Photosynthesis stimulators based on hybrid carbon nanoparticles, related methods of preparation and related uses as nanobiostimulants and nanofertilizers in agricultural crops

Abstract

The present invention belongs to the field of agrochemicals. It relates to the preparation of nanocomposites based on carbon dots with luminescent and nutrient transport properties that, upon application to the leaves and / or roots of plants, stimulate photosynthesis and also physiological responses that increase growth and productivity. Such responses are characteristic of materials classified in the literature as biostimulants and / or biofertilizers.

Description

[Technical Field]

[0001] [Field of the Invention]

[01] This patent relates to the field of agrochemicals. The invention relates to the preparation of carbon dot-based nanocomposites with luminescent and nutrient transport properties that, upon application to plant leaves and / or roots, stimulate photosynthesis and also stimulate physiological responses that increase growth and productivity. Such responses are characteristic of materials classified in the literature as biostimulants and / or biofertilizers.

[0002] [Prior art]

[02] Continuous population growth, along with unstoppable urbanization, a shrinking rural workforce, and adaptation to climate change, are putting pressure on agriculture and other productive sectors to overcome the challenges of increasing food demand and sustaining production on limited agricultural areas (JAGGARD; QI; OBER, Philosophical Transactions of the Royal Society B: Biological Sciences, 2010).

[0003]

[03] According to the United Nations (UN), the world population will exceed 9 billion by 2050, and food production will have to increase by 50-70%. At the same time, demand for water and energy will increase proportionately (KEATING et al., Global Food Security, 3, 125-132, 2014).

[0004]

[04] The Food and Agriculture Organization of the United Nations (FAO) has expressed concern about the depletion and degradation of agricultural land, the dramatic decline in water resources, and their impacts on food security and the integrity of environmental systems (PRASAD; BHATTACHARYYA; NGUYEN, Frontiers in microbiology, 8, 1014, 2017). For example, demand for grains, both for food and animal feed, could exceed the current 2.1 billion tons and reach 3 billion tons by 2050 (KEATING et al., Global Food Security, 3, 125-132, 2014).

[0005]

[05] Such a bleak picture urges a global effort to develop alternative ways to meet the growing global demand for food, energy, and water without placing a strain on natural resources (Müller et al., Nature, 490, 25–257, out. 2012).

[0006]

[06] Historically, agriculture has been the most stable and important sector, providing raw materials for the food and animal feed industries for centuries. The Green Revolution of the 1960s helped significantly increase global food supplies, as agriculture benefited from various technological innovations introduced by the biotechnology and chemical sectors. However, the heavy reliance on synthetic pesticides and fertilizers that followed the Green Revolution has resulted in negative impacts on environmental systems and public health (EUROPEAN PUBLIC HEALTH ALLIANCE, Agriculture and Public Health: Impacts and pathways for better coherence 2016).

[0007]

[07] The overuse of pesticides and fertilizers contributes to the contamination of environmental systems as well as the eutrophication of aquatic environments and soils (Tilman et al., Nature, 490, 254-257, 2002; Bhat, Asia Pacific Journal of Clinical Nutrition, 17, 91-94, 2008). Pesticide residues have been found in fresh water sources (Albuquerque et al., Environmental science. Processes & impacts, 18, 779-787, 2016), fruits, processed foods (Cabras, Angioini, J. Agric. Food Chem., 48, 967-973, 2000), and breast milk (Bedi et al., Science of the Total Environment, 463-464, 720-726, 2013).

[0008]

[08] Martin et al. (2018) observed an association between cancer and exposure to pesticides and suggested that monitoring of people's exposure to pesticide residues is necessary, as pesticides may significantly contribute to the pathogenesis of malignant colorectal cancer neoplasms (Martin et al., Chemosphere 209, 623-631, 2018).

[0009]

[09] In addition to the use of pesticides, another agricultural practice that contributes to soil degradation is irrigation systems. Nearly half of agricultural production is now irrigated crops. Such practices can contribute to soil acidification, accelerated mineral extraction rates, or salt formation in the soil, which ultimately leads to the abandonment of such cultivated land (PRESLEY et al., Journal of the Soil Science Society of America, 68, 1916-1926 2004).

[0010]

[10] Therefore, the development of sustainable agricultural technologies is progressing, and in this context, nanotechnology has emerged as a possible solution to overcome the shortcomings of conventional agriculture, as it appears to be able to increase productivity and at the same time reduce the environmental burden (BHATTACHARYYA; NGUYEN, Frontiers in microbiology, 8, 1014, 2017).

[0011]

[11] In recent years, a gradual input of nanomaterials in agriculture has been observed for slow-release fertilizers, pesticides and herbicides, sensors, and plant growth and germination (GOGOS; KNAUER; BUCHELI, Journal of Agricultural and Food Chemistry, 60, 9781-9792, 2012; VISHWAKARMA et al., Nanomaterials in Plants, Algae, and Microorganisms, 1, 473-500, 2017; MULLEN, Nature Nanotechnology, 14, 515-516, 2019). Several reports indicate an increasing trend in scientific papers and patent applications on nanotechnology in agriculture, mainly related to disease management and crop protection (FRACETO et al., Frontiers in Environmental Science, 4, 20, 2016). Furthermore, nanomaterials are already being used for plant breeding and genetic engineering purposes (AMENTA et al., Regulatory Toxicology and Pharmacology, 73, 463-476 2015).

[0012]

[12] Many studies have proposed the beneficial role of nanotechnology in the agricultural sector in several different ways (KUMAR et al., Journal of Environmental Science and Technology, 16, 2175-2184, 2019). Recent reports have highlighted the applicability of effective and efficient methods of applying nanofertilizers and the economic returns they can bring, as well as the benefits and limitations associated with the use of inorganic nanofertilizers in industry (RALIYA et al., Metallomics, 7, 1584-1594, 2018).

[0013]

[13] Nevertheless, the use of nanotechnology in agricultural applications remains minimal compared to other industrial sectors (CHANDRIKA et al., Nanotechnology Prospects and Constraints in Agriculture, pp. 159-186, 2018). The specific implementation of nanotechnology in agriculture has gaps that need to be filled, such as ensuring safety for the general public. Other limitations of nanotechnology in agricultural and food development include regulatory issues and public opinion (FRACETO et al., Frontiers in Environmental Science, 4, 20 2016).

[0014]

[14] Furthermore, the majority of studies considering the use of nanofertilizers have focused on the transport of micronutrients (zinc, copper, iron, and manganese), while the advanced transport of macronutrients (potassium, nitrogen, sulfur, calcium, and magnesium) has been neglected (RALIYA et al., Metallomics, 7, 1584–1594, 2018).

[0015]

[15] Among the nanostructures already used in the agribusiness sector, inorganic nanomaterials (metal oxides and metal nanoparticles (NPs)) account for 55% of the total applications in this sector, nanocapsules account for 26%, nanocomposites correspond to 7%, and carbon-based nanomaterials (carbon nanotubes, black carbon and fullerenes) are investigated in only 6% of the reported technologies (PETERS et al., Trends in Food Science & Technology, 54, 155-164 2016).

[0016]

[16] Metal nanoparticles can also be used to control plant disease and stress resistance. For example, silver nanoparticles have shown good results against several plant pathogens, suggesting promising possibilities in agriculture (Worral et al., Agronomy, 8, 258, 2018). However, several studies have shown that these nanomaterials may cause bioaccumulation of soil aggregates and nanoparticles and nutrient transfer through the food chain (Figure 1) (DE LA TORRE ROCHE et al., Environmental Science and Technology, 49, 11866-11874, 2015; UNRINE et al., Environmental Science and Technology, 46, 9753-9760, 2012; RALIYA et al., Metallomics, 7, 1584-1594, 2018), and may even induce changes in the soil biological balance (GE; SCHIMEL; HOLDENA, Applied and Environmental Microbiology, 78, 6749-6758, 2012).

[0017]

[17] The bioaccumulation of nanopesticide residues in humans and the environment is a challenge that must be addressed, as it inevitably leads to a gradual buildup of such additives in the production chain. It is necessary to take into account all the levels at which these residues may be found and to determine the safety parameters of such inputs, thus ensuring that the environmental burden is minimized.

[0018]

[18] In this context, the present invention surpasses all technologies using metal nanoparticles in that it is based on carbon nanoparticles or carbon dots, which are completely metabolized by plants and do not transfer up the food chain, i.e., no nutrients are transferred.

[0019]

[19] Furthermore, metal nanoparticles have an effect on soil microorganisms. Ge et al. (2012) evaluated the effect of TiO2 and ZnO nanoparticles on soil bacterial populations using DNA-based fingerprinting analysis (GE; SCHIMEL; HOLDENA, Applied and Environmental Microbiology, 78, 6749-6758, 2012). The results showed that these materials induced changes in the soil biological balance. A reduction in the populations of Rhizobiales, Bradyrhizobiaceae, and Vibradyrhizobium (nitrogen-fixing bacteria) and a positive effect on the populations of Sphingomonadaceae and Streptomycetaceae were observed (GE; SCHIMEL; HOLDENA, Applied and Environmental Microbiology, 78, 6749-6758, 2012).

[0020]

[20] Carbon nanomaterials are therefore seen as promising alternatives for developing sustainable agriculture because they are low-toxicity, biocompatible, and non-bioaccumulative. Carbon nanomaterials not only function as fertilizers but also have shown beneficial effects on seed germination and plant growth (ZHENG et al., ACS Omega, 2, 3958-3965, 2017).

[0021]

[21] Carbon dots are a class of paracrystalline spherical nanoparticles with dimensions less than 10 nm, consisting of highly ordered polyaromatic domains (cores) encapsulated by amorphous carbon interfaces (CHOI et al., Chemistry - An Asian Journal, 13, 586-598, 2018). Carbon dots were first isolated in 2004 during the purification process of single-walled carbon nanotubes (XU et al., Journal of the American Chemical Society, 120, 12737-12737, 2004).

[0022]

[22] Numerous methods for preparing carbon dots have been described in the prior art.

[0023]

[23] Liu et al. (2016) employed an electrochemical method to synthesize carbon dots by using graphite electrodes as a carbon source (LIU et al., Analyst, 141, 2657–2664, 2016). The researchers used graphite rods as the cathode and anode in an electrochemical cell containing sodium hydroxide / ethanol as the electrolyte solution. Current flow through the electrochemical circuit resulted in the formation of graphite rod tips, thus producing carbon dots with different luminescence properties (wide wavelength range). Because the carbon dots obtained by the authors were polydisperse, differences in luminescence behavior may be due to mixed diameters and distinct surface defects. Another drawback of this method is the low product yield, the high cost of the graphite rods, and the need for a purification step. It is important to emphasize that the above-mentioned aspects make commercial applications of electrolytically produced carbon dots impractical.

[0024] Electrochemical routes have also been described (RAMILA DEVI; VIGNESH KUMAR; SUNDRAMOORTHY, Journal of the Electrochemical Society, 165, G3112-G3119, 2018), and various electrolyte solution compositions have been proposed to achieve other properties. However, obvious limitations of electrochemical methods are the large particle size distribution and morphology, which usually require subsequent separation and purification of the resulting materials at the end of the synthesis (RAMILA DEVI; VIGNESH KUMAR; SUNDRAMOORTHY, Journal of the Electrochemical Society, 165, G3112-G3119, 2018).

[0025]

[25] Soot, produced during several reaction processes requiring a carbon source, such as combustion or burning, has been used as a precursor for the synthesis of carbon dots. These materials / by-products are introduced into a reflux system with concentrated acid (e.g., nitric acid), followed by size separation of relatively small nanoparticles (LIU et al., Angewandte Chemie, 46, 6473–6475, 2007). The strong oxidizing properties of the acid are important for the decomposition of carbon aggregates in the soot, which then reacts with the carbon colloids to produce oxygen-rich and nitrogen-rich surface defects, along with the fluorescent emission of the particles. However, the use of such soot-based or carbon-rich by-product materials has resulted in low yields of carbon dots and the formation of large amounts of toxic residues.

[0026]

[26] Hydrothermal treatment of carbon sources has become the most common procedure for producing carbon dots. This approach allows the generation of nanoparticles from various sources, such as molecules and residues (DE YRO et al., AIP Conference Proceedings, 2083, 0200007, 2019).

[0027]

[27] The essence of the hydrothermal synthesis of carbon dots is a process that begins with high-temperature-induced nucleation / condensation of carbon molecules, which act as building blocks for the formation of graphite cores. Another important feature of the hydrothermal synthesis scheme is that some residues from the carbon reagents are retained on the surface of the graphite core, thereby imparting various controllable functional and optical properties to the particles (WANG et al., Analytical Methods, 10, 2775-2784, 2018).

[0028]

[28] In contrast to the aforementioned methods, the hydrothermal route produces particles with a narrow size distribution and consistent luminescence behavior. However, limiting factors are related to the synthesis timing, which is generally high, and the use of an internal pressure control system results in low yields.

[0029]

[29] De Medeiros et al. (2019) obtained carbon dots via microwave-assisted hydrothermal synthesis. Microwave irradiation replaces the heat source used in conventional hydrothermal methods. Carbon dots produced by microwave heating emit strong luminescence both in solution and in the solid phase. The amount of energy released during the process is quite high and can be controlled within the device itself, significantly reducing the synthesis time. The resulting particles are similar to those obtained via hydrothermal methods, but the yield is lower and is limited by the strength of the equipment used in the synthesis (DE MEDEIROS et al., Journal of Materials Chemistry C, 7, 7175-7195, 2019). The aforementioned aspects hinder the large-scale production of carbon dots via hydrothermal reactions.

[0030]

[30] High-temperature carbonization to produce carbon dots has been carried out in solvents other than water. Researchers at the Max-Planck Institute for Polymerforschung have shown that highly luminescent carbon dots can be produced by thermal treatment of a carbon source, such as citric acid, dispersed in a non-coordinating solvent, such as octadecene and an organic solvent like 1-hexadecylamine (WANG et al., Chemistry of Materials, 22, 4528–4530, 2010). Conversely, the residues from the synthesis hinder the increased production of carbon dots under such conditions.

[0031]

[31] While the aforementioned techniques are effective in producing carbon dots, they share the obstacle of producing large quantities of material with uniform and selectable characteristics. Even if carbon dots are produced in high yields, the synthesis faces problems of reproducibility and quality of the resulting material, as do electrochemical and hydrothermal processes. It is noteworthy that the method proposed by the present invention differs from the aforementioned methods in that it enables large-scale production of carbon dots, does not use hydrothermal methods, and is free of toxic reagents or residues. In the present invention, carbon dot production is carried out via a continuous flow reaction in a closed circuit, which ensures reproducibility and high yields. The proposed method recycles all water and residues and does not generate greenhouse gases.

[0032]

[32] It is important to emphasize that the validation of carbon dots in biological environments requires caution, since the raw materials and synthetic conditions, in addition to the carbon dot fabrication technology, are factors that affect the surface composition, functionality, and biological activity.

[0033]

[33] In a few examples, hydrothermal methods have been described to produce carbon dots containing fragments of precursor molecules on their surfaces. For example, Zhao et al. (2017) reported that carbon dots produced by pyrolysis of egg yolk oil exhibited anti-hemorrhagic activity similar to the drug hemocoagulase (Zhao, Y., Zhang, Y., Liu, X. et al., Scientific Reports, 7, 4452, 2017).

[0034]

[34] In Chinese Patent Application Publication No. 103980894, carbon dots prepared from folic acid by a hydrothermal route were able to selectively recognize tumor cells by specifically interacting with cell membrane proteins (folate receptors) that interact with folic acid. Each carbon dot exhibited all of the morphological and physicochemical characteristics similar to other nanoparticles prepared from other precursors. Nevertheless, the presence of folic acid fragments on the surface made these carbon dots unique and eligible for patent protection.

[0035]

[35] In another example, carbon dots produced by pyrolysis of chicken egg membranes were able to selectively recognize DNA and RNA fragments (Pramanik et al., PCCP, 20, 20476, 2018). It is noteworthy that carbon dots produced by pyrolysis of other carbon sources did not show the selective ability to recognize nucleic acids.

[0036]

[36] In WO 2018 / 160142, carbon dots produced by condensation of polyoxyethylene in acidic conditions resulted in nanomaterials with antibiotic properties and were incorporated into inert polymer films. Once again, the morphological and physicochemical characteristics are similar to those of carbon dots produced with other carbon sources, although the surface properties of the particles were responsible for the specific biological activity that allowed them to be protected.

[0037]

[37] Therefore, even though the morphological and physicochemical aspects usually suggest that carbon dots are only carbon spheres, the synthesis process and carbon source affect the surface composition and biological properties of carbon dots.

[0038]

[38] The use of carbon dots as fertilizer in agriculture has been proposed in a paper published by Zheng et al. (2017), who disclosed the use of pollen as a precursor to nanoparticles (ZHENG et al., ACS Omega, 2, 3958–3965, 2017). Tests were conducted on hydroponically grown lettuce, with a concentration of 20 mg L -1 The results show that plants treated with the solution grow by approximately 50%. The results do not suggest a selective interaction with proteins or enzymes responsible for plant growth. The authors simply propose using carbon dots as a fertilizer.

[0039]

[39] In a study published by Li et al. (2018), five carbon dots produced by electrolysis of graphite showed unique surface compositions and unique responses in rice (LI et al., ACS, Applied Biomaterials, 1, 663-672, 2018).

[0040]

[40] Significant differences in plant growth effects were noted among particles, revealing that each particle has unique characteristics, even if they are similar in size and physicochemical properties. Furthermore, the synthesized carbon dots were observed to interact with plant genetic material and promote the expression of the growth gene Os06g32600. It was further observed that the carbon dots interacted with the Rubisco protein, which is responsible for the fixation of atmospheric CO2 (LI et al., ACS, Applied Biomaterials, 1, 663-672, 2018).

[0041]

[41] In all the above, the present invention goes beyond other technologies in that it deals with carbon dots obtained by a synthetic organocatalysis process specifically developed to have specific properties that mimic reactions similar to those of plant hormones that specifically interact with cell membrane proteins, promoting accelerated plant growth and transporting macro- and micronutrients directly into plant cells.

[0042]

[42] The carbon dots described in this patent act as biostimulants, keeping stomata open for longer periods of time and promoting accelerated growth of the test medium, enhancing nutrient absorption, improving water use by plants, and increasing photosynthesis rates.

[0043]

[43] The biological activity of the carbon dots proposed herein is unique and suggests that the action of the particles on plant metabolism occurs at a molecular level, similar to the action of plant hormones such as auxin, gibberellins, and cytokinins, among others.

[0044]

[44] In all of the above, the development of biostimulants with plant hormone-mimetic activity that activates the cell membrane protein H-ATPase, as disclosed in the present invention, is unexpected in the prior art.

[0045] The present invention can be more easily understood based on Figures 1 to 19, the description of which follows. [Brief explanation of the drawings]

[0046] [Figure 1] Illustrates the eutrophication process of food chains by metal and metal oxide nanoparticles. [Figure 2] A scheme for producing biostimulants from the developed method is shown. [Figure 3] 1 shows the Fourier transform infrared spectrum of carbon dots synthesized by the developed method. [Figure 4] The formation of carbon dots and the retention of surface groups from the synthetic precursors are shown. [Figure 5] The absorption spectrum of the carbon dots in the UV-Vis range and the excitation-emission spectrum between 360 nm and 620 nm are shown. [Figure 6] The absorption spectra of chlorophyll a and b are shown. [Figure 7] A shows the XPS spectrum and biostimulant composition by measuring the macronutrients nitrogen, phosphorus, and potassium (N, P, K). B shows the high-resolution spectrum of phosphorus. C shows the microscopic image and high-resolution transmission electron microscopy image (HRTEM) and D shows the particle size distribution. [Figure 8] 1 shows the zeta potential (Z) and hydrodynamic diameter as a function of the volume (mL) of nutrient solution added to carbon dots at a concentration of 50 mg / L in a 10 ml volume. [Figure 9] The plant distribution in the four blocks P1 (white) and P2 (blue) is shown. [Figure 10] The distribution of samples from group P1 treated with various concentrations of carbon dots is shown 17 days (A), 28 days (B), 42 days (C), and 56 days (D) after the start of treatment. [Figure 11] The sample distributions for group P2 treated with various concentrations of carbon dots are shown 17 days (A), 28 days (B), 42 days (C), and 56 days (D) after the start of treatment. [Figure 12] Treatment distribution in the greenhouse is shown for groups B1, B2, B3, and B4. [Figure 13] The effect of treatment with carbon dots at various concentrations on photosynthetic rate (right) and water use efficiency (left) for blocks B1 and B2 of 'Finestra' tomato. [Figure 14] The effect of treatment with carbon dots at various concentrations on photosynthetic rate (right) and water use efficiency (left) for blocks B3 and B4 of 'Finestra' tomatoes is shown. [Figure 15] 1 shows the effect of treatment with carbon dots at various concentrations on water use efficiency in hydroponic lettuce crops. [Figure 16] Figure 1 shows the effect of carbon dot concentration on root dry mass (RDM) of maize seedlings. [Figure 17] 1 illustrates a corn cultivation system in a Leonardo vase with application of carbon dots via the nutrient solution. [Figure 18] The effect of a 100 mg / L concentration of carbon dots on liquid photosynthesis (A), stomatal conductance (B), transpiration (C), and water use efficiency (D) is shown. [Figure 19] 1 shows the percentage increase or decrease of nutrients in corn plants by using Hoagland's solution containing carbon dots.

[0047] [Detailed Description of the Invention]

[65] The present invention relates to carbonaceous photosynthesis stimulators (carbon dots) with photoluminescent properties used as biostimulants and biofertilizers that, once applied to plant leaves or roots, can induce physiological responses that enhance plant growth. The physiological responses are observed in photosynthetic variables, plant root characteristics, and in the uptake of essential micro- and macronutrients for the growth cycle.

[0048]

[66] Furthermore, the products of the present invention exhibit high solubility in water, biodegradability, are completely metabolized by plants and therefore do not bioaccumulate, their use reduces the adverse effects of nutrient transfer in the food chain of conventional nanoparticles, and are non-toxic. For this very reason, they can reduce the environmental burden in terms of soil and water contamination compared to traditional pesticides, and therefore offer high availability as inputs.

[0049]

[67] Nanomaterials act as organic carbon sources and carriers of micro- and macronutrients in their structure. During the bottom-up synthesis process, the size of the nanomaterials can be controlled, and therefore the activation of active sites in their structure can be controlled. Through surface engineering techniques, these active sites are created and tend to receive nutrients and / or stimulants that are more efficiently transported to most types of crops, facilitating plant biocompatibility with the nanocomposites and their metabolism.

[0050]

[68] The nanomaterials presented in this technique are obtained by a flow-through method using a combination of reactors according to Figure 2. After the synthesis process, the material is subjected to another drying operation to facilitate packaging.

[0051]

[69] One aspect of the present invention relates to a method for producing carbon dots, which is a versatile method that allows the use of different carbon-rich matrices as starting materials, such as glutamine, organic acids, citric acid, tartaric acid, oleic acid, malic acid, fumaric acid, isocitric acid, corn starch, etc. The method can also be performed in a co-processing manner by using alternative sources that have the same functional groups and short chains, preferably citric acid and tartaric acid, and can provide the appropriate reagent composition, composite.

[0052]

[70] The proposed method is faster than the conventional route (hydrothermal route) because it does not require a long stabilization period to initiate the carbonization process and particle growth that occurs in the hydrothermal route. It is also a low-cost method because it can be produced in a continuous process. Water is used as a solvent, and the synthesis does not produce toxic waste at the end of the process.

[0053]

[71] The method used to synthesize carbon dots is a process involving physicochemical transformations. The route used is considered chemically environmentally friendly, as it uses water as a solvent and the synthesis does not produce toxic residues.

[0054]

[72] Generally, the above aspects can be understood by a process for obtaining carbon dots that consists of catalytic chemical reactions and flow-through methods.

[0055]

[73] Nanoparticles are obtained from different carbon sources, namely citric acid, glucose, amino acids, and agricultural wastes such as peels, pulp, and animal waste. Mainly citric acid is used for partial acid-base neutralization in a basic solution, which is the source of hydroxyl functional groups used to generate salts from the acids, resulting in the formation of ions (OH) in solution form. - The electrostatic repulsion induced by the base contributes to the formation of several nucleation points. The base solution can be calcium hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, aniline, metallamine, urea and thiourea, preferably ammonium hydroxide.

[0056]

[74] An acid solution, which is a carbon source, and a base solution, which is a functional group source, are prepared and introduced into a flow system.

[0057]

[75] Using an injection pump and valves, the solution is injected into a mixer, then transferred again to a second mixing vessel to receive recycles and dopants (micro- and macronutrients or stimulants). After passing through the mixer, the reagents are pumped into a piston flow tube reactor (PFR) equipped with a valve to prevent the discharge of material and maintain a constant pressure inside the reactor, which is heated to 100-250°C, preferably 150°C, thereby avoiding volatilization of the reagents.

[0058]

[76] The PFR reactor must have a diameter reduction of only a few millimeters and be made of low-roughness materials to reduce the pressure drop at the material outlet. Another important factor in this process is maintaining a turbulent fluid outlet regime, as the reactor must be housed in a greenhouse to heat the mixture, all of which are governed by Bernoulli's principle.

[0059]

[77] The reactor must be heated between 100 and 250 °C, depending on the concentration of the materials used. The pressure inside the reactor is controlled by an output valve to prevent the solution from boiling. The pressure varies from reaction to reaction, depending on the density of the salts involved. Because the pressure is high, the reactor materials must be able to withstand pressures up to 5 bar. The material flow from the first reactor, the tube reactor, is discharged into a similarly heated, stirred continuous stirred tank reactor (CSTR). The reactor discharge flows through a hydrocyclone density differential separation system or a decanter, which allows the final product to be removed by overflow, and then returns downstream to the reflux stream.

[0060]

[78] The material is refluxed until optimal operating conditions are established. Because the density of the formed product is lower than that of the precursor salt solution, the product can be isolated by this principle: remove the supernatant, recycle the bottom material, insert it into the front of the reactor, and add the initial reagent stream. The stabilization process, in which the material is refluxed, takes 0.5 to 2 hours, preferably 1 hour, at which point the material density begins to change and product begins to form.

[0061]

[79] The yield of the process after a two-hour cycle is 95% (713 g). This amount of material is sufficient to cover 24 hectares.

[0062]

[80] This product may be stored in a liquid state or may undergo a drying process in which the material expands its surface area so that its volume increases by approximately five times.

[0063]

[81] This carbon dot manufacturing technology has scalability that allows for viable conversion to industrial scale without adversely affecting the quality of the carbon dots formed. Tests conducted on a 100-fold scale-up of bench-scale carbon dot production have shown that identical properties and quality can be produced and maintained. Given the demands for low-environmental-impact manufacturing, such as those required by the agricultural inputs industry, such a technology is unconventional and meets emerging current manufacturing requirements.

[0064]

[82] Versatility is also one of the main features of the developed manufacturing platform, allowing for a variety of compositions and properties aimed at meeting the demands of the production chain.

[0065]

[83] Carbon dots produced by this process exhibit high water solubility, as confirmed by the infrared spectrum shown in Figure 3, due to the presence of hydrophilic groups in the precursor molecules during synthesis.

[0066]

[84] The surface groups of nanoparticles, in addition to being hydrophilic, have an ionic complex potential in solution (Figure 4). Both macronutrients with high demand by plants (potassium, nitrogen, calcium, phosphorus, magnesium, and sulfur) and micronutrients with low demand (manganese, boron, zinc, iron, copper, molybdenum, and chlorine) can be added to the process to prepare carbon dots complexed with surface groups after synthesis.

[0067]

[85] Furthermore, they are biodegradable materials offering high applicability as agricultural inputs.

[0068]

[86] Therefore, another aspect of the present invention relates to the use of carbon dot-based nanoformulations as potent photosynthetic stimulants, biostimulants, and organo-mineral biofertilizers. The technology presented here is completely non-toxic, biocompatible, non-bioaccumulative, and sustainably produced using green nanotechnology principles. Test results showed an approximately 60% increase in photosynthetic efficiency, a 50% increase in water use efficiency, and a 50% increase in root mass. Nanomaterials manufactured and doped with essential chemical elements simultaneously provide nutrients, improve plant physical aspects, and reduce the negative effects of conventional fertilizer use. It is important to emphasize that the presented technology overcomes limitations related to economic feasibility, scalability, environmentally friendly manufacturing, and safety that currently hinder the sustainable use of nanotechnology in agriculture.

[0069]

[87] The use of carbon dots as photosynthetic stimulators is due to their spectroscopic properties. The introduction of nitrogenated aromatic reagents promotes the formation of C=N / C=O bonds, which are responsible for the multistate absorption and emission in the blue / green / red region (Figure 5).

[0070]

[88] Chlorophyll a and b exhibit two absorption bands centered in the spectral regions corresponding to the blue (about 440 nm) and red (about 650 nm) but show extremely low spectral response in the green region (Figure 6). The synthesized carbon dots exhibit a strong absorption in the green region (532 nm) and an emission centered at 645 nm. Due to increased exposure to sunlight, the adoption of such a spectrum results in increased agricultural productivity.

[0071] [Example]

[89] The following examples are presented to illustrate the best mode of carrying out the present invention. It should be noted that the present invention is not limited to the cited examples, but can be used for all the applications described or any other equivalent modifications.

[0072] Example 1: Synthesis of carbon dots

[90] Agricultural applications require larger quantities than laboratory applications. Therefore, process and manufacturing scaling was implemented in a pilot setup, as shown in Figure 1. As shown in Figure 1, 500 g of glucose and 550 mL of ammonium hydroxide were dissolved in 1000 mL of water and introduced into a flow-through system. The operating temperature was set at 150 °C, and the yield of the final product after a 2-hour cycle was 95% (713 g). This amount of material is sufficient for application to 24 hectares.

[0073] Example 2: Synthesis of macronutrient-containing hybrid carbon dots

[91] Initially, the process did not include a second mixer, but due to recycling needs and nutrient input requirements, a second mixer was added after the first mixer to receive the acid and base flows. In this way, recycling and dopant inputs are calculated to achieve stoichiometric ratios. This allows for the production of both pure and hybrid carbon dots, with the widest variety of dopants added to the second mixer.

[0074]

[92] Figure 7a shows the XPS spectrum and composition of a sample containing macronutrients. The high-resolution XPS spectrum in Figure 7b indicates the incorporation of phosphorus (P) into the carbon matrix. The bands centered at 131.25 and 132.25 eV correspond to -C3-PO and C-PO3 groups, respectively. Figures 7c and 7d show images of spherical nanoparticles with an average diameter of 5 nm. This suggests that phosphorus can be directly transported into the plant cellular machinery and that the nutrient bioutilization process, similar to that of nitrogen nutrients, depends on the plant's metabolism of nanomaterials. The nanoparticles simultaneously function as photosynthetic stimulants, biostimulants / biofertilizers, and macronutrient sources (organo-mineral fertilizers). Furthermore, the nanoparticles are completely water-soluble and non-toxic, advantageously reducing the environmental burden caused by excessive fertilizer use in soil. It is noteworthy that no similar technology had been reported in the literature prior to the publication of this invention.

[0075] Example 3: Evaluation of nutrient carrier potential

[93] The nutrient carrier potential of carbon dots is determined by the carboxyl groups (COO) present mainly on the surface in solution. - It has been investigated that repulsion forces favor a colloid-stabilized system because metals, classified as micronutrients and macronutrients, are positively charged in solution and are attracted by the carboxyl groups in the particles.

[0076]

[94] To analyze the surface behavior and stability of carbon dots upon the addition of ions, measurements of the zeta potential (Z) and hydrodynamic diameter were performed with increasing doses of nutrient solution. A 10 mL volume of carbon dots with a concentration of 50 mg / L was compared to a standard Hoagland solution (1 / 2 strength). In Figure 8, the behavior of the zeta potential and hydrodynamic diameter can be observed with increasing numbers of ions added.

[0077]

[95] As expected, the decreased potential indicates that metal cations bind to the surface, resulting in a gradual decrease in potential. However, as observed in Figure 8, the curvilinear behavior tends to remain constant from a certain volume. Even at high ionic strength, the particles exhibit stability, as confirmed by the small changes in hydrodynamic radius. These characteristics suggest that carbon dots can be used in conjunction with standard nutrient solutions to facilitate nutrient transport and avoid nutrient leaching, which occurs in conventional agriculture. Colloidal stability also strengthens the commercialization potential of such technology as photosynthesis stimulators, biostimulants / biofertilizers, and liquid organo-mineral fertilizers.

[0078] Example 4: Agricultural testing

[96] The pepper crop (Capsicum annuum) is chosen to demonstrate the effects of nanomaterials on plant growth, as it has received much criticism regarding pesticide residues in the fruit.

[0079]

[97] The effect of carbon dots on the production efficiency of bell peppers was set in a 2 × 4 factorial scheme, where 2 is the number of applications (dividing one application number into four) and 4 is the dose of carbon dots at 0, 25, 50, and 100 mg.L. -1 The study was conducted in a randomized block design with four replicates, resulting in a total of 32 test units.

[0080]

[98] The experimental units were 5.0 dm² filled with an inert substrate and a plant mixture. 3 The plants are kept in plastic containers. Nutrients and water are provided by fertilization irrigation, with 400 mL of the total nutrient solution applied four times a day.

[0081]

[99] Bell pepper seeds were sown in germination trays, and after 27 days, the young seedlings were transplanted into 5-liter containers pre-filled with an inert substrate consisting of turf, vermiculite, organic waste, agricultural organic waste, and lime. Thirty-two seedlings within 19 days of transplanting were selected for initiation of carbon dot application, with size and growth criteria being similar for all seedlings at the time of selection.

[0082]

[0100] The applied carbon dot concentrations (0–100 mg L -1 ) is defined from the previous test. The maximum amount of solution applied in the mass quantification of carbon dots described below is 200 mL per plant with each concentration. The plants were divided into two groups, P1 and P2, and the plants classified as P1 were given a single dose of solution (200 mL), while the plants in P2 were given gradually increasing doses (50 mL) every 15 days until they reached the maximum solution volume (200 mL). Figure 9 shows the plants divided into four blocks to confirm the randomness of the treatment.

[0083]

[0101] Plants were placed in a glasshouse and irrigated uniformly four times daily. The carbon dot solution was sprayed onto the leaves in a manner that allowed excess solution to run off into the planting substrate.

[0084]

[0102] To measure the effect of carbon dots on plant allometry, a millimeter tape measure is used to measure size along the test. On the first day, before application begins, initial size measurements are taken and collected, followed by the application of the first concentration. Group P1 receives a 200 mL volume on each plant, while Group P2 receives a 50 mL initial dose. The size collection process is carried out every two weeks, on the same day as application to Group P2, until a 200 mL volume is achieved.

[0085]

[0103] Equation 1 is used to calculate the relative growth of plants, and "h final ” represents measurements taken after treatment application, and “h initial " represents measurements taken before treatment application.

[0086]

number

[0087]

[0104] The collected data are tabulated in a spreadsheet for the application of statistical tests. The first statistical test used is the Shapiro-Wilk normality test, which indicates non-normality of the distribution of the collected data. Secondly, the Kruskal-Wallis test, which uses non-parametric statistics and independent samples, is used to examine the results (Figure 10). From this perspective, comparison of mean values ​​is the most appropriate method to identify statistical differences and the conclusions observed.

[0088]

[0105] The effect of concentration and number of carbon dots along the physiological development of plants is shown by the statistical data of groups P1 and P2 separately.

[0089]

[0106] The statistical analysis performed showed that for group P1 (Fig. 10), a single application of 200 mL per plant at the very beginning of the study, followed by 100 mg L 56 days after application, -1 Treatment with a concentration of 0 mg L -1 It can be concluded that there is a statistical difference between the other treatments, especially the control treatment, i.e., no nanobiostimulant applied (0; 0 mg.L -1 ) when a high concentration of carbon dots was used (100 mg.L -1 ) results in greater relative growth of bell peppers.

[0090]

[0107] Conversely, when analyzing the results of group P2 when a volume of 50 mL was applied at each concentration every 2 weeks until a volume of 200 mL was achieved (Figure 11), 25 mg L -1 Treatment with a concentration of 0 mg L -1 Statistical differences between the control treatment at 42 and 56 days are observed. The low concentration and multiple applications contribute in a more effective way to the relative growth when compared to the control concentration and other treatments.

[0091] Example 4 : Effect of carbon dots on photosynthetic efficiency of 'Finestra' tomato

[0108] The effect of carbon dots on photosynthetic efficiency is demonstrated using a portable photosynthesis system that employs an infrared gas analyzer. The key to these measurements is the relationship between CO2 fixation and water loss by leaf transpiration through the stomata. The specific response of both CO2 and water in the infrared is used to develop a sensor that detects infrared during gas exchange (Infrared Gas-Exchange Analyzer - IRGA).

[0092]

[0109] Changes in CO2 and water vapor can be monitored simultaneously through the leaf, allowing for accurate and integrated measurements of in vivo liquid photosynthesis and sample transpiration when illuminated by a device in a reading chamber that clamps the leaf intact, and also mitochondrial respiration rate and residual transpiration when the sample is in the dark environment of the reading chamber.

[0093]

[0110] The effect of carbon dots on photosynthesis has been observed in 'Finestra' tomatoes, a first generation hybrid developed by Embrapa Vegetables.

[0094]

[0111] Tomato seedlings are transplanted into vases 25 days after sowing, and fruit harvesting begins 60 days after transplanting.

[0095]

[0112] 48 days after sowing, five trays containing seedlings were selected to begin biostimulant applications. The concentrations used in this study were: 0 mg L -1 (water only), 0.5 mg L -1 , 2.5 mg L -1 , 5 mg L -1 and 10 mg·L -1 The solution is sprayed onto the leaves, allowing excess solution to run off into the substrate. The substrate used to fill the trays and vases is made up of biostabilized pine bark, vermiculite, charcoal dust, water, and foamed phenolic.

[0096]

[0113] The first dose application to the seedlings is done after emergence of true leaves while still in the germination trays. A single dose of 250 mL is sprayed equally onto each treatment tray, and this procedure is repeated for three more consecutive days.

[0097]

[0114] From the pre-treated trays, each treatment (0; 0.5; 2.5; 5.0; 10 mg L -1 ) and transplanted into 15-liter vases containing substrate. The seedlings were randomly distributed into four blocks, resulting in four replicates per block for each treatment and a total of 80 seedlings. Blocks B1 and B2 were exposed to light in the greenhouse, while blocks B3 and B4 were covered with shade cloth (Figure 12).

[0098]

[0115] Equation 1 given in the previous example is used to calculate relative growth.

[0099]

[0116] After transplanting into vases, the seedlings are allowed to mature in the substrate for 15 days, after which applications are resumed. At the tomato plant's growth stage, a 500 mL dose is applied onto the leaves, allowing contact between the solution and the substrate. Treatments of the same dose (concentration) are separated and pooled together to ensure equal spraying. The treatments are then redistributed to their original benches, taking great care not to mix up vases with shade cloths with those without shade cloths, when returning them to the benches. The application procedure is repeated four more times, weekly, always with a single dose.

[0100]

[0117] Data collection is carried out in the morning after application has ceased to allow physiological studies of the mechanism and intensity of photosynthetic efficiency and transpiration throughout the day. Readings are saved for all treatments and, after analysis is complete, data are extracted for statistical testing according to the variables of interest provided by the instrument.

[0101]

[0118] Figures 13 and 14 show the effect of the treatment. The first statistical test employed is the Shapiro-Wilk normality test, which indicates the non-normal distribution of the collected data. Then, non-parametric statistics, the independent samples Kruskal-Wallis test, is used to evaluate the results. In that respect, the comparison of means is the most appropriate method to identify statistical differences and conclusions of the observations made.

[0102]

[0119] The combined effect of carbon dot concentration and natural light on photosynthesis in 'Finestra' tomato plants is shown by comparing the shade cloth treatments (B3 and B4) with the non-shade cloth treatments (B1 and B2). As previously explained, carbon dots have detectable absorption / emission and emission properties in the chlorophyll absorption process.

[0103]

[0120] Data obtained in blocks B1 and B2 were 2.5 mg L -1 and 5 mg·L -1 This shows that treatment with a concentration of 100 mg / kg of sucrose tends to increase the photosynthetic rate (Figure 13).

[0104]

[0121] Water use efficiency is not directly obtained by IRGA, but is a parameter obtained by dividing photosynthetic value (Pho) by stomatal conductance (Ci). Thus, the water use efficiency in treatments B1 and B2 was also 2.5 mg L -1 and 5 mg·L -1 (Figure 13) The concentration tends to increase.

[0105]

[0122] Data collected in blocks B3 and B4, both protected by shade cloth (Figure 14), showed that when carbon dots were applied, the photosynthetic rate was lower than that of the control treatment (0 mg L -1 ), but water use efficiency is increasing (Figure 14).

[0106]

[0123] The difference between the shaded and unshaded blocks indicates that the luminescent behavior of the carbon dots occurs only when it is illuminated by an external light source.

[0107] Example 5: Effect of carbon dots on photosynthetic efficiency of lettuce crops

[0124] The effect of carbon dots on photosynthesis of lettuce (Lactuca sativa) grown in a hydroponic system is also demonstrated. Hydroponic crops are a cultivation technique in which the soil or substrate is replaced by a solution containing all the nutrients essential for plant growth.

[0108]

[0125] The hydroponic system allows the seedlings to be gradually irrigated with the nutrient solution from the roots down, thus eliminating the need for spraying and using the hydroponic system itself as a means of distributing the carbon dots to the plants.

[0109]

[0126] Three independent benches were used, connected to three containers containing independent hydroponic solutions. Each container contained a volume of 1000 liters of solution that was periodically added during the circulation of the system. Hoagland's modified nutrient solution, essential for plant growth, was prepared for all containers, with three carbon dot concentrations of 0 mg / L. -1 ;20mg.L -1 and 50 mg.L -1 The nutrient solution itself is used as a means of distributing the carbon dots.

[0110]

[0127] Seedlings are obtained and distributed equally among three benches in a greenhouse and undergo an adaptation period during which they are irrigated with a pre-prepared nutrient solution. Carbon dots are diluted in containers and introduced into the circulation 21 days after transplanting the lettuce seedlings.

[0111]

[0128] Data collection is performed on 10 lettuce plants for each treatment. Measurements are performed 15 days after the start of carbon dot application, and reading conditions are saved for all treatments. After analysis is complete, data are extracted for statistical testing, as in Example 4.

[0112]

[0129] Figure 15 shows the data obtained by IRGA. The difference is in the higher concentration treatment of 50 mg.L. -1 and control treatment 0 mg.L -1 These observations contribute to the fact that photosynthetic efficiency is achieved when application is done on leaves, but even without promoting changes in photosynthesis, carbon dots can promote better use of water during photosynthesis.

[0113] Example 6: Effect of carbon dot concentration on the roots of maize seedlings

[0130] Corn seedlings were sonicated in a 10% NaClO4 solution for 10 minutes and rinsed five times with distilled water for sterilization. They were then placed sterilely on germination paper and germinated in a BOD chamber at 28°C in the dark. Two days after germination, seedlings that had rooted approximately 1-1.5 cm were soaked in 0 mg L -1 , 10 mg.L -1 , 20 mg.L -1 , 40 mg.L -1 , 80 mg.L -1 and 160 mg.L -1 The plants were then transferred to a hydroponic system in plastic vases containing Hoagland nutrient solution (25% strength) supplemented with 1000 mg of carbon dots. After 5 days of growth, the plants (n = 9) were harvested and evaluated for root dry weight (RDW). The results were analyzed by regression to determine the most efficient carbon dot concentration.

[0114]

[0131] To calculate the most effective carbon dot concentration, a regression analysis was performed, and the model shown best fit the data. The regression data followed a normal distribution (Figure 16).

[0115]

[0132] A significant quadratic model is shown, with a stepwise inhibition at low concentrations and with increasing concentrations of carbon dots used (Figure 16). The most efficient concentration was 100 mg L -1 estimated to be carbon dots, resulting in approximately 75% more RDM compared to the control treatment.

[0116] Example 7: Effect of carbon dots on photosynthetic rate, gas exchange in leaves and nutrient content in maize seeds

[0133] Once the regression analysis of the different doses of carbon dots was completed, as outlined in Example 6, the carbon dot effect was compared with the control (0 mg L -1 ) and the most effective dose observed (100 mg.L -1 ) is shown in contrast to

[0117]

[0134] After transferring approximately 1-1.5 cm of rooted seeds to a Leonardo vase (Figure 17) containing 0.5 kg of sand (0.5-0.84 mm) sterilized with 10% HCl solution on top, the electrical conductivity was measured to be <5 mS.cm. -1 Rinse with distilled water until the concentration of the leonardo vase is 0 or 100 mg.L. -1 Carbon dots added 0.5dm 3 Hoagland nutrient solution (25% strength) is placed in the seedlings. The seedlings are repotted and every week the solution is replaced by a freshly prepared solution of increasing concentrations of 50, 75 and 100% strength.

[0118]

[0135] After 28 days of growth, leaf gas exchange parameters such as liquid photosynthesis, stomatal conductance, and transpiration were measured at 6 cm depth using an IRGA-type photosynthesis system. 2 Measurements are performed on plants (n=4) with a standard leaf chamber. Water use efficiency (WUE) is calculated as the ratio of liquid photosynthesis and transpiration. The CO2 concentration in the gas exchange chamber is maintained at 400 μmol.mol. -1 and the measurement was performed at 500 μmol.m -2 .s -1 The calculations were carried out under a photosynthetic flux density of 1000 kJ / s. The calculations were carried out between 9:00 and 11:00 am. The average temperature throughout the experiment was approximately 28°C, and the relative humidity was between 50 and 60%.

[0119]

[0136] For nutrient analysis, the plants are harvested, heat-treated at 65°C until a constant weight is reached, and then ground to a fine powder using a knife mill (2 mm mesh sieve). The resulting material is analyzed for Ca, Mg, S, Fe, Cu, Mn, Zn, and Ni content by microwave plasma atomic emission spectroscopy after digestion of 0.5 g of the composition with HNO3 and HClO4 at 180°C. Total phosphorus is estimated by the ascorbic acid / molybdenum blue acid method after digestion of the composition with H2SO4 and HO2 by colorimetry at 660 nm.

[0120]

[0137] Leaf gas exchange and nutrient content are analyzed by F-test of analysis of variance.

[0121]

[0138] The addition of carbon dots resulted in an increase in liquid photosynthesis (p<0.05) (Figure 18a), but no change in stomatal conductance or transpiration rate (Figures 18b and 18c, respectively).

[0122]

[0139] Water use efficiency (WUE) is an increasing demand in agriculture worldwide. In this regard, the use of plant stimulants should be evaluated as a valuable tool, as they act as root architecture modifiers and inducers of processes that affect plant primary and secondary metabolism. WUE represents the plant's ability to optimize carbon uptake and minimize water loss, thus resulting in a competitive advantage.

[0123]

[0140] Carbon dots significantly increased WUE by 22% (Figure 18d). This increase in parameter is characteristic of stimulant application to plants and highlights the ability of carbon dots to regulate root growth by increasing the plant's ability to absorb water from the soil.

[0124]

[0141] The contents of all analyzed nutrients change significantly due to the presence of carbon dots in the nutrient solution (Figure 19). Among them, only a decrease is observed for Mg (8.7%). Phosphorus, Ca, and Zn show relatively small increases of 7.8, 8.3, and 13.0%, respectively. The contents of sulfur and Ni show moderate increases of 26.6 and 55.5%. The largest increases are observed for Mn, Cu, and Fe, with their contents reaching 70.2, 82.1, and 82.6%, respectively.

[0125]

[0142] Nutrient content data indicates that carbon dots act as photosynthetic stimulators and nanobiostimulants, improving the uptake of some nutrients.

Claims

1. A method for producing a photosynthesis stimulant, the photosynthesis stimulant comprising hybrid carbon dots having luminescent properties and nutrient transport properties and water, the hybrid carbon dots containing membrane protein H ＋ -activating ATPase, the hybrid carbon dots are formed from a carbon-rich matrix selected from the group consisting of glutamine, organic acids, citric acid, tartaric acid, oleic acid, malic acid, fumaric acid, isocitric acid, and corn starch; The hybrid carbon dots are formed in a continuous flow-through system in which an acid and base solution is injected into a mixing vessel by an injection pump, followed by retransfer to a second mixing vessel for receiving recycles and dopants (micro- and macronutrients or stimulants), and then pumped into a piston-flow tube reactor equipped with a valve that controls the discharge of the material and maintains a constant pressure and temperature between 100 and 250°C therein, maintaining the process in a regime of turbulent fluid outflow. The material stream exiting the tube reactor is then discharged into a continuous stirred tank reactor under a similar heating pattern, the reactor discharge stream is passed through a separation system that removes the supernatant and recycles the bottom material, inserted before the reactor, an initial reagent stream is added, and the reflux process is repeated until product is formed. Method for producing photosynthesis stimulant.

2. 2. The method for producing a photosynthesis stimulator according to claim 1, wherein the hybrid carbon dots are doped with macronutrients selected from the group consisting of potassium, nitrogen, calcium, phosphorus, magnesium and sulfur, or with micronutrients selected from the group consisting of manganese, boron, zinc, iron, copper, molybdenum and chlorine.

3. 2. The method for producing a photosynthesis stimulant according to claim 1, wherein the formation of the hybrid carbon dots is by a partial acid-base neutralization reaction, and the base solution used contains calcium hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, aniline, methylamine, urea, or thiourea.

4. The method for producing a photosynthesis stimulant according to claim 1, wherein water is used as the solvent.

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