An ionic organic conjugated microporous polymer-sphaeroides-like Rhodobacterium biohybrid and its preparation method and application

Abstract

The present invention belongs to the technical field of ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrids, and particularly relates to an ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid, its preparation method, and application. An ionic organic conjugated microporous polymer and Rhodobacter sphaeroides biohybrid are constructed under anaerobic and carbon-free conditions. The constructed organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid fixes CO2 and produces carotenoids under anaerobic conditions. The ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid constructed by the present invention has a high CO2 fixation capacity under anaerobic conditions, uses CO2 as the sole carbon source for metabolism, and can efficiently synthesize carotenoids.

Description

Technical Field

[0001] The present invention belongs to the technical field of ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrids, and particularly relates to an ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid and a preparation method and application thereof. Background Art

[0002] Unrestricted consumption of fossil fuels has increased atmospheric CO2 concentrations, exacerbating the global energy crisis and global warming. There is a global consensus on developing technologies for CO2 resource utilization, using clean energy, and promoting the transition from a fossil fuel energy system to one dominated by green energy. The catalytic reduction of CO2 to high-value-added carbon-containing chemicals can reduce dependence on CO2, and thus on fossil fuels.

[0003] Microorganisms have long been attractive in the field of microbial biosynthesis due to their ability to convert renewable carbon sources into high-value chemicals through a wide range of metabolic pathways. It is well known that microorganisms can utilize carbon sources such as CO₂ or glucose for biosynthesis through metabolic pathways such as the Calvin cycle and the tricarboxylic acid cycle. However, the low efficiency of natural microbial photosynthesis is primarily due to the following: ① The photosystem can only absorb visible light (λ = 400-700 nm) and has weak absorption of green light (λ = 500-600 nm); ② Photoprotection mechanisms and other factors reduce the efficiency of light energy capture; ③ The transfer and utilization efficiency of light energy is low; and ④ The carbon fixation efficiency of the carbon-carbon bond (CBB) cycle is low. As a result, the efficiency of light-to-chemical energy conversion in natural photosynthesis is typically less than 1%.

[0004] Organic conjugated microporous polymers are an emerging class of functional materials with inherent pore structures, constructed from fully conjugated polymer networks. They combine the high specific surface area of porous materials with the excellent optoelectronic properties of conjugated polymers. These materials offer advantages such as diverse synthetic pathways, low density, rich pores, and controllable surface chemical properties, showing promising applications in a wide range of fields, including gas adsorption, sensing, catalysis, and energy storage. However, existing organic conjugated microporous polymers suffer from poor dispersibility and stability due to their highly cross-linked networks and long-range π-π conjugated rigid structures. These materials are difficult to further modify and cannot achieve perfect binding with microorganisms, severely limiting their scope of application. Summary of the Invention

[0005] In order to solve the technical problem of the lack of biohybrids for high-efficiency conversion of sunlight in the prior art, the present invention provides an ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] An ionic organic conjugated microporous polymer and Rhodobacter sphaeroides are combined to form an ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid under anaerobic and carbon-free conditions; the constructed organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid fixes CO2 and produces carotenoids under anaerobic conditions.

[0008] The preparation method of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid comprises the following steps: synthesizing the ionic organic conjugated microporous polymer using 1,1'-bis(2,4-dinitrophenyl)-4,4'-dichlorobipyridinium and N,N,N',N'-tetrakis(p-aminophenyl)-p-phenylenediamine as raw materials; and constructing the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid with the ionic organic conjugated microporous polymer and Rhodobacter sphaeroides under oxygen-free and carbon-free conditions.

[0009] Furthermore, the preparation method of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid specifically comprises the following steps:

[0010] (a) 0.05 mmol of 1,1'-bis(2,4-dinitrophenyl)-4,4'-dichlorobipyridyl and 0.025 mmol of N,N,N',N'-tetrakis(p-aminophenyl)-p-phenylenediamine were added to 50 mL of N,N-dimethylformamide and stirred until completely dissolved. The mixed solution was then placed in a reactor for reaction and finally post-treated to obtain an ionic organic conjugated microporous polymer. The specific reaction mechanism is as follows:

[0011]

[0012] (b) dissolving the obtained ionic organic conjugated microporous polymer in a carbon-free culture medium, placing the mixed solution in an anaerobic culture tube, exchanging the air in the anaerobic culture tube with a mixed gas of N2 and CO2 with a volume content ratio of 80%:20%, and inoculating OD 600 =0.1 of the Rhodobacter sphaeroides bacterial liquid was added to an anaerobic culture tube to construct an ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid.

[0013] Furthermore, in step (a), the reaction temperature is 160° C. and the reaction time is 30 h; the post-treatment method is to cool the reactor to room temperature, collect the purple precipitate by centrifugation, wash, and dry to obtain an ionic organic conjugated microporous polymer.

[0014] Furthermore, before the reaction in step (a), the air in the reactor can be replaced with an inert gas.

[0015] Another object of the present invention is to provide an application of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid, wherein CO2 in the N2 and CO2 mixed gas is used as the sole carbon source, and the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid fixes the carbon source CO2 and produces carotenoids through photoautotrophic metabolism under anaerobic conditions.

[0016] Furthermore, during the process of fixing the carbon source CO2 and generating carotenoids by the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid, carotenoids are qualitatively detected by the color change of the extract after the reaction; and carotenoids are quantitatively detected by measuring the corresponding absorbance value of the carotenoids in the extract after the reaction.

[0017] Specifically, after the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid has reacted for several days, 10 mL of the culture medium of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid can be taken, the solid obtained by centrifugation can be dissolved with an acetone solution, the acetone solution can be ultrasonicated in an ultrasonic machine, and then the supernatant can be obtained by centrifugation; the absorbance value of the supernatant in the wavelength range of 330-850 nm can be measured to quantitatively determine the carotenoids.

[0018] The ionic organic conjugated microporous polymer prepared by the present invention using 1,1'-bis(2,4-dinitrophenyl)-4,4'-dichlorobipyridinium and N,N,N',N'-tetra(p-aminophenyl)-p-phenylenediamine as raw materials has enhanced dispersibility in water due to the introduction of ionic bonds. After combining with bacteria, it does not aggregate into blocks, and has a stable structure and good dispersibility. Moreover, the ionic organic conjugated microporous polymer is tightly bound to the Rhodobacter sphaeroides through electrostatic interaction. The two can use CO2 as the sole carbon source for metabolism, and generate carotenoids by fixing CO2 under anaerobic conditions. The photogenerated electrons of the ionic organic conjugated microporous polymer described in the present invention are transferred into the cell through the protein on the Rhodobacter sphaeroides, participating in the regeneration of NADPH and increasing the NADPH / NADP ratio. + The ratio of CO2 to carotenoids is improved, thereby promoting the Calvin cycle and mevalonate pathway, increasing the amount of CO2 fixed and the efficient synthesis of carotene. The schematic diagram of the principle of the biohybrid provided by the present invention to fix the carbon source CO2 to produce carotenoids is as follows Figure 5 shown.

[0019] The ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid provided by the present invention, as well as its preparation method and application, successfully achieves efficient CO2 fixation and efficient carotenoid synthesis compared with existing biosynthetic carotenoid methods and simple Rhodobacter sphaeroides carotenoid production methods, and has high specificity for the product carotenoid; while solving the energy crisis, it can create higher economic benefits.

[0020] Secondly, compared with traditional genetic engineering methods, the preparation process of ionic organic conjugated microporous polymer-sphaeroides biohybrids is simple, low-cost, stable, mild reaction conditions, and not easily affected by environmental factors.

[0021] Finally, compared with existing materials, the present invention selects ionic organic conjugated microporous polymers that have better binding ability with bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a SEM image of the organic conjugated microporous polymer provided in Example 1 of the present invention;

[0023] Figure 2 This is an SEM image of the Rhodobacterium sphaeroides provided in Example 2 of the present invention;

[0024] Figure 3 This is an SEM image of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid provided in Example 2 of the present invention;

[0025] Figure 4 The absorbance value of the carotenoid produced by the systems a and b provided in Example 3 of the present invention within the wavelength range of 330-850 nm;

[0026] Figure 5 This is a schematic diagram of the principle of fixing carbon source CO2 to produce carotenoids by the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid provided by the present invention. DETAILED DESCRIPTION

[0027] The present invention discloses an ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid and its preparation method and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0028] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0029] The Rhodobacter sphaeroides described in the present invention was purchased from Beina Biotechnology Co., Ltd., originated from the United States, and was deposited in the American Type Culture Collection with the number ATCC49419.

[0030] The total carotenoid content (μg / L) described in the examples of the present invention can be calculated by the following formula:

[0031]

[0032] In the formula, A is the reading of the acetone extract at a wavelength of 480 nm;

[0033] D is the dilution factor;

[0034] V is the volume of acetone added during extraction;

[0035] W is the total volume of the extracted sample;

[0036] 0.16 is the uniform extinction coefficient of carotenoids.

[0037] Example 1 Synthesis of organic conjugated microporous polymers

[0038] Take 0.05mmol 1,1'-bis(2,4-dinitrophenyl)-4,4'-dichlorobipyridyl and 0.025mmol N,N,N',N'-tetrakis(p-aminophenyl)-p-phenylenediamine in a container, then add 50mL N,N-dimethylformamide and stir until completely dissolved. Then, place the mixed solution in a reactor and heat it to 160℃ for 30h. Then, wait for the reactor to cool to room temperature, collect the purple precipitate by centrifugation, wash it, and dry it to obtain an organic conjugated microporous polymer. The SEM image of the obtained organic conjugated microporous polymer is shown in the figure below. Figure 1 As shown, Figure 1 It shows that the present invention successfully synthesizes the ionic organic conjugated microporous polymer.

[0039] Example 2 Preparation Method of Ionic Organic Conjugated Microporous Polymer-Rhodobacterium sphaeroides Biohybrid

[0040] The organic conjugated microporous polymer prepared in Example 1 was dissolved in a carbon-free culture medium, and the mixed solution was placed in an anaerobic culture tube. The air in the anaerobic culture tube was exchanged with a mixed gas of N2 and CO2 with a volume content ratio of 80%:20%. The OD 600 = 0.1 of the spherical red bacteria liquid into the anaerobic culture tube to form a relatively stable and evenly dispersed suspension, and finally successfully prepared the ionic organic conjugated microporous polymer-spherical red bacteria biological hybrid. Figure 2 As shown; the SEM imaging of the prepared ionic organic conjugated microporous polymer-spherical red bacteria biohybrid is shown as Figure 3 As shown; Figure 3 It shows that the ionic organic conjugated microporous polymer prepared by this invention and the sphaeroides successfully synthesize a biohybrid with a stable structure and no aggregation.

[0041] Effect Example: Comparison of the effect of fixing carbon source CO2 to produce carotenoids

[0042] Experiment A: The ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid prepared in Example 2 of the present invention was allowed to react for several days. The CO2 in the N2 and CO2 mixture served as the sole carbon source, and the CO2 was converted by the Rhodobacter sphaeroides into carotenoids. Subsequently, 10 mL of the culture medium of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid was centrifuged and dissolved in 1 mL of acetone. The acetone solution was sonicated for 1 hour, and then centrifuged to obtain the supernatant. The absorbance of the supernatant was measured within the wavelength range of 330-850 nm to quantitatively determine the carotenoid content.

[0043] Experiment b: The air in an anaerobic culture tube was exchanged with a mixture of N2 and CO2 at a volume ratio of 80%:20%. A Rhodobacter sphaeroides bacterial culture at an OD600 of 0.1 was inoculated into the anaerobic culture tube in a carbon-free medium. The reaction was allowed to proceed for several days. The CO2 in the N2 / CO2 mixture served as the sole carbon source, and the Rhodobacter sphaeroides converted the CO2 into carotenoids. Subsequently, 10 mL of the bacterial culture was centrifuged and the resulting solid was dissolved in 1 mL of acetone. The acetone solution was sonicated for 1 hour, and the supernatant was centrifuged. The absorbance of the supernatant was measured within the wavelength range of 330-850 nm to quantitatively determine the carotenoid content.

[0044] The results of quantitative determination of carotenoids by the systems of Examples a and b are as follows Figure 4 As shown, Figure 4 In FIG. 1 , a is the absorbance of the carotenoid produced by the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid provided by the present invention, and b is the absorbance of the carotenoid produced by Rhodobacter sphaeroides alone. Figure 4In the experiments, the extracts of systems a and b showed two characteristic peaks between 400 and 550 nm, which are characteristic absorption peaks of carotenoids. The carotenoid production of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid in system a was significantly higher than that of Rhodobacter sphaeroides alone. Calculations ultimately revealed that the carotenoid production of system a was 46.69 μg / L, while that of system b was 28.38 μg / L. The carotenoid yield obtained by the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid provided by the present invention was 64.62% higher than that of carotenoids produced by Rhodobacter sphaeroides alone. Compared to carotenoids produced by Rhodobacter sphaeroides alone, the carotenoid production by the biohybrid provided by the present invention achieved efficient CO2 fixation and efficient carotenoid synthesis. The ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid constructed by the present invention can fix CO2 and generate carotenoids under anaerobic conditions, has a high fixation capacity for CO2, uses CO2 as the sole carbon source for metabolism, and can efficiently synthesize carotenoids.

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid, characterized in that: An ionic organic conjugated microporous polymer and Rhodobacter sphaeroides are combined to form an ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid under anaerobic and carbon-free conditions; the constructed organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid fixes CO2 and produces carotenoids under anaerobic conditions; The preparation method of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid comprises: synthesizing the ionic organic conjugated microporous polymer using 1,1'-bis(2,4-dinitrophenyl)-4,4'-dichlorobipyridinium and N,N,N',N'-tetrakis(p-aminophenyl)-p-phenylenediamine as raw materials; and constructing the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid with the ionic organic conjugated microporous polymer and Rhodobacter sphaeroides under oxygen-free and carbon-free conditions.

2. The method for preparing a biohybrid according to claim 1, wherein: The preparation method of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid specifically comprises the following steps: (a) 0.05 mmol of 1,1'-bis(2,4-dinitrophenyl)-4,4'-dipyridyl dichloride and 0.025 mmol of N,N,N',N'-tetrakis(p-aminophenyl)-p-phenylenediamine were added to 50 mL of N,N-dimethylformamide and stirred until completely dissolved. The mixed solution was then placed in a reactor for reaction and finally post-treated to obtain an ionic organic conjugated microporous polymer. (b) dissolving the obtained ionic organic conjugated microporous polymer in a carbon-free culture medium, placing the mixed solution in an anaerobic culture tube, exchanging the air in the anaerobic culture tube with a mixed gas of N2 and CO2 with a volume content ratio of 80%:20%, inoculating a sphaeroides red bacterium liquid with an OD600 of 0.1 into the anaerobic culture tube, and constructing an ionic organic conjugated microporous polymer-sphaeroides red bacterium biohybrid.

3. The method for preparing the biohybrid according to claim 2, wherein: In step (a), the reaction temperature is 160° C. and the reaction time is 30 h. The post-treatment method is to cool the reactor to room temperature, collect the purple precipitate by centrifugation, wash it, and dry it to obtain an ionic organic conjugated microporous polymer.

4. The method for preparing a biohybrid according to claim 2 or 3, wherein: Before the reaction in step (a), the air in the reaction vessel is replaced with an inert gas.

5. A use of the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid according to claim 1, characterized in that: CO2 in the mixed gas of N2 and CO2 serves as the sole carbon source, and the ionic organic conjugated microporous polymer-Rhodobacterium sphaeroides biohybrid fixes the carbon source CO2 and produces carotenoids through photoautotrophic metabolism under anaerobic conditions.

6. The use according to claim 5, characterized in that After the ionic organic conjugated microporous polymer-Rhodobacter sphaeroides biohybrid fixes the carbon source CO2 and generates carotenoids, the carotenoids are qualitatively detected by the color change of the extract after the reaction; and the carotenoids are quantitatively detected by measuring the corresponding absorbance value of the carotenoids in the extract after the reaction.

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