and TPy-C12.

[0074] The specific procedures are as follows: Prepare 1.0 μM solutions of TPy-C8, TPyD, and TPy-C12 molecules in 2 mL of BG11 solvent. After self-assembly in the dark for 30 min, add 50 µM of the DCFH-DA probe. Irradiate the solutions for 120 s with photosynthetic light intensity (PLight) and strong light (SLight), respectively. Measure the fluorescence emission spectra of the solutions under excitation light at 488 nm, and plot the highest emission peak intensity (PLight: 1200 µmol·m⁻¹). -2 ·s -1 SLight: 1000 W·m -2 ).

[0075] The results are as follows Figure 11As shown, under photosynthetic light intensity (PLight), the ROS production capacity of the three molecules followed the order: TPy-C8 > TPy-C12 ≈ TPyD. Under strong light (SLight) irradiation, the ROS production capacity of the three AIE molecules significantly increased, with the order being: TPy-C8 > TPyD > TPy-C12. This indicates that compared to TPyD, the TPy-C8 molecule, containing an 8-carbon hydrophobic chain, produces a large amount of ROS under both photosynthetic light intensity and strong light irradiation, suggesting that TPy-C8 still exhibits significant biotoxicity under photosynthetic light intensity. The TPy-C12 molecule, with a 12-carbon hydrophobic chain, shows a similar ROS production capacity to TPyD under photosynthetic light intensity, but its ROS production capacity is lower under strong light, thus performing poorly in the subsequent membrane lysis and lipid extraction process.

[0076] (2) Comparison of biotoxicity of TPy-C8, TPyD and TPy-C12

[0077] The specific procedure is as follows: Take 25 mL of Chlorella proteoglycans (OD2000) and... 680 = 0.3), added 1.0 μM TPy-C8, TPyD and TPy-C12 molecules, and incubated in an artificial climate chamber for 12 days. The OD of the solution was measured daily using a UV-Vis spectrophotometer. 680 The values ​​are calculated, and a line graph is plotted.

[0078] like Figure 12 As shown, 1.0 μM TPyD molecules helped promote the division and growth of Chlorella proteoglycans, while TPy-C12 molecules had almost no effect on the growth of Chlorella proteoglycans. However, TPy-C8 molecules, which have a strong ROS-generating capacity, significantly inhibited the growth of Chlorella proteoglycans, indicating that they have significant biotoxicity.

[0079] In summary, compared to other molecules, TPyD molecules containing 10 carbon atom hydrophobic chains can promote efficient solar energy conversion during photosynthesis, while still meeting the requirements for membrane disruption under high light intensity, and have the potential to promote the growth of Chlorella proteoglycans and improve lipid extraction efficiency.

[0080] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A compound, characterized in that: The structural formula of the compound is: ； Wherein, G is selected from straight-chain saturated alkanes with 4 to 16 carbon atoms; X - Selected from anions with one charge; R is independently selected from hydrogen, hydroxyl, amino or alkyl.

2. The compound according to claim 1, characterized in that: The structure of the compound is as follows: 。 3. The application of the compound according to claim 1 or 2 in a method that integrates the promotion of microalgal lipid accumulation, algal cell flocculation and lipid extraction.

4. The application as described in claim 3, characterized in that: This includes adding the compound to a microalgae culture system for cultivation to promote microalgae growth and lipid accumulation.

5. The application as described in claim 3, characterized in that: This includes adding the compound to a microalgae culture system to promote microalgae flocculation.

6. The application as described in claim 3, characterized in that: This includes adding the compound to a microalgae culture system and irradiating it with light to promote the generation of ROS by the compound, which disrupts the microalgae structure and improves lipid extraction efficiency.

7. The application as described in claim 4, characterized in that: The concentration of the compound added is 0.2-5.0 µM.

8. The application as described in claim 5, characterized in that: The concentration of the compound added is greater than 5 µM, and the flocculation time is greater than 1 hour.

9. A method integrating the promotion of microalgal lipid accumulation, algal cell flocculation, and lipid extraction, characterized in that: include, Adding the compound described in claim 1 or claim 2 to a microalgae culture system and cultivating it promotes microalgae growth and lipid accumulation. Adding the compound according to claim 1 or claim 2 promotes microalgae flocculation; Applying SLight illumination generates ROS that disrupt the microalgal structure, improving lipid extraction efficiency and achieving a combined effect of promoting microalgal lipid accumulation, algal cell flocculation, and lipid extraction.

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