Cyanobacteria-based carbon dot material, and preparation method and application thereof

Abstract

The application discloses a preparation method of carbon dot material based on blue bacteria, and the carbon dot material is prepared by taking blue bacteria photosystem pigment as raw material. The semiconductor fluorescent carbon dot prepared by the method has stable properties, has wide spectrum absorption to sunlight, can convert ultraviolet light, visible light and infrared light into red light, and has good photoelectric performance. The application further discloses a preparation method of a blue bacteria-based semiconductor fluorescent carbon dot-blue bacteria hybrid, and application of the carbon dot material based on blue bacteria in crop production.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a carbon dot material based on cyanobacteria, a method for preparing the carbon dot material based on cyanobacteria, and its applications. Background Technology

[0002] Semiconductor fluorescent carbon dots are carbon materials with excellent light absorption, light conversion, and photoelectric properties, and are widely used in biomedicine, sensors, and optoelectronic devices. The properties of semiconductor fluorescent carbon dots are influenced by factors such as their structure, size, internal doping, and surface modification at the microscopic level; and by the raw materials, synthesis methods, and synthesis processes at the macroscopic level.

[0003] Biomass has a wide range of sources. Due to its abundant raw materials and low toxicity, more and more research and applications are using plant or animal products as raw materials to synthesize semiconductor fluorescent carbon dots. Through searching, it was found that biomass such as tea leaves, silkworm excrement, oil pressing residue, livestock and poultry blood, flower-based Chinese medicinal materials, loofah sponge, and Agrimonia pilosa are used to prepare semiconductor fluorescent carbon dots. In addition, through literature search, it was found that biomass such as fruit peels, tree bark, straw, natural products, and microbial cells are used to prepare semiconductor fluorescent carbon dots. In terms of synthesis process, the direct use of these biomass generally requires a relatively cumbersome cleaning and crushing process. Although these biomass sources are abundant, they have the following shortcomings: (1) there are large differences between batches of biomass, and the properties are unstable; (2) a relatively complex non-customized pretreatment process is generally required for subsequent synthesis, which is time-consuming, labor-intensive, and energy-intensive; (3) it is difficult to precisely control the raw materials from the front end through the modern genetic modification technology, and the properties are not easy to control; (4) it is difficult to cultivate these raw materials on a large scale under relatively fixed conditions; (5) the acquisition of these raw materials is not cost-free. Meanwhile, in terms of synthesis processes, the final raw materials used in existing patents generally do not possess photoelectric properties and rarely involve large-scale green biomanufacturing research and application.

[0004] Therefore, developing methods and processes for preparing fluorescent carbon dots that possess photoelectric properties, are readily available on a large scale, have low production costs, are stable, have easily controllable properties, and are environmentally friendly is key to the industrial application of bio-based fluorescent carbon dot materials.

[0005] Material-microbe hybrids utilize semiconductor materials to efficiently absorb light energy and convert it into NAD(P)H and ATP, which bacteria can use to provide energy for intracellular biosynthesis. This technology holds immense promise for applications in intelligent and green biomanufacturing. Material-microbe hybrids have been widely demonstrated and applied in heterotrophic chassis microorganisms. However, their application in autotrophic chassis microorganisms is currently limited, and their mechanism of action remains unclear. Therefore, developing material-autotrophic chassis microorganism hybrids that do not require the addition of organic carbon sources and elucidating their mechanisms of action are crucial for further technological advancements in material-microbe hybrids within the field of intelligent and green biomanufacturing.

[0006] In recent years, researchers have combined artificially synthesized light absorbers with natural biocatalytic systems (enzymes or whole-cell catalysts) to construct material-bio hybrids for photocatalysis, applying them to the efficient and highly specific conversion of light energy into chemical energy. This new technology has been named "semi-artificial photosynthesis." Currently, many studies and applications of these semi-artificial hybrids rely on heterotrophic microbial chassis cells. However, research and applications of semi-artificial photosynthetic hybrids targeting autotrophic chassis whose energy can be entirely derived from light are relatively few. Moreover, these studies and applications mainly target autotrophic microbial chassis that are not genetically modified or are difficult to modify.

[0007] For example, Wang Shu et al. applied water-soluble organic polymer semiconductor materials such as PFP or PBF to ungenetically modified WT7942 cyanobacteria or Chlorella proteoglycans, thereby improving the photosynthetic efficiency of the corresponding microorganisms.

[0008] Other studies have utilized carbon and MoS2 materials to improve the photosynthetic efficiency of Chlorella proteoglycans. Recently, research has also used InP materials to increase ethylene production by natural Chlorella vulgaris. Meanwhile, studies have applied gold nanoparticles to engineered cyanobacteria that produce glycerol, resulting in a 14.3% increase in glycerol production.

[0009] In these research cases, the material-microbe hybrids of autotrophic chassis currently face two main problems that urgently need to be solved:

[0010] (1) Develop low-cost, low-toxicity, and highly biocompatible semiconductor materials;

[0011] (2) A light-energy autotrophic microbial chassis with simple genetic manipulation.

[0012] Semiconductor fluorescent carbon dot materials, as a type of carbon-based nanomaterial, have significant advantages over metal-based materials in terms of biocompatibility and low cost, while cyanobacterial genetic manipulation is simple. Combining these two technologies to construct carbon dot-cyanobacterial hybrids holds promise for further technological advancements in material-microorganism hybrids.

[0013] Photosynthesis, the process by which plants, algae, and cyanobacteria use solar energy to convert carbon dioxide and water into sugars, provides food and energy for virtually all life on Earth. Although photosynthetic organisms have evolved highly efficient light-harvesting systems with high quantum efficiency, overall photosynthetic efficiency remains low (e.g., 0.2-1% for crop plants). In natural photosynthesis, light is absorbed by chlorophyll molecules within the photosystem. Because chlorophyll is primarily sensitive to visible light, the photosystem can only intercept about 40% of incident solar energy. Further energy loss occurs due to weak absorption of green light and reflection and transmission. Improving photosynthesis is essential to feed a growing population, especially considering the harmful effects of climate change and the reduction of arable land. Attempts to improve photosynthesis include genetic manipulation of light-harvesting antennae, engineering ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) to enhance its activity and specificity, improving the regenerative capacity of the carbon reduction cycle, and reconnecting photorespiration to prevent carbon dioxide release. However, these efforts face challenges: firstly, only a small number of photosynthetic organisms are suitable for complex genetic engineering. Furthermore, chlorophyll-based light harvesting and charge separation are kinetically limited and susceptible to light damage, thus hindering the efficiency of photosynthesis.

[0014] The use of photosynthetic biohybrids, combining materials and plants, offers another solution for the efficient utilization of solar energy. In such hybrid systems, materials supplement light absorption, thereby reducing the adverse effects of sunlight on natural photosynthetic systems. Currently, the application of semiconductor fluorescent carbon dots in agricultural production mainly utilizes carbon dots as blue or red light conversion materials to improve the photosynthetic efficiency of plants. However, the carbon dot materials used have not been precisely optimized for the composition of sunlight, the light utilization preferences of plants, and the characteristics of the optical electron transport chain in the plant photosynthetic system, resulting in limited performance and low efficiency. Summary of the Invention

[0015] The primary objective of this invention is to provide a method for preparing carbon dot materials based on cyanobacteria, thereby overcoming the shortcomings of existing methods for preparing semiconductor fluorescent carbon dot materials, which are cumbersome and have poor stability.

[0016] A second objective of this invention is to provide a carbon dot material based on cyanobacteria prepared by the above method.

[0017] The third objective of this invention is to provide a method for preparing cyanobacterial-based semiconductor fluorescent carbon dot-cyanobacterial hybrids, which addresses the shortcomings of existing technologies in preparing microbial hybrids, such as low biocompatibility and inapplicability to autotrophic chassis microorganisms.

[0018] A fourth objective of this invention is to provide an application of carbon dot materials based on cyanobacteria.

[0019] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted:

[0020] A method for preparing carbon dot materials based on cyanobacteria includes the following steps:

[0021] S1. Cyanobacteria are cultured in a photofermenter under light conditions. After the culture is completed, the bacteria settle naturally, the bacterial enrichment liquid is collected, and then the culture medium is removed by centrifugation to collect the cyanobacterial cells.

[0022] S2. Add solvent to the collected cyanobacterial cells for extraction, filter, retain the liquid, and obtain cyanobacterial photosystem pigments;

[0023] S3. Semiconductor fluorescent carbon dot materials were synthesized using the extracted cyanobacterial photosystem pigments as raw materials.

[0024] In this invention, the culture medium used for cyanobacterial culture is BG11 medium, which consists of: 1.5 g / L sodium nitrate (NaNO3), 40 mg / L dipotassium hydrogen phosphate (K2HPO4), 36.6 mg / L magnesium sulfate (MgSO4), 27.2 mg / L calcium chloride (CaCl2), 6 mg / L citric acid, and 6 mg / L ferric ammonium citrate. ferriccitrate), 1 mg / L disodium ethylenediaminetetraacetate (EDTANa2), 20 mg / L sodium carbonate (Na2CO3), 2.86 mg / L boric acid (H3BO3), 1.81 mg / L manganese chloride tetrahydrate (MnCl2·4H2O), 0.22 mg / L zinc sulfate (ZnSO4), 0.39 mg / L sodium molybdate dihydrate (Na2MoO4·2H2O), 0.08 mg / L copper sulfate pentahydrate (CuSO4·5H2O), 0.0409 mg / L cobalt chloride hexahydrate (CoCl2·6H2O).

[0025] In this invention, the cyanobacteria include one or more of PCC.7942, UTEX.2973, PCC.6803, and PCC.7120.

[0026] In this invention, the pH of the culture medium is maintained at 7-12, and the culture time is until OD (dose-to-temperature ratio) is reached. 730 Accumulate to reach 3-20.

[0027] In this invention, the light used during the cultivation process is sunlight or LED light.

[0028] Furthermore, the light intensity is 1-6 mW / cm². 2 Preferably, the light intensity is 3 mW / cm². 2 .

[0029] In this invention, the solvent is anhydrous ethanol, and the weight-to-volume ratio of the collected cyanobacterial cells to the solvent is 0.01-1 g / mL. Preferably, the weight-to-volume ratio of the collected cyanobacterial cells to the solvent is 0.1 g / mL.

[0030] Furthermore, the extraction is carried out under dark conditions with stirring for 2-12 hours and a stirring speed of 200-1500 rpm.

[0031] Furthermore, the extraction time was 4 hours, and the stirring speed was 800 rpm.

[0032] In this invention, filtration is performed using a 0.22μm nylon filter membrane.

[0033] In this invention, semiconductor fluorescent carbon dot materials are synthesized using a solvothermal method in step S3.

[0034] Furthermore, a solvothermal method was used to synthesize semiconductor fluorescent carbon dot materials. The specific process is as follows: cyanobacterial photosystem pigments were added to a reaction vessel lined with polytetrafluoroethylene, calcined, filtered, and evaporated to obtain the crude product, which is the semiconductor fluorescent carbon dot material.

[0035] Furthermore, the calcination temperature is 100-200℃, and the calcination time is 2-10 hours.

[0036] Preferably, the calcination temperature is 150°C and the calcination time is 4 hours.

[0037] A carbon dot material based on cyanobacteria was prepared by the above method.

[0038] A method for preparing a cyanobacterium-based semiconductor fluorescent carbon dot-cyanobacterium hybrid includes the following steps:

[0039] S1. Preparation of carbon dot materials based on cyanobacteria;

[0040] S2. The carbon dot material based on cyanobacteria prepared in S1 is prepared into a solution, added to the cyanobacteria culture, incubated, and then fermented. The supernatant is collected, separated and purified to obtain the cyanobacteria-based semiconductor fluorescent carbon dot-cyanobacteria hybrid.

[0041] In this invention, the concentration of the cyanobacterial-based carbon dot material solution is 10-200 mg / L.

[0042] Furthermore, the concentration of the cyanobacterial-based carbon dot material solution is 100 mg / L.

[0043] In this invention, the OD of the cyanobacterial culture solution 730 In 1-10. Preferably, OD 730 The value is 3.

[0044] In this invention, the cyanobacterial culture solution is either wild-type or engineered cyanobacterial culture solution.

[0045] In this invention, the incubation is carried out at 25-42°C with LED white light intensity of 1-6 mW / cm². 2 Incubate for 10-60 minutes on a shaker at a speed of 50-220 rpm.

[0046] Furthermore, incubation was carried out at 30°C with LED white light intensity of 1.35 mW / cm². 2 Incubate for 30 minutes in a shaker at 150 rpm.

[0047] In this invention, the fermentation culture is carried out at 25-42℃ with LED white light intensity of 1-6 mW / cm². 2 Shaking speed 50-220 rpm, OD of cyanobacterial solution 730 Fermentation culture under conditions 1-10.

[0048] Furthermore, the fermentation culture was carried out at 30°C with LED white light intensity of 1.35 mW / cm². 2 Shaking speed 150 rpm, OD of cyanobacterial solution 730 The value is 3.

[0049] Application of a carbon dot material based on cyanobacteria in crop production or in microbial culture and manufacturing.

[0050] Furthermore, the crop production application involves dissolving cyanobacteria-based carbon dot materials to obtain a solution, which is then sprayed onto the leaves of the crops.

[0051] Furthermore, spraying should be done every two days, based on the amount of liquid that runs off the leaves.

[0052] The present invention has the following beneficial effects:

[0053] (1) The present invention is based on the preparation method of carbon dot materials of cyanobacteria. The cyanobacterial cells cultured on a large scale are used as the initial raw material for the synthesis of semiconductor fluorescent carbon dots. Compared with traditional biomass, the source of raw materials is fixed, the properties of raw materials are stable, the batch differences are small, the culture is convenient, and the properties can be kept stable, which is conducive to the stable properties of the synthesized semiconductor fluorescent carbon dots.

[0054] (2) The method of the present invention uses cyanobacterial photosystem pigments with photoelectric properties as direct raw materials for the synthesis of semiconductor fluorescent carbon dot materials, which is beneficial to synthesizing semiconductor fluorescent carbon dot materials with better photoelectric properties, thereby enhancing the light conversion and photoelectric properties of the synthesized carbon dot materials. At the same time, it is convenient to modify the synthesized semiconductor fluorescent carbon dot materials at the front end (genetic engineering modification to modify the properties of pigments), the middle end (modifying pigments by adding additional additives), and the back end (surface modification of synthesized carbon dots).

[0055] (3) The method of the present invention can greatly improve the production efficiency of cyanobacterial biomass through large-scale continuous cultivation, realize the reuse of resources, and reduce the difficulty and cost of pretreatment before biomass utilization.

[0056] (4) The carbon dot material based on cyanobacteria prepared in this invention is stable, has a broad spectrum of absorption of sunlight, can convert ultraviolet, visible and infrared light into red light, and has good photoelectric properties (good photocurrent and ability to degrade organic matter).

[0057] (5) This invention applies cyanobacterial-based semiconductor fluorescent carbon dot materials to agricultural production. Targeting plants' preferences for absorbing and utilizing sunlight, the semiconductor fluorescent material used can convert ultraviolet, visible, and infrared light into red light, which is highly efficient for plant utilization. This expands the absorption spectrum and improves utilization efficiency. The material can penetrate plant cells, and the photogenerated electrons produced under sunlight excitation can directly enter the plant's photosystem's optical electron transport chain. Furthermore, through the material's light conversion and photoelectric synergistic effect, the photosynthetic efficiency of plants is more effectively improved. Attached Figure Description

[0058] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0059] Figure 1 This is a flowchart of the preparation method of carbon dot materials based on cyanobacteria according to the present invention;

[0060] Figure 2 This describes the growth of key performance indicators of cyanobacteria in the cyanobacterial-based semiconductor fluorescent carbon dot-glycerol-producing cyanobacterial hybrid of the present invention.

[0061] Figure 3 This is a diagram illustrating the mechanism of photogenerated electron transfer at carbon dots in the hybrid of this invention;

[0062] Figure 4 This is a photograph of the hybrid fermentation process simulating sunlight conditions according to the present invention.

[0063] Figure 5 This invention relates to the principle of how cyanobacterial-based semiconductor fluorescent carbon dots improve the efficiency of plant photosynthesis.

[0064] Figure 6 This is a characterization of the light absorption, light conversion, and photoelectric properties of the cyanobacterial-based semiconductor fluorescent carbon dots of the present invention;

[0065] Figure 7 This invention relates to the effect of cyanobacterial-based semiconductor fluorescent carbon dots on the growth phenotype of Arabidopsis thaliana. Detailed Implementation

[0066] Cyanobacteria are prokaryotic autotrophic photosynthetic microorganisms that can be cultured on a large scale using photofermenters and in a culture medium containing only a small amount of nutrients under sunlight. The light reaction of photosynthesis in cyanobacteria mainly relies on a photosystem containing pigments, and the pigments of the cyanobacterial photosystem are good raw materials for the synthesis of potential photoelectric materials. As prokaryotes, cyanobacteria are easy to genetically manipulate, and in recent years many genetic tools and elements have been developed to regulate their properties and functions.

[0067] Based on this, the present invention utilizes a low-cost, large-scale culturing process of cyanobacteria (or engineered cyanobacteria) using solar energy to extract their photosystem pigments on a large scale, and then synthesizes a novel semiconductor fluorescent carbon dot based on photosystem pigments, thereby achieving low-cost, green, and precise synthesis of semiconductor fluorescent carbon dot materials.

[0068] Example 1

[0069] like Figure 1 The method for preparing carbon dot materials based on cyanobacteria, as shown, includes the following steps:

[0070] S1. Cyanobacteria are cultured in a photofermenter under light conditions. After the culture is completed, the bacteria settle naturally, the bacterial enrichment liquid is collected, and then the culture medium is removed by centrifugation to collect the cyanobacterial cells.

[0071] The specific process is as follows:

[0072] The selected cyanobacterial culture equipment was a photofermenter with a total volume of 50L and an effective fermentation volume of 40L. The selected culture medium was BG11 medium, which consisted of: 1.5 g / L sodium nitrate (NaNO3), 40 mg / L dipotassium hydrogen phosphate (K2HPO4), 36.6 mg / L magnesium sulfate (MgSO4), 27.2 mg / L calcium chloride (CaCl2), 6 mg / L citric acid, and 6 mg / L ammonium ferric citrate. The culture medium contains: 1 mg / L disodium ethylenediaminetetraacetate (EDTANa2), 20 mg / L sodium carbonate (Na2CO3), 2.86 mg / L boric acid (H3BO3), 1.81 mg / L manganese chloride tetrahydrate (MnCl2·4H2O), 0.22 mg / L zinc sulfate (ZnSO4), 0.39 mg / L sodium molybdate dihydrate (Na2MoO4·2H2O), 0.08 mg / L copper sulfate pentahydrate (CuSO4·5H2O), and 0.0409 mg / L cobalt chloride hexahydrate (CoCl2·6H2O). This culture medium does not require strict sterilization and can be used directly after preparation.

[0073] The cyanobacteria selected for cultivation were PCC.7942, UTEX.2973, PCC.6803, PCC.7120, and engineered bacteria derived from them.

[0074] By starting OD 730 The absorbance of the bacterial culture at 730 nm is 0.5. Cyanobacteria are inoculated; in this invention, the cultivation of cyanobacteria is a large-scale continuous process. The light used for cultivation is sunlight or LED (light intensity 3 mW / cm²). 2 Light. The culture medium can be reused; only water and a small amount of essential nutrients need to be added at a certain rate. Depending on production needs, when the OD of the cultured cyanobacteria reaches a certain level... 730 When the bacterial concentration reaches 3-20, stop the continuous aeration auxiliary equipment of the photofermenter and allow the bacteria to settle naturally. After 4-6 hours of natural settling, collect approximately 5-10 L of bacterial enrichment solution from the bottom of the fermenter. After further centrifugation (centrifugation speed not exceeding 5000 rpm), the culture medium is thoroughly removed, and the cyanobacterial cells are collected for pigment extraction. The supernatant collected after centrifugation is combined with the original photofermenter. During the culture process, due to water evaporation, 5% 10x (10 times the composition of the above-mentioned BG11 medium) of BG11 medium is added daily. The pH of the medium is maintained between 7 and 12 during the culture process.

[0075] S2. Add solvent to the collected cyanobacterial cells for extraction, filter, retain the liquid, and obtain cyanobacterial photosystem pigments;

[0076] The specific process is as follows:

[0077] The collected cyanobacterial cells were subjected to pigment extraction treatment at a ratio of 1g wet weight cyanobacteria / 10ml anhydrous ethanol. After 4 hours of dark treatment with stirring at 800rpm, the mixture was filtered through a 0.22μm nylon filter membrane. The filtrate was used for the solvothermal synthesis of semiconductor fluorescent carbon dot materials.

[0078] S3. Semiconductor fluorescent carbon dot materials were synthesized using the extracted cyanobacterial photosystem pigments as raw materials.

[0079] Semiconductor fluorescent carbon dot materials were synthesized using a solvothermal method, and the specific process is as follows:

[0080] The filtrate obtained in S2 was transferred to a 50 ml reaction vessel lined with polytetrafluoroethylene and calcined in an oven at 150°C for 4 hours. Subsequently, the resulting mixture was filtered again through a 0.22 μm nylon membrane. The ethanol in the filtrate was evaporated and recovered for reuse. The crude product obtained after ethanol recovery is the cyanobacterial-based semiconductor fluorescent carbon dot material, which can be further separated and purified as needed.

[0081] Example 2

[0082] A method for preparing a cyanobacterium-based semiconductor fluorescent carbon dot-cyanobacterium hybrid includes the following steps:

[0083] S1. Prepare carbon dot materials based on cyanobacteria according to the method of Example 1;

[0084] S2. The cyanobacteria were engineered and cultured.

[0085] The specific process is as follows:

[0086] The transformation method for the cyanobacterium *Synechococcus* PCC.7942 was based on the method used in Susan Golden's laboratory, with some modifications. The specific steps are as follows: Collect 1.5 mL of *Synechococcus* PCC7942 cell culture medium (OD200). 730=Approximately 1) to a 2mL EP centrifuge tube, centrifuge at 5000g for 5min, remove the supernatant and resuspend in 0.75mL of 10mM NaCl solution, centrifuge at 5000g for 5min, then resuspend the cells in 100μL of fresh BG11, add 400ng (usually 3-5μl) of DNA and mix well, wrap the EP tube with aluminum foil to protect it from light, and place it in a 30℃ incubator with shaking overnight. Spread the Synechococcus cell solution onto BG11 plates containing the corresponding antibiotics (20μg / ml kanamycin, 15μg / ml chloramphenicol, 20μg / ml spectinomycin), and incubate at 30℃ and 55μmol of photons m-2s-1 light intensity for about 5 days until transformants grow (if a large number of transformants are found later, the volume of cyanobacteria and the amount of plasmid used at the beginning can be reduced accordingly). The PCR method is used to identify whether the target fragment is correctly inserted into the genome of the transformant. Transformants with the target fragment correctly inserted are cultured on plates with antibiotics. After about 4 generations, a recombinant strain with a pure genotype that has completely inserted the target gene can be obtained. Its genotype can then be identified by PCR.

[0087] Engineered cyanobacteria with initial OD 730 Inoculate 0.2 g of the culture medium into Erlenmeyer flasks and culture. Add appropriate antibiotics as needed. The basic culture conditions are BG11 medium, 30°C, and LED white light intensity of 1.35 mW / cm². 2 The shaker speed is 150 rpm, and the inoculation volume does not exceed 30% of the volume of the Erlenmeyer flask used. After 12 hours of incubation, add inducing agents such as IPTG as needed. After another 24-36 hours of incubation, the OD of cyanobacteria will... 730 Once the cyanobacteria have grown to approximately 1.0-1.5, collect them and wash them 1-2 times with fresh culture medium, then resuspend them in fresh culture medium to an OD value. 730 Reserved for 1, 3 and 6.

[0088] S3. The carbon dot material based on cyanobacteria prepared in S1 is prepared into a solution, added to the cyanobacteria culture, incubated, and then fermented. The supernatant is collected, separated and purified to obtain the cyanobacteria-based semiconductor fluorescent carbon dot-cyanobacteria hybrid.

[0089] The specific process is as follows:

[0090] The semiconductor fluorescent carbon dots synthesized by S1 were prepared into a solution of the required concentration and added to the S2 relative to OD. 730 The hybrid was placed in a cyanobacterial culture and then immersed in an LED white light source at 30°C with a light intensity of 1.35 mW / cm². 2Incubate for 30 minutes on a shaker at 150 rpm. After 30 minutes, place the heterozygote in a fermentation environment at 30°C, 150 rpm on a shaker, and appropriate light intensity. Collect the supernatant as needed for fermentation, and analyze the product content using mass spectrometry. Further separation and purification of the desired product is then performed. In this step, the concentration of added semiconductor fluorescent carbon dots and the OD of the bacterial culture used are considered. 730 The optimal conditions for these key factors are selected, see [link / reference]. Figure 4-6 The optimal assembly conditions for the hybrid were finally determined to be an LED white light intensity of 1.35 mW / cm². 2 The concentration of semiconductor fluorescent carbon dots added was 100 mg / L, and the OD of the cyanobacterial solution was... 730 The value is 3.

[0091] Figure 2 This relates to the use of carbon dots to improve the photosynthetic efficiency of cyanobacteria. Among other things, Figure 2 a) The effect of adding different amounts (10 mg / L, 40 mg / L, 80 mg / L, 160 mg / L) of carbon dots on the growth of cyanobacteria was investigated. The results showed that the carbon dots had the greatest promoting effect on the growth of cyanobacteria when the addition amount was 80 mg / L. Figure 2 b shows the effect of adding different amounts (10 mg / L, 40 mg / L, 80 mg / L, 160 mg / L) of carbon dots on the oxygen release rate of cyanobacteria. The results show that the higher the concentration of carbon dots, the greater the promoting effect on the oxygen release rate of cyanobacteria. Figure 2 c represents the promoting effect of carbon dots on the oxygen release rate of cyanobacteria under different light intensities. Figure 2 d-2e shows the effect of carbon dots on the photosynthetic parameters of cyanobacteria. The results indicate that the photosynthetic parameters were all upregulated to some extent. Figure 2 f-2g shows the effect of carbon dots on the concentration of intracellular metabolites in cyanobacteria. The results indicate that the addition of carbon dots increased the concentration of intracellular NADP+ and NADPH. Figure 2 h is a schematic diagram of the glycerol metabolic pathway produced by engineered cyanobacteria. Figure 2 The i-2j results showed that carbon dots could increase the activity of Rubisco enzyme in cyanobacteria and increase the glycerol production of cyanobacteria. Figure 2 k represents different ODs 730 The effect of carbon dots on the fold increase of glycerol production by cyanobacteria under certain conditions was investigated, and the results showed that OD 730 When the value is 3, carbon dots have the greatest effect on increasing glycerol production by cyanobacteria.

[0092] Figure 3 The carbon dots shown enhance the efficiency of photosynthesis based on photosensitization and light conversion. Figure 3 a represents the site of action for DCMU, DBMIB, and PMA to suppress the optical electron transport chain; Figure 3b shows that the addition of DCMU, DBMIB, and PMA respectively increased the glycerol yield by a factor of 1, indicating that the photogenerated electrons generated by the carbon dots enter the optical electron transport chain from PQ.

[0093] Figure 4 The study investigated the effect of carbon dots on cyanobacterial glycerol production under simulated sunlight conditions. Results showed that under simulated sunlight conditions, the proportion of glycerol produced by cyanobacteria increased by 24.1% compared to LED light sources.

[0094] In summary, the hybrid cyanobacteria exhibited significantly enhanced growth rate, oxygen evolution rate, photosynthetic parameters of the photosystem, intracellular NADP+ and NADPH concentrations, and Rubisco carbon fixation enzyme activity. When this hybrid was applied to glycerol-producing cyanobacteria, glycerol production was increased by up to 2.2 times.

[0095] Example 3

[0096] Application of a cyanobacterial-based carbon dot material prepared in Example 1 in crop production.

[0097] Specifically, the light absorption, light conversion, and photoelectric properties of the semiconductor fluorescent carbon dot material synthesized in Example 1 were tested using a UV spectrophotometer, a fluorescence spectrometer, an electrochemical workstation, and nuclear magnetic resonance analysis, respectively. The results are shown in [Figure number missing]. Figure 5 and Figure 6 .

[0098] Figure 5 This is a schematic diagram illustrating the principle of enhancing photosynthetic capacity in cyanobacteria and higher plants using carbon dot materials based on cyanobacteria. Results show that the semiconductor fluorescent carbon dots synthesized in this invention enhance glycerol production and plant growth based on the photoelectric effect and light conversion of photosensitizers.

[0099] Figure 6 a is a TEM image of the carbon dots, and the average size of the synthesized carbon dots is 3.5 nm; Figure 6 b shows the UV-Vis absorption spectra of carbon dots and cyanobacteria, indicating that carbon dots have a relatively wide absorption spectral range. Figure 6 c is the fluorescence spectrum of carbon dots, which shows that carbon dots can convert ultraviolet, visible and infrared light into red light of about 680nm, which is preferred by cyanobacteria and plant photosynthesis. Figure 6 d is the XPS characterization of carbon dots, showing that the conduction band of carbon dots is -1.72 eV and the valence band is 0.09 eV; Figure 6 e is the photocurrent characterization diagram of carbon dots and hybrids, which shows that carbon dots can generate photocurrent, and the photocurrent of hybrids is greater than that of individual cyanobacteria and individual carbon dots. Figure 6 f is a graph showing the effect of carbon dots degrading phenol, indicating that carbon dots can degrade phenol through charge separation.

[0100] Experiments have shown that the semiconductor fluorescent carbon dot material used in this invention has excellent light absorption, light conversion, and photoelectric properties. It has broad-spectrum absorption of sunlight, can convert ultraviolet, visible, and infrared light into red light with high photosynthetic efficiency in plants, and has excellent photoelectric properties (good photocurrent and ability to degrade organic matter).

[0101] Arabidopsis thaliana was cultivated and planted under appropriate culture conditions. The cyanobacterial-based carbon dot material prepared in Example 1 was dissolved in ethanol to a carbon dot concentration of 100 mg / L and sprayed onto the surface of Arabidopsis thaliana. The spraying was repeated every two days for two weeks, with the liquid running off the leaves as the standard. Results are shown below. Figure 7 ,in Figure 7 a is a fluorescent image of carbon dots sprayed starting on day 7, indicating that the carbon dots remain on the surface of plant leaves; Figure 7 b is a picture of Arabidopsis thaliana growth on day 14, which visually shows that the growth of Arabidopsis thaliana was significantly improved after spraying with 50 mg / L and 100 mg / L carbon dots. Figure 7 c. Figure 7 Figure d shows the statistics of fresh weight and leaf area of ​​Arabidopsis thaliana after spraying with different concentrations of carbon dots on day 14. It shows that after spraying with 50 mg / L and 100 mg / L carbon dots, the fresh weight and leaf area of ​​Arabidopsis thaliana were significantly increased. Specifically, the fresh weight of Arabidopsis thaliana increased by 80% and the leaf area increased by 39%. This indicates that carbon dots improved photosynthetic efficiency and enhanced the growth of Arabidopsis thaliana.

[0102] The invention allows for the modification of the raw materials for synthesizing semiconductor fluorescent carbon dots using synthetic biology techniques at the front end, resulting in semiconductor fluorescent carbon dot materials that enhance photosynthesis. Furthermore, the synthesized semiconductor fluorescent carbon dot materials can be further modified to improve their performance. This allows the semiconductor fluorescent carbon dot materials to be used in various agricultural production scenarios.

[0103] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for preparing a cyanobacterium-based semiconductor fluorescent carbon dot-cyanobacterium hybrid, characterized in that, Includes the following steps: S1. Preparation of carbon dot materials based on cyanobacteria using the preparation method of cyanobacterial photosystem pigments; The method for preparing the cyanobacterial photosystem pigment includes the following steps: Cyanobacteria were cultured in a photofermenter under light conditions. After the culture was completed, the bacteria were allowed to settle naturally, and the bacterial enrichment solution was collected. The culture medium was then removed by centrifugation, and the cyanobacterial cells were collected. Solvent was added to the collected cyanobacterial cells for extraction, the mixture was filtered, and the liquid was retained to obtain cyanobacterial photosystem pigments. Semiconductor fluorescent carbon dot materials were synthesized using extracted cyanobacterial photosystem pigments as raw materials; S2. The cyanobacteria are engineered and cultured. S3. The carbon dot material based on cyanobacteria prepared in S1 is prepared into a solution, added to the cyanobacteria culture, incubated, and then fermented. The supernatant is collected, separated and purified to obtain the cyanobacteria-based semiconductor fluorescent carbon dot-cyanobacteria hybrid. The OD of the cyanobacterial culture 730 In 1-10.

2. The preparation method according to claim 1, characterized in that, The cyanobacteria include one or more of PCC. 7942, UTEX.2973, PCC. 6803, and PCC. 7120.

3. The preparation method according to claim 2, characterized in that, The pH of the culture medium was maintained at 7-12, and the incubation time was until OD (October Expiratory Time). 730 Accumulate to reach 3-20.

4. The preparation method according to claim 3, characterized in that, The solvent is anhydrous ethanol, and the weight-to-volume ratio of the collected cyanobacterial cells to the solvent is 0.01-1 g / ml; the extraction is carried out under dark conditions with stirring for 2-12 hours and a stirring speed of 200-1500 rpm.

5. The preparation method according to claim 1, characterized in that, Semiconductor fluorescent carbon dot materials were synthesized using a solvothermal method. The specific process is as follows: cyanobacterial photosystem pigments were added to a reaction vessel lined with polytetrafluoroethylene, calcined, filtered, and evaporated to obtain the crude product, which is the semiconductor fluorescent carbon dot material.

6. The preparation method according to claim 5, characterized in that, The calcination temperature is 100-200℃, and the calcination time is 2-10 hours.

7. The application of a carbon dot material based on cyanobacteria in crop production, characterized in that, The method for preparing the cyanobacterial-based carbon dot material includes the following steps: Cyanobacteria were cultured in a photofermenter under light conditions. After the culture was completed, the bacteria were allowed to settle naturally, and the bacterial enrichment solution was collected. The culture medium was then removed by centrifugation, and the cyanobacterial cells were collected. Solvent was added to the collected cyanobacterial cells for extraction, the mixture was filtered, and the liquid was retained to obtain cyanobacterial photosystem pigments. Semiconductor fluorescent carbon dot materials were synthesized using extracted cyanobacterial photosystem pigments as raw materials.

Images