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

Abstract

The present invention belongs to the field of agrochemicals. The present invention relates to the preparation of nanocomposites based on carbon dots with the properties of light emission and nutrient carrying, which stimulate photosynthesis when applied to the leaves and / or roots of plants, and stimulate physiological responses to enhance development and productivity. Such responses are characteristic of materials classified in the literature as biostimulants and / or biofertilizers.

Description

Technical Field

[0001] This patent belongs to the field of agrochemicals. The invention relates to the preparation of nanocomposites based on carbon dots with the properties of luminescence and nutrient carrying, which stimulate photosynthesis when applied to the leaves and / or roots of plants and also stimulate physiological responses to enhance development and productivity. Such responses are characteristic of materials classified as biostimulants and / or biofertilizers in the literature. Background Art

[0002] Continued population growth, coupled with uncontrolled urbanization, puts pressure on agriculture and other productive sectors to overcome the challenges of growing food demand, sustainable production in limited agricultural areas, and a declining rural workforce and adaptation to climate change (JAGGARD; QI; OBER, Philosophical Transactions of the Royal Society B: Biological Sciences, 2010).

[0003] According to the UN (United Nations), the world population will exceed 9 billion people in 2050, and food production will have to increase by 50-70%. At the same time, the demand for water and energy will increase proportionally (KEATING et al., Global Food Security, 3, 125-132, 2014).

[0004] FAO (Food and Agriculture Organization of the United Nations) has expressed concern about the depletion and degradation of agricultural land, the sharp decline in water sources and their impact on food security and ecosystem protection. (PRASAD; BHATTACHARYYA; NGUYEN, Frontiers inmicrobiology, 8, 1014, 2017) For example, by 2050, the demand for grains for food and animal feed could reach 3 billion tons, compared to the current 2.1 billion tons (KEATING et al., Global Food Security, 3, 125-132, 2014).

[0005] This pessimistic scenario has prompted worldwide efforts to develop alternative methods to meet the growing global demand for food, energy and water without increasing pressure on natural resources (MUELLER et al., Nature, 490, 254–257, out. 2012).

[0006] Historically, agriculture is the most stable and important sector, responsible for providing raw materials for the food and animal feed industries for centuries. The Green Revolution of the 1960s greatly increased the global food supply as agriculture benefited from various technological innovations introduced by the biotechnology and chemical sectors. However, the heavy reliance on synthetic pesticides and fertilizers after the Green Revolution had negative impacts on ecosystems and public health (EUROPEAN PUBLIC HEALTHALLIANCE, Agriculture and Public Health: Impacts and pathways for better coherence 2016).

[0007] The misuse of pesticides and fertilizers has caused the pollution of ecosystems and 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 freshwater resources (ALBUQUERQUE et al., Environmental science. Processes & impacts, 18, 779-787, 2016), fruits, processed foods (CABRAS, ANGIONI, J. Agric. Food Chem. 48, 967-973 2000) and even human breast milk (BEDI et al., Science of The Total Environment, 463-464, 720-726, 2013).

[0008] Martin et al. (2018) observed a link between cancer and exposure to pesticides and argued that pesticides may play an important role in the etiology of malignant colon cancer tumors, and therefore it is necessary to monitor the level of exposure to pesticide residues in the human population (Martin et al., Chemosphere 209, 623–631, 2018).

[0009] In addition to the use of pesticides, another agricultural practice that leads to soil degradation is the irrigation system. Currently, almost half of agricultural production comes from irrigated crops. This frequent practice can lead to soil acidification and accelerate the extraction rate of minerals in the soil or salt layers, which ultimately lead to the abandonment of such planting areas (PRESLEY et al., Soil Science Society of America Journal, 68, 1916-1926 2004).

[0010] Therefore, the development of sustainable agricultural technologies has been growing, and in this context, nanotechnology has emerged as a potential solution to overcome the shortcomings of traditional agriculture as it is able to increase productivity while reducing the impact on the environment (BHATTACHARYYANGUYEN, Frontiers in microbiology, 8, 1014, 2017).

[0011] In the past few years, it has been observed that nanomaterials are gradually being put into agriculture for controlled 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 show that scientific papers and patent applications in the field of agricultural nanotechnology are on the rise, mainly for disease management and crop protection (FRACETO et al., Frontiers in Environmental Science, 4, 202016). In addition, nanomaterials have been used for plant breeding and genetic engineering purposes (AMENTA et al., Regulatory Toxicology and Pharmacology, 73, 463–476 2015).

[0012] Many studies have described the beneficial effects of nanotechnology in the agricultural sector in several different ways (KUMAR et al., Journal of Environmental Science and Technology, 16, 2175-21842019). In recent reports, the advantages and limitations of using inorganic nanofertilizers in industry, as well as the effective and efficient way of applying nanofertilizers and the applicability of their economic returns (RALIYA et al., Metallomics, 7, 1584–1594, 2018) are emphasized.

[0013] Nevertheless, the use of nanotechnology in agricultural applications is still negligible compared to other industrial sectors (CHANDRIKA et al., Nanotechnology Prospects and Constraints in Agriculture, 159-186, 2018). The specific implementation of nanotechnology in agriculture has a gap that needs to be filled to ensure the safety of the overall population. Other limitations of nanotechnology in agriculture and food development are regulatory issues and public opinion (FRACETO et al., Frontiers in Environmental Science, 4, 20 2016).

[0014] Furthermore, the vast majority of research on the use of nanofertilizers has focused on the delivery of micronutrients (Zn, Cu, Fe, Mn), while the smart delivery of macronutrients (K, N, S, Ca, and Mg) has been neglected (RALIYA et al., Metallomics, 7, 1584–1594, 2018).

[0015] Among the nanostructures that have been used in the agribusiness sector, inorganic nanomaterials (metal oxide nanoparticles and metal nanoparticles (NPs)) account for 55% of all applications in the sector, while nanocapsules account for 26%, nanocomposites account for 7%, and the development of carbon-based nanomaterials (carbon nanotubes, carbon black and furelene) accounts for only 6% of the reported technologies (PETERS et al., Trends in Food Science & Technology, 54, 155–164 2016).

[0016] Metal nanoparticles can also be used to control plant diseases and stress tolerance. For example, silver nanoparticles have shown excellent effects against several plant pathogens, thus indicating their promising potential in agriculture (Worrall et al. Agronomy, 8, 258, 2018). However, some studies have shown that these nanomaterials may present bioaccumulation and trophic transfer of soil particles and nanoparticles 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), in addition, it may also cause changes in the balance of soil biota (GE; SCHIMEL; HOLDENA, Applied and Environmental Microbiology, 78, 6749–6758, 2012).

[0017] The bioaccumulation of nano-agrochemical residues in humans and the environment is an issue to be addressed, since this accumulation is bound to increase with the gradual increase of such additives in the production chain, and it is necessary to consider all the levels at which these residues may be present and also determine the safety parameters of such inputs, thus ensuring the minimum impact on the environment.

[0018] In this context, the present invention is superior to all technologies using metal nanoparticles, since it is based on carbon nanoparticles or carbon dots, which are completely metabolized by plants and are not transferred in the food chain, ie there is no nutrient transfer.

[0019] In addition, metal nanoparticles have an impact on soil microorganisms. Ge et al. (2012) evaluated the effects of TiO 2 and ZnO nanoparticles on soil bacterial communities (GE; SCHIMEL; HOLDENA, Applied and Environmental Microbiology, 78, 6749–6758, 2012). The results obtained showed that these materials caused changes in the balance of soil biota. A decrease in the populations of Rhizobiales, Bradyrhizobiaceae and Bradyrhizobia (diazotrophs) was observed, as well as a positive effect on the populations of Sphingomonasaceae and Streptomycesaceae (GE; SCHIMEL; HOLDENA, Applied and Environmental Microbiology, 78, 6749–6758, 2012).

[0020] For this reason, carbon nanomaterials have become a promising candidate for developing sustainable agricultural alternatives due to their low toxicity, biocompatibility, and non-bioaccumulative properties. In addition to being used as fertilizers, carbon nanomaterials have shown beneficial effects on seed germination and enhanced plant growth (ZHENG et al., ACS Omega, 2, 3958–3965, 2017).

[0021] Carbon dots are a class of quasi-crystalline spherical nanoparticles with a size less than 10 nm and composed of highly ordered polyaromatic domains (cores) surrounded by amorphous carbon boundaries (CHOI et al., Chemistry-An Asian Journal, 13, 586-598, 2018). Carbon dots were first isolated in 2004 during the purification of single-walled carbon nanotubes (XU et al. Journal of the American Chemical Society, 120, 12737-12737, 2004).

[0022] Many methods for preparing carbon dots have been described in the prior art.

[0023] Liu et al. (2016) adopted 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 cathodes and anodes of electrochemical cells and sodium hydroxide / ethanol as electrolyte solutions. The passage of current through the electrochemical circuit resulted in the formation of graphite rod fragments, resulting in carbon dots with different luminescence characteristics (different wavelengths). Since the carbon dots obtained by the authors are polydisperse, the differences in luminescence behavior can be attributed to mixed sizes and obvious surface defects. Another disadvantage of this method is the low product yield, the high cost of graphite rods, and the need for a purification stage. It must be emphasized that the above aspects make the commercial application of carbon dots produced by electrolysis unfeasible.

[0024] Electrochemical approaches have also been described, (RAMILA DEVI; VIGNESH KUMAR; SUNDRAMOORTHY, Journal of the Electrochemical Society, 165, G3112–G3119, 2018) where changes in the electrolyte solution composition were proposed to obtain other properties. However, an obvious limitation of electrochemical methods is the large particle size distribution and morphology, which usually requires subsequent separation and purification of the obtained material at the end of the synthesis (RAMILA DEVI; VIGNESH KUMAR; SUNDRAMOORTHY, Journal of the Electrochemical Society, 165, G3112–G3119, 2018).

[0025] Soot produced in multiple reaction processes involving carbon sources (e.g., combustion or burning) is also used as a precursor for synthesizing carbon dots. These materials / byproducts are placed in a reflux system with concentrated acid (e.g., nitric acid) and then size-separated for smaller nanoparticles (LIU et al., Angewandte Chemie, 46, 6473-6475, 2007). The strong oxidizing properties of the acid are essential for the dissolution of carbon aggregates in the soot; the acid further reacts with the carbon colloid to produce oxygen-rich and nitrogen-rich surface defects associated with the fluorescence emission of the particles. However, such soot-based or carbon-rich byproduct materials result in low yields of carbon dots and form a large amount of toxic residues.

[0026] Hydrothermal treatment of carbon sources has become the most common procedure for carbon dot production. This method enables the production of nanoparticles from a variety of sources (such as molecules and residues) (DE YRO et al., AIP Conference Proceedings, 2083, 0200007, 2019).

[0027] The essence of the hydrothermal synthesis of carbon dots is a process that starts with high temperature induced nucleation / condensation of carbon molecules, which serve as building blocks for the formation of graphite cores. Another important feature of the hydrothermal synthesis scheme is that some residues from the carbon reagent remain on the surface of the graphite core, thereby providing the particles with a variety of controllable functional and optical properties (WANG et al., Analytical Methods, 10, 2775–2784, 2018).

[0028] Compared to the previously mentioned methods, the hydrothermal route shows particles with narrow size distribution and uniform luminescence properties. However, limiting factors are related to the generally higher synthesis times and low yields limited by the use of systems that control the internal pressure.

[0029] De Medeiros et al. (2019) obtained carbon dots by microwave-based hydrothermal synthesis. Microwave radiation replaced the heat source used in traditional hydrothermal methods. The carbon dots produced by microwave heating emit high luminescence in both solution and solid phase. The synthesis time is greatly reduced because the energy released throughout the process is quite high and can be controlled within the device itself. The particles obtained are similar to those obtained by hydrothermal methods, although the yield is reduced and limited by the strength of the accessories used in the synthesis (DEMEDEIROS et al., Journal of Materials Chemistry C, 7, 7175–7195, 2019). The above aspects hinder the large-scale production of carbon dots by hydrothermal reactions.

[0030] High-temperature carbonization for making carbon dots has been performed in solvents other than water. Researchers at the Max Planck Institute for Polymer Research have shown that heat treatment of carbon sources (e.g., citric acid) dispersed in non-coordinating solvents (e.g., organic solvents octadecene and 1-hexadecene) produces highly luminescent carbon dots (WANG et al., Chemistry of Materials, 22, 4528–4530, 2010). In turn, under such conditions, residues from the synthesis become an obstacle to increasing the yield of carbon dots.

[0031] The aforementioned techniques, while effective for the production of carbon dots, all have obstacles in producing large quantities of materials with uniform and selectable characteristics. As in electrochemical and hydrothermal methods, even those techniques with high yield synthesis face difficulties in the reproducibility and quality of the resulting materials. It is worth mentioning that the method proposed in the present invention allows for large-scale production of carbon dots and is different from the aforementioned methods because no hydrothermal method is used, no toxic reagents or residues. In the present invention, the production of carbon dots is carried out by a continuous flow reaction in a closed loop, which ensures reproducibility and high yield. The proposed method reuses all water and residues and does not produce greenhouse gases.

[0032] In addition to Cdot production techniques, it is important to emphasize that exploring Cdots in biological environments requires caution, as the raw materials and conditions of synthesis are factors that affect the surface composition, functionality, and bioactivity.

[0033] Several examples show that hydrothermally generated carbon dots contain fragments of precursor molecules on their surface. For example, in a paper reported by Zhao et al. (2017), carbon dots generated by pyrolysis of egg yolk oil exhibited anti-hemorrhagic activity similar to the drug hemocoagulase (Zhao, Y., Zhang, Y., Liu, X. et al. Scientific Report, 7, 4452, 2017).

[0034] In patent application CN103980894A, carbon dots produced from folic acid by a hydrothermal route are able to selectively recognize tumor cells by specifically interacting with plasma membrane proteins (folate receptors) that interact with folic acid. Individual carbon dots exhibit all morphological and physicochemical characteristics similar to other nanoparticles produced from other precursors. Nevertheless, the presence of folic acid fragments on the surface makes them unique and eligible for patent protection.

[0035] In another example, carbon dots produced by pyrolysis of egg membranes were able to selectively recognize DNA and RNA fragments (Pramanik et al. PCCP, 20, 20476, 2018). It is worth mentioning that carbon dots produced by pyrolysis of other carbon sources did not show the selective ability to recognize nucleic acids.

[0036] In international patent application WO / 2018 / 160142, carbon dots produced by the condensation of polyethylene oxide under acidic conditions produce nanomaterials with antimicrobial properties and are incorporated into inert polymer membranes. Again, the morphological and physicochemical characteristics are similar to those of carbon dots produced from other carbon sources, although the surface characteristics of the particles lead to specific biological activities that protect them.

[0037] Therefore, although the morphological and physicochemical aspects indicate that Cdots are usually just carbon spheres, the synthesis process and carbon source affect the surface composition and biological properties of Cdots.

[0038] The use of carbon dots as fertilizers in agriculture was proposed in a paper published by Zheng et al. (2017), which disclosed the use of pollen as a precursor for nanoparticles (ZHENG et al., ACS Omega, 2, 3958–3965, 2017). Experiments were conducted in hydroponic lettuce and showed that the use of 20 mg·L -1 Plants treated with the solution grew about 50 percent more. The results did not indicate a selective interaction with proteins or enzymes responsible for plant growth. The authors simply proposed the use of the carbon dots as a fertilizer.

[0039] In a study published by Li et al. (2018), five carbon dots produced by graphite electrolysis exhibited unique surface compositions and different responses in rice (LI et al., ACS, Applied Biomaterials, 1, 663-672, 2018).

[0040] Significant differences in plant growth activity between particles were described, demonstrating that each particle has unique characteristics even though the size and physicochemical characteristics are similar. In addition, the synthetic carbon dots were observed to interact with plant genetic material, promoting the expression of the growth gene Os06g32600. It was further observed that the C dots interacted with the gene responsible for fixing atmospheric CO 2 RuBisCo protein interaction (LI et al., ACS, Applied Biomaterials, 1, 663-672, 2018).

[0041] In summary, the present invention is superior to other technologies because it deals with carbon dots with specific features that mimic responses similar to those of plant hormones that specifically interact with plasma membrane proteins and is obtained through a specially developed synthetic organocatalytic approach to promote accelerated plant growth and direct delivery of macro- and micronutrients into plant cells.

[0042] The carbon dots described in this patent act as biostimulants, enhancing nutrient uptake, improving water use by plants, and increasing photosynthesis rates as it keeps stomata open longer and promotes accelerated growth of test cultures.

[0043] The proposed bioactivity of C-dots is unique and suggests that the particulate role in plant metabolism occurs at a molecular level similar to the level of plant hormones such as auxins, Guiberlines, and cytokinins.

[0044] With all the above, the development of biostimulants as disclosed in the present invention having the activity of mimicking plant hormones activating the plasma membrane protein H-ATPase was not foreseen in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] based on Figures 1 to 19 , the present invention can be more easily understood, which is described as follows:

[0046] Figure 1 Illustration of the eutrophication process of the food chain caused by metal nanoparticles and metal oxide nanoparticles.

[0047] Figure 2 A scheme for biostimulant production according to the developed method is shown.

[0048] Figure 3 Fourier transform infrared spectra of carbon dots synthesized by the developed method are shown.

[0049] Figure 4 The formation of carbon dots produced by synthetic precursors and the retention of surface groups are shown.

[0050] Figure 5 The absorption spectrum of carbon dots in the UV-visible region and the excitation emission spectrum in the range of 360 to 620 nm are shown.

[0051] Figure 6 The absorption spectra of chlorophyll a and b are shown.

[0052] Figure 7 XPS spectra and biostimulant composition by calculation of macronutrients nitrogen, phosphorus and potassium (N, P, K) are shown in A. High resolution spectra of phosphorus are shown in B. Microscopic images and high resolution transmission electron microscopy images (HRTEM) are shown in C, and particle diameter distribution is shown in D.

[0053] Figure 8 Zeta potential (ζ) and hydrodynamic diameter are shown as a function of volume (mL) of nutrient solution to which a volume of 10 ml of carbon dots at a concentration of 50 mg / L was added.

[0054] Fig. 9 The distribution of plants in four zones P1 (white) and P2 (blue) is shown.

[0055] Fig.10The sample distributions of the P1 group treated with different concentrations of carbon dots on the 17th day (A), 28th day (B), 42nd day (C), and 56th day (D) after the start of treatment are shown.

[0056] Fig.11 The sample distribution of the P2 group treated with different concentrations of carbon dots on the 17th day (A), 28th day (B), 42nd day (C), and 56th day (D) after the start of treatment is shown.

[0057] Fig.12 The distribution of treatments in the plant room for groups B1, B2, B3 and B4 is shown.

[0058] Fig.13 The effect of different concentrations of carbon dots on the photosynthesis rate (right) and water use efficiency (left) of 'Finestra' tomatoes in zones B1 and B2 is shown.

[0059] Fig.14 The effects of different concentrations of carbon dots on the photosynthetic rate (right) and water use efficiency (left) of 'Finestra' tomatoes in zones B3 and B4 are shown.

[0060] Fig.15 The treatment effects of different concentrations of carbon dots on the water use efficiency of hydroponic lettuce crops are shown.

[0061] Fig.16 The effect of carbon dots concentration in root dry mass (RDM) of maize seedlings is shown.

[0062] Fig.17 Diagram showing a corn crop system in a Leonard vase with carbon dots applied via nutrient solution.

[0063] Fig.18 Shown are the concentration effects of 100 mg / L carbon dots on liquid photosynthesis (A), stomatal conductance (B), transpiration (C), and water use efficiency (D).

[0064] Fig.19 Shown is the percentage of nutrient increase or decrease in corn plants by using Hoagland's solution with carbon dots. DETAILED DESCRIPTION

[0065] The present invention relates to a carbon-containing photosynthesis stimulator (carbon dots) with photoluminescent properties, which is used as a biostimulant and biofertilizer because once applied to the leaves or roots of plants, it can induce a physiological response that promotes plant growth. The physiological response is observed in photosynthetic variables, plant root characteristics, and the absorption of micro- and macronutrients necessary for the development cycle.

[0066] In addition, the product of the present invention exhibits high water solubility and biodegradability; can be completely metabolized by plants and therefore does not bioaccumulate; its use reduces the negative impact of nutrient transfer of conventional nanoparticles in the food chain; and it is non-toxic. For this reason, it reduces the environmental impact associated with soil and water pollution compared to traditional agrochemicals, making it have a higher potential for use as an input.

[0067] Nanomaterials act as organic carbon sources and as carriers of micro- and macronutrients anchored in their structure. In a bottom-up synthesis process, the size of the nanomaterials is controlled and therefore the activation of active sites in their structure is controlled. Through surface engineering expertise, these active sites are structured and receptive to nutrients and / or stimulants, which will be delivered to the most diverse types of crops with greater efficiency due to the bioaffinity of plants to the nanocomposites and their easy metabolism.

[0068] The nanomaterials shown in this technology are Figure 2 The material was subjected to another drying operation for better storage after the synthesis process.

[0069] One form of the invention relates to a method for producing carbon dots, which is a universal method because it allows the use of different carbon-rich substrates as raw materials, such as glutamine, organic acids, citric acid, tartaric acid, oleic acid, malic acid, fumaric acid, isocitric acid, corn starch; and can also be processed in a coordinated manner by using alternative sources that can provide appropriate reagent components, composite materials with the same functional groups and short chains, preferably citric acid and tartaric acid.

[0070] The proposed method is a rapid method compared to the conventional route (hydrothermal route) because long stabilization time is not required to initiate the carbonization process and particle growth as 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 no toxic waste is generated at the end of the synthesis process.

[0071] The method used in the synthesis of carbon dots is one that involves physicochemical transformations. The route used is considered chemically green as it uses water as a solvent and no toxic residues are produced in the synthesis.

[0072] Generally, the modality can be understood as a process of obtaining carbon dots consisting of a catalytic chemical reaction and a flow process.

[0073] Nanoparticles were obtained from different carbon sources (i.e., citric acid, glucose, amino acids, and agricultural wastes such as peels, pulp, and animal manure); citric acid was mainly used because of the partial neutralization of the acid-base reaction, in which a base solution (a source of hydroxyl functional groups) is used to form a salt from the acid and to generate the ions (OH) in solution. - ) induced electrostatic repulsion promotes the formation of multiple nucleation sites. The alkaline solution used can be calcium hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, aniline, metal amine, urea and thiourea, preferably ammonium hydroxide.

[0074] Prepare the acid solution (carbon source) and base solution (functional group source) and place them into the flow system.

[0075] Using metering pumps and valves, the solution is metered into a mixer, which is then redirected to a second mixing vessel that receives recycle and dopants (micro and macro nutrients or stimulants). After passing through the mixer, the reagents are pumped into a plug flow tubular reactor (PFR) equipped with a valve that limits the material output and maintains a constant pressure inside it, thus avoiding evaporation of the reagents when the reactor is heated to 100° C. to 250° C., preferably 150° C.

[0076] The PFR reactor must have a reduced diameter, just a few millimeters, and be made of low-roughness materials to reduce the pressure drop during the outflow of the material. Another key factor in the process is to maintain the turbulent hydrodynamic runoff regime; all of this is calculated using the Bernoulli equation, as the reactor must be placed in a greenhouse to heat the mixture.

[0077] The reactor must be heated to 100-250°C, depending on the concentration of the materials used. The pressure inside the reactor is controlled by an outlet valve, thus preventing the solution from boiling; the pressure varies from reaction to reaction, depending on the density of the salts involved. The pressure is high, so the reactor materials must withstand pressures of up to 5 bar. The material flow coming out of the first reactor (tubular reactor) is discharged into a continuous stirred tank reactor (CSTR), which is also heated and has stirring action. The outlet flow of the reactor passes through a separation system that works either by hydrocyclone density difference or a decanter that allows the removal of the end product by overflow and the return of the underflow stream to the reflux.

[0078] It is necessary to reflux the material until the optimal operating conditions are established. The density of the product formed is lower than that of the precursor salt solution, so the product can be separated by this principle, removing the supernatant and recycling the bottom material, which is inserted before the reactor and added to the initial reagent stream. The process before the stabilization process in which the material is kept refluxed takes 0.5 to 2 hours, preferably 1 hour; from this time on, the material density begins to change and the product begins to take shape.

[0079] The yield of the process was 95% (713 g after a 2-hour cycle). These quantities of material were sufficient for 24 hectares.

[0080] The product can be stored in a liquid state, or it can go through a drying process in which the surface area of ​​the material expands to approximately five times its volume.

[0081] The technology used to produce the carbon dots has scaling characteristics, which allows its feasible transition to industrial scale without negatively affecting the quality of the formed carbon dots. In trials conducted by scaling up the production of laboratory-produced C dots by 100 times, it was shown that the same performance and quality can be produced and maintained. Under the high production demand with low environmental impact, such as required by the agricultural input industry, this technology is innovative and combines with the current new production needs.

[0082] Versatility is also one of the main characteristics of the developed production platform, which allows its composition and characteristics to be varied to meet the needs of the production chain.

[0083] The carbon dots produced by this method show high water solubility due to the presence of hydrophilic groups in the precursor molecules during synthesis, which can be Figure 3 The infrared spectrum shown is used for verification.

[0084] In addition to the hydrophilic properties, the nanoparticle surface groups have ion complexing potential in solution ( Figure 4 ). Both macronutrients that plants require in high amounts (potassium, nitrogen, calcium, phosphorus, magnesium, and sulfur) and micronutrients that plants require in low amounts (manganese, boron, zinc, iron, copper, molybdenum, and chlorine) can be incorporated into the preparation of carbon dots when complexed via surface groups after synthesis.

[0085] Apart from this, they are biodegradable materials, which endows them with higher application potential as agricultural inputs.

[0086] Therefore, another aspect of the invention relates to the use of carbon dot-based nanoformulations as powerful photosynthesis stimulants, biostimulants and organo-mineral biofertilizers. The technology presented in this document is completely non-toxic, biocompatible, non-bioaccumulative and sustainably produced by using green nanotechnology principles. Test results show an increase in photosynthesis efficiency of about 60%, a 50% increase in water use efficiency and a 50% increase in root mass. Nanomaterials produced and doped with basic chemical elements simultaneously provide nutrients and improve the physiological condition of plants and reduce the adverse effects of traditional use of fertilizers. It must be emphasized that the technology presented overcomes the limitations related to economic feasibility, production scale, environmental production and safety, which are obstacles to the sustainable use of nanotechnology in agriculture.

[0087] The use of carbon dots as photosynthesis stimulators stems from their spectral properties. The introduction of nitrogen-containing aromatic reagents (which promote the formation of C=N / C=O bonds) is responsible for the multi-state absorption and emission in the blue / green / red region ( Figure 5 ).

[0088] Chlorophyll a and b show two absorption bands centered in the spectral regions corresponding to blue (~440 nm) and red (~650 nm), but show very low spectral response to the green region ( Figure 6 ). The synthesized carbon dots showed strong absorption in the green region (532 nm) and emission centered at 645 nm. This spectral adaptation leads to increased culture productivity due to better utilization of sunlight.

[0089] Example

[0090] The following examples are presented to illustrate the best embodiment of the invention. It is worth mentioning that the invention is not limited to the cited examples but can be used for all applications described or any other equivalent variants.

[0091] Example 1: Carbon dot synthesis

[0092] The amount required for agricultural use is higher than that used in the laboratory. Figure 1 The method and production scale were introduced in the pilot configuration shown. 500 g of glucose and 550 mL of ammonium hydroxide were dissolved in 1000 mL of water and placed in Figure 1 In the flow-through system shown. The operating temperature was set at 150°C and the yield of the final product was 95% (713 g after a 2 hour cycle). These amounts of material were sufficient for 24 hectares.

[0093] Example 2: Synthesis of Hybridized Carbon Dots Containing Macronutrients

[0094] Initially, the process was not provided with a second mixer, but with the need for recirculation and the need for input nutrients, a second mixer was added after the first mixer receiving the acid and base streams. Thus, the recirculation and dopant dosages were calculated to achieve a stoichiometric ratio. This allowed the generation of pure C-dots and hybridized C-dots, where the most diverse dopants were added to the second mixer.

[0095] Figure 7 a shows the XPS spectra and composition of samples containing macronutrients. Figure 7 The high-resolution XPS spectrum in b shows that phosphorus (P) is incorporated into the carbon matrix. The bands centered at 131.25 and 132.25 eV correspond to -C 3 -PO and -C-PO 3 Group. Figure 7C and d show the image of the nanoparticle with spherical shape and average size of 5nm.This result shows that phosphorus can be directly transported to plant cell mechanism, and the nutrient bioavailability process is similar to the bioavailability process of nutrient nitrogen, which depends on the metabolism of plant to nano material.This nano material serves as photosynthesis stimulant, biostimulant / biofertilizer and serves as the source of macronutrient (organo-mineral fertilizer) simultaneously.In addition, it is completely water-soluble, nontoxic, and advantageously reduces the environmental impact caused by excessive use of fertilizer in soil.It is worth pointing out that, up to the disclosure of the present invention, similar technology is not reported in the document.

[0096] Example 3: Evaluation of Nutrient Carrier Potential

[0097] The potential of carbon dots as nutrient carriers was studied because in solution, the carboxyl groups (COO - ) imparts a negative charge, thus imparting a repulsion that favors the colloidal stability of the system. Metals classified as micro- and macronutrients carry a positive charge in solution and can be attracted to the carboxyl groups in the particles.

[0098] To analyze the surface behavior and stability of C-dots when ions were added, the zeta potential (ζ) and hydrodynamic diameter were measured with increasing amounts of nutrient solution added. 10 mL of C-dots with a concentration of 50 mg / L were calibrated with standard Hoagland solution (1 / 2 strength). Figure 8 In the experiment, the behavior of zeta potential and hydrodynamic diameter can be observed as more ions are added.

[0099] As expected, the potential decreases, indicating that positive metal ions bind to the surface, resulting in a gradual decrease in potential. However, starting from a certain volume, the curve behavior tends to remain constant, as shown in Figure 8 The particles exhibited stability even at high ionic strengths, as evidenced by the narrow variation in hydrodynamic diameter. These properties suggest that Cdots can be used with standard nutrient solutions to facilitate nutrient transport and avoid nutrient leaching that occurs in conventional agriculture. Colloidal stability also enhances the potential for commercialization of technologies such as photosynthesis stimulators, biostimulants / biofertilizers, and liquid organo-mineral fertilizers.

[0100] Example 4: Agronomic Trial

[0101] A sweet pepper crop (Capsicum annuum) was chosen to show the effects of nanomaterials on plant growth because this crop has received a lot of criticism regarding agrochemical residues in the fruit.

[0102] The effect of carbon dots on the production coefficient of bell pepper was established in a factorial scheme of 2 × 4, with two application uses (single application and four instalments) and four carbon dot concentrations ranging from 0, 25, 50 and 100 mg.L -1 The experiment adopted a randomized block design and was repeated four times, with a total of 32 experimental units.

[0103] The experimental units were 5.0 dm each filled with a mixture of inert substrate and plants. 3 Add nutrients and water by fertilizing, adding 400mL of complete nutrient solution four times a day.

[0104] The bell pepper seeds were sown in germination trays and after 27 days, the grown seedlings were transplanted into 5-liter containers pre-filled with an inert matrix composed of turf, vermiculite, organic waste, agro-industrial organic waste and limestone. Thirty-two seedlings were selected within 19 days of transplantation to begin the application of carbon dots; at the time of selection, all seedlings were of the same size and growth criteria used.

[0105] The applied carbon dot concentration (0 to 100 mg.L -1 ) was determined according to the previous experiment. For each concentration of each plant, the maximum solution volume used for subsequent carbon dot mass quantification was 200 mL. The plants were divided into two groups, P1 and P2, where plants classified as P1 received a single dose of solution (200 mL), while plants of P2 received stepwise doses (50 mL) every 15 days until the maximum solution volume (200 mL) was reached. Fig. 9 The division of plants into four plots to check treatment randomization is shown.

[0106] The plants were placed in a glass greenhouse and irrigated evenly four times a day. The carbon dot solution was sprayed on the leaves so that excess solution could flow freely into the plant substrate.

[0107] To measure the effect of carbon dots on the relative growth of plants, the size was measured along the test direction with a tape measure graduated in millimeters. On the first day, before the start of the application, the initial size was measured and collected, and then the first concentration was applied. The P1 group received a volume of 200 mL in each plant, while the P2 group received a first dose of 50 mL. The size collection process was repeated every two weeks on the same day after the application of the P2 group until a volume of 200 mL was reached.

[0108] Equation 1 is used to calculate the relative growth of plants, where “h 最终 ” represents the measurement after application of the treatment, “h 初始 ” represents measurements taken before application of treatment.

[0109] Equation 1:

[0110] The collected data were spreadsheeted for statistical testing. The first statistical test used was the Shapiro-Wilk normality test, which indicated the non-normal distribution type of the collected data. The results were then evaluated using non-parametric statistics (Kruskall-Wallis test for independent samples). Fig.10 ). Given this, comparing means is the most appropriate way to determine statistical differences and conclusions about the observations made.

[0111] The effects of the concentration and number of C dots on plant physiological growth were shown by separate statistical data for the P1 and P2 groups.

[0112] From the statistical analysis performed, it can be concluded that for the P1 group ( Fig.10 ), only after a single application of 200 mL per plant at the beginning of the test, and 56 days after application, the concentration was 100 mg·L -1 The treatment and concentration were 0 mg·L -1 There was a statistical difference between the control treatment and the other treatments, especially the control treatment (i.e., no nano-biostimulant was applied (0, 0 mg.L -1 )) compared to the control group, when a higher concentration of carbon dots (100 mg.L -1 ), bell peppers have greater relative growth.

[0113] In contrast, when the results of the P2 group were analyzed ( Fig.11 ), where 50 mL of each concentration was administered every two weeks until a volume of 200 mL was reached, with concentrations of 25 mg·L observed after 42 and 56 days -1 Treatment and 0mg·L -1 When compared to the control concentration and other treatments, the smaller concentrations applied in split doses promoted relative growth in a more efficient manner.

[0114] Example 4: Effect of carbon dots on photosynthesis rate of 'Finestra' tomato

[0115] The effect of the carbon dots on the rate of photosynthesis was shown using a portable photosynthesis system that uses an infrared gas analyzer. The key point in these measurements is CO 2 Relationship between assimilation and water loss through leaf transpiration through stomata. 2 The specific response of water in the infrared was used to develop a sensor that reads infrared light during gas exchange (infrared gas exchange analyzer – IRGA).

[0116] CO 2and water vapor changes can be monitored simultaneously through the leaf, thus providing an accurate and integrated in vivo measurement of liquid photosynthesis and sample transpiration when illuminated by the apparatus in a reading chamber that clamps the leaf without damaging it, and also providing mitochondrial respiration rate and residual transpiration when the sample is in a dark environment within the reading chamber.

[0117] In the ‘Finestra’ tomato (developed by Embrapa Vegetables 1 The effect of carbon dots on photosynthesis was observed in hybrids.

[0118] Tomato seedlings were transplanted into vases 25 days after sowing, and fruits were harvested starting 60 days after transplanting.

[0119] 48 days after sowing, 5 trays with seedlings were selected to start applying biostimulants. The concentrations of the test were as follows: 0 mg·L -1 (water only); 0.5mg·L -1 ; 2.5mg·L -1 ; 5mg·L -1 and 10mg·L -1 The solution was sprayed onto the leaves so that excess solution could flow into the substrate. The substrate used to fill trays and vases was made from bio-stabilized pine bark, vermiculite, charcoal powder, water and phenolic foam.

[0120] The first dose was applied on the seedlings after the emergence of true leaves while still in the germination trays. A single dose of 250 mL was sprayed evenly on the trays for each treatment and the process was repeated for three consecutive days.

[0121] Twenty seedlings from each treatment (0; 0.5; 2.5; 5.0; 10 mg·L) were selected from the previously treated plates. -1 ) were transplanted into 15-liter vases with substrate. The seedlings were randomly distributed in the four plots, so for each treatment, each plot was counted four times, for a total of 80 seedlings. Plots B1 and B2 were exposed to greenhouse light, while plots B3 and B4 were covered with shade cloth ( Fig.12 ).

[0122] Equation 1 shown in the previous examples was used to calculate relative growth.

[0123] After transplanting to the vase, the seedlings were left to mature in the substrate for 15 days before application was resumed. For the growth stage that the tomato plants were in, 500 mL of the dose was applied to the leaves, with possible contact between the solution and the substrate. Treatments (concentrations) of the same dose were separated and grouped for uniform spraying of the solution. The treatments were then redistributed to their respective initial benches, taking care not to mix the vases with shade cloths with those without shade cloths when returning to the benches. The application process was repeated four or more times a week with a single dose.

[0124] Data collection was performed in the morning after the application was stopped and physiological studies were performed on the mechanisms and intensity of photosynthesis rate and transpiration during the day. Reading conditions were saved for all treatments and at the end of the analysis, data were extracted for applying statistical tests on the variables of interest provided by the equipment.

[0125] Fig.13 and 14 The effect of the treatment is shown. The first statistical test used was the Shapiro-Wilk normality test, which indicated the non-normal distribution type of the collected data. Then, non-parametric statistics (Kruskall-Wallis test for independent samples) were used to evaluate the results. In view of this, comparing the means became the most appropriate way to determine the statistical differences and conclusions of the observations made.

[0126] The combined effects of C-dot concentration and natural light on photosynthesis in 'Finestra' tomato plants were shown by comparing the groups treated with shade cloth (B3 and B4) and the groups treated without shade cloth (B1 and B2). As explained earlier, C-dots have light absorption / luminescence and emission properties, which can be studied through the absorption process of light by chlorophyll.

[0127] The data obtained for area B1 and area B2 showed that the concentration was 2.5 mg·L -1 and 5mg·L -1 The treatments showed a tendency to increase the photosynthesis rate ( Fig.13 ).

[0128] Water use efficiency is not a parameter directly provided by IRGA, but can be calculated by dividing the photosynthesis value (Pho) by the stomatal conductance (C i ) is obtained. Therefore, the concentration in the B1 and B2 regions is 2.5 mg·L -1 and 5mg·L -1 The water use efficiency of the treatment also tended to increase ( Fig.13 ).

[0129] Data collected from areas B3 and B4 (both protected by shade cloth) Fig.14) highlighted the fact that where carbon dots were applied, the photosynthesis rate was lower than that of the control treatment (0 mg·L -1 ). However, water use efficiency is improved ( Fig.14 ).

[0130] The difference between the light-shielded and non-light-shielded areas indicates that the carbon dots luminescent behavior occurs only when illuminated by an external light source.

[0131] Example 5: Effect of carbon dots on the photosynthesis rate of lettuce crops

[0132] The effects of the carbon dots on photosynthesis of lettuce (Lactuca sativa) grown in a hydroponic system were also shown. Hydroponics is a growing technique in which the soil or substrate is replaced with a medium containing all the nutrients necessary for plant growth.

[0133] The hydroponic system allows the seedlings to be gradually irrigated with the nutrient solution through the roots. Thus, spraying is excluded and the hydroponic system itself is used as the distribution vehicle for the carbon dots to the plants.

[0134] Three separate workstations connected to three reservoirs with separate hydroponic solutions were used. Each reservoir had a volume of 1000 liters of solution that was periodically circulated into the system. Hoagland's modified nutrient solution (necessary for plant growth) was prepared for all reservoirs and three concentrations of carbon dots were measured, 0 mg.L -1 ; 20mg.L -1 and 50mg.L -1 The nutrient solution itself was used as the carbon dot distribution carrier.

[0135] Seedlings were obtained and passed an acclimatization period in the greenhouse where they were evenly distributed in three workbenches and irrigated with a previously prepared nutrient solution. The carbon dots were diluted in a reservoir and put into circulation 21 days after the subsequent transplantation of lettuce seedlings.

[0136] Data collection was performed on ten lettuce seedlings in each treatment. Measurements were taken 15 days after the start of carbon dot application and the reading conditions for all treatments were saved. After the analysis was completed, data were extracted for statistical tests as in Example 4.

[0137] Fig.15 Data obtained by IRGA are shown. Only in the higher concentration treatment 50 mg.L -1 and control treatment 0mg.L -1 These observations contribute to the fact that when applied on leaves, photosynthesis efficiency was achieved, however, even in the absence of changes that promote photosynthesis, carbon dots can promote better water use during photosynthesis.

[0138] Example 6: Effect of carbon dot concentration on the root system of corn seedlings

[0139] Corn seedlings with 10% NaClO 4 The solution was sonicated for 10 minutes and rinsed 5 times with distilled water for sterilization. They were then aseptically placed on germination paper and germinated in a dark, 28°C BOD chamber. Two days after germination, rooted seedlings of approximately 1-1.5 cm were transferred to a hydroponic system in a plastic vase containing 10 mg.L -1 , 10mg.L -1 , 20mg.L -1 , 40mg.L -1 , 80mg.L -1 and 160mg.L -1 After 5 days of growth, plants (n=9) were harvested and root dry weight (RDW) was assessed. The results were analyzed by regression, and the most effective carbon dot concentration was calculated.

[0140] In order to calculate the most effective carbon dot concentration, regression analysis was performed and the model shown in this paper is the best fit for the data. The regression data follows a normal distribution ( Fig.16 ).

[0141] A significant quadratic model was shown, with an increase at lower concentrations and a gradual inhibition at higher concentrations of the used carbon dots ( Fig.16 The most effective concentration is estimated to be 100 mg.L -1 C-dots, resulting in an approximately 75% increase in RDM compared to the control treatment.

[0142] Example 7: Effects of carbon dots on photosynthesis rate, gas exchange in leaves and nutrient content in corn seeds ring

[0143] After regression analysis with different doses of carbon dots, in the control group (0 mg.L -1 ) and the most effective dose observed as outlined in Example 6 (100 mg.L -1 ), the influence of carbon dots is shown.

[0144] Transfer rooted seeds about 1-1.5 cm into a Leonard vase ( Fig.17 ), the top of the vase contained 0.5 kg of sterilized 10% HCl solution, followed by rinsing with distilled water until the conductivity was <5 mS.cm -1 sand (0.5-0.84mm). At the bottom of the Leonard vase, place 0.5dm 3 Supplemented with 0 or 100 mg.L -1Hoagland nutrient solution (25% strength) of carbon dots. Seedlings were transplanted and the solution was replaced weekly with a newly produced solution but with increased concentrations to 50%, 75% and 100% concentrations.

[0145] After 28 days of growth, the 2 The IRGA-type photosynthesis system with a standard leaf chamber measured parameters of gas exchange in leaves of plants (n=4), such as liquid photosynthesis, stomatal conductance and transpiration. Water use efficiency (WUE) was calculated as the ratio between liquid photosynthesis and transpiration. CO in the gas exchange chamber 2 The concentration was set to 400 μmol.mol -1 , and measured at 500 μmol.m -2 .s -1 The calculations were performed between 9 am and 11 am. The average temperature during the experiment was about 28°C and the relative humidity was 50% to 60%.

[0146] For nutritional analysis, plants were harvested and heat treated at 65 °C until constant weight and further ground to a fine powder (2 mm mesh) using a knife mill. 3 and HClO 4 After decomposing 0.5 g of the composite material, the Ca, Mg, S, Fe, Cu, Mn, Zn and Ni contents of the obtained material were analyzed by microwave plasma atomic emission spectrometry. 2 SO 4 and H 2 O 2 After disintegration of the composites, total phosphorus was estimated colorimetrically at 660 nm by the ascorbic acid / molybdic acid method.

[0147] Leaf gas exchange and nutrient content were analyzed by ANOVA with F test.

[0148] Addition of carbon dots resulted in an increase in liquid photosynthesis (p<0.05)( Fig.18 a). However, stomatal conductance and transpiration rate did not change (respectively Fig.18 b and 18c).

[0149] There is an increasing demand for water use efficiency (WUE) in world agriculture. In this regard, the use of plant stimulants should be considered a valuable tool, as these stimulants act as modifiers of root architecture and inducers of primary and secondary metabolic processes that impair plants. WUE represents the ability of plants to optimize carbon uptake and minimize water losses, thus generating a competitive advantage.

[0150] Carbon dots significantly increased WUE by 22% ( Fig.18d). The increase in this parameter is characteristic of the addition of plant stimulants and highlights the ability of carbon dots to regulate root growth by increasing the ability of plants to absorb water from the soil.

[0151] Due to the presence of carbon dots in the nutrient solution, the contents of all nutrients analyzed changed significantly ( Fig.19 ). Among them, only magnesium was observed to decrease (8.7%). Phosphorus, Ca and Zn showed smaller increases, in order of 7.8; 8.3% and 13.0%, respectively. Sulfur and Ni contents showed moderate increases of 26.6% and 55.5%. The largest increases were observed for Mn, Cu and Fe, reaching 70.2; 82.1; 82.6%, respectively.

[0152] Nutrient content data showed that carbon dots, acting as photosynthesis stimulators and nanobiostimulators, improved the absorption of some nutrients.

Claims

1. A method for producing a photosynthesis stimulant, characterized in that The method produces carbon dot nanoparticles through partial acid-base neutralization reaction, wherein the formation of carbon dot nanoparticles occurs in a continuous circulation system, in which acid and base solutions are injected into a first mixing container through a metering pump, and then directed to a second mixing container receiving a recycled material flow and a dopant, and then pumped into a piston flow tubular reactor with a valve, the valve restricts the material output and maintains a constant pressure and a temperature of 100-250° C. inside, wherein the process is maintained in a state of turbulent fluid dynamic runoff; then, the material flow coming out of the tubular reactor is discharged into a continuous stirred tank reactor under the same heating mode, the output flow of the continuous stirred tank reactor passes through a separation system, in which the supernatant is removed and the material at the bottom is recycled; the recycled material flow re-enters the second mixing container together with the feed reagent flow containing the acid and base solution; the reflux process is continued until the product is formed.

2. The method for producing a photosynthesis stimulator according to claim 1, characterized in that The adulterants are micronutrients and macronutrients.

3. The method for producing a photosynthesis stimulator according to claim 1, characterized in that The carbon dot nanoparticles 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.

4. The method for producing a photosynthesis stimulator according to claim 1, characterized in that The alkaline solutions used include: calcium hydroxide, potassium hydroxide, ammonium hydroxide, aniline, metallic amines, urea and thiourea.

5. The method for producing a photosynthesis stimulator according to claim 1, characterized in that The method uses water as the solvent in a continuous flow-through system.

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