Fertilizer additive based on sulphate lignin modification and its preparation method and use

Abstract

The application discloses a kind of fertilizer additive based on sulphate lignin modification and its preparation method and use.The fertilizer additive is to sulphate lignin hydrothermal alkali cutting and obtains a water-soluble lignin-based material.This material is as a kind of polyphenol form lignin-based natural polymer compound, with superior metal chelation effect, can be used as a new type of natural polymer trace element chelation material, improve the growth of plant under the condition of trace element deficiency and promote plant growth.Take iron as an example, the mechanism of HSAL promoting plant growth is that the unique high-phenolic aromatic polymer structure on water-soluble HSAL can efficiently chelate and enrich the non-physiological active iron ions in the plant root environment and promote the efficient absorption and utilization of plants, so as to regulate the growth environment of plants and the form of trace elements in plants.This process further promotes the absorption of trace elements in plants and the distribution efficiency in vivo, so as to realize the promotion of plant growth.

Description

Technical Field

[0001] This invention belongs to the field of fertilizer additives, specifically relating to a fertilizer additive based on sulfate-modified lignin, its preparation method, and its application. Background Technology

[0002] The long-term excessive application of traditional nitrogen, phosphorus, and potassium fertilizers not only causes soil acidification and even compaction, damaging soil aggregate structure, but also depletes micronutrients in the soil, leading to a decline in soil biodiversity. The overall soil fertility and the marginal effect of traditional fertilizer inputs decrease year by year, seriously endangering the sustainable and green development of agriculture.

[0003] Lignin, the most abundant natural aromatic polymer on Earth, accounts for nearly one-third of the carbon reserves in nature and is one of the main structural components of plant cell walls. Lignin is a network polymer composed of three types of phenylpropane monomers (p-hydroxyphenyl (H-type), guaiacolyl (G-type), and syringyl (S-type)) linked by covalent bonds of β-O-4, β-β, β-5, and 5-5. The linkage form and bond type ratio of lignin are highly susceptible to influences such as plant variety, sampling location, planting latitude and longitude, altitude, and light intensity. Accurate description of the chemical structure of lignin has long been a challenge in the field of basic lignin research.

[0004] In recent years, a large number of studies have moved beyond the precise description of the overall chemical structure of lignin, viewing it as a renewable resource with extremely abundant reserves in nature, and thus pursuing its high-value development and utilization. Currently, the high-value development of lignin both domestically and internationally mainly falls into two categories: energy utilization for converting lignin into aviation-grade fuel, and resource utilization for obtaining chemical raw materials, synthetic chemical products, carbon fibers, etc. Firstly, regarding the exploration of energy utilization focusing on the high-carbon structure of lignin, compared to the current simple drying and incineration for heat recovery in biomass refining plants, this conversion attempt can significantly improve the combustion quality and calorific value conversion efficiency of lignin. However, it is essentially still burning lignin and does not reduce carbon emissions from industrial lignin sources. Secondly, research on catalytically cracking industrial lignin into lignin monomers and then separating and purifying them for use as chemical raw materials faces challenges. Because the connections between lignin structural units are mainly ether bonds and carbon-carbon bonds with high bond energies, the complete cracking process requires a large amount of energy, and the subsequent separation and purification processes are complex. Compared to chemical raw materials refined from existing petrochemical resources, the overall cost is extremely high, making large-scale application unlikely in the short term.

[0005] Lignin has a stable chemical structure and typically exists in soil as humus during natural decomposition, requiring a long time to fully degrade. With the growing emphasis on carbon neutrality, the biomass refining industry, which uses lignocellulose as a raw material to produce biofuels and other products, has developed rapidly. Simultaneously, the scale of industrial lignin emissions, as industrial solid waste, has also increased dramatically. Industrial lignin is not being fully utilized, being simply incinerated as a low-calorific-value fuel to recover heat energy, placing a heavy burden on the environment. How to transform and develop high-value-added utilization of these ever-increasing lignin-based industrial wastes, enabling industrial lignin to participate in the ecological carbon cycle as a carbon sink and reducing the massive carbon emissions from simple incineration, has become a major bottleneck for the sustainable development of the current biomass refining industry.

[0006] Lignin sulfate (also known as Clason lignin) is a type of lignin remaining from the refining industry, a classic method for efficiently and cost-effectively obtaining water-soluble sugars from lignocellulosic biomass through concentrated sulfuric acid hydrolysis. This process yields lignins for subsequent fermentation and bioethanol production. Compared to industrial lignins such as alkali lignin emitted from the pulping industry, lignin sulfate possesses a highly condensed chemical structure and extremely low chemical reactivity, and is insoluble in water and most organic solvents. The efficient and high-value development and utilization of lignin sulfate remains a significant industrial challenge and constraint in the acid-hydrolyzed lignocellulosic biorefining industry.

[0007] In summary, considering the structural characteristics of lignin sulfate and the development trends of the biomass refining industry, a novel natural polymeric fertilizer additive based on lignin sulfate-modified materials has been developed. This additive can effectively increase soil organic matter content and alleviate plant growth inhibition caused by deficiencies in micronutrients, particularly iron, and organic matter in the soil. It will not only significantly alleviate the problem of large amounts of lignin-based waste generated during biomass refining but also contribute to the green, efficient, and sustainable development of agriculture in my country. Summary of the Invention

[0008] The purpose of this invention is to provide a novel method for preparing a natural polymeric fertilizer additive based on industrial lignin modification. This invention exhibits superior plant growth-promoting effects and can be recycled or naturally degraded without causing environmental pollution. Furthermore, this invention is convenient to process and produces, and has low costs.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing fertilizer additives based on sulfated lignin modification, wherein sulfated lignin is subjected to hydrothermal alkaline cutting treatment in a strong alkaline solution at 200-300°C for 1-6 hours, and after the treatment is completed, impurity removal treatment is performed to obtain fertilizer additives.

[0011] As a preferred embodiment of the first aspect above, the sulfated lignin is obtained by grinding coniferous wood chips into powder, then processing the wood powder using the Klassen process to separate the acid-insoluble portion, and finally washing it to neutral and drying it; preferably, the coniferous wood is Chinese fir.

[0012] As a preferred embodiment of the first aspect, the impurity removal process involves continuously rinsing the lignin after hydrothermal alkali cutting in running water and then fully filtering and replacing it with a semi-permeable membrane with a molecular weight cutoff of 3500 to remove inorganic salts and organic molecular fragments that can pass through the membrane.

[0013] As a preferred embodiment of the first aspect mentioned above, after hydrothermal alkali cutting and desiccation, sulfated lignin needs to be freeze-dried to obtain a fertilizer additive in the form of a dry powder.

[0014] In a second aspect, the present invention provides a fertilizer additive based on sulfate-modified lignin prepared by any of the preparation methods described in the first aspect above.

[0015] Thirdly, the present invention provides a use of the fertilizer additive as described in the second aspect above, specifically: applying it as a fertilizer additive that chelates metal elements to a plant culture medium lacking a single trace metal element to restore the growth phenotypic defects caused by the deficiency of the plant; or applying it as a fertilizer additive that chelates metal elements to a plant culture medium with normal nutrient supply to increase the content of trace metal elements in the plant and promote plant growth.

[0016] As a preferred embodiment of the third aspect above, the plant culture medium lacking a single trace element is a culture medium lacking a single iron, a single calcium, a single magnesium, a single zinc, or a single copper; preferably, the culture medium is 1 / 2 MS medium, and the plant is Arabidopsis thaliana.

[0017] Fourthly, the present invention provides a use of the fertilizer additive as described in the second aspect above, specifically: applying it as a fertilizer additive with iron ion chelation effect to a single iron-deficient Arabidopsis culture medium to promote the absorption of iron by Arabidopsis, increase the chlorophyll content of Arabidopsis leaves that are yellowing due to iron deficiency, and restore the leaf growth phenotype of Arabidopsis.

[0018] Fifthly, the present invention provides a use of the fertilizer additive as described in the second aspect above, specifically: applying it as a fertilizer additive with iron ion chelation effect to rice culture medium that is either iron-deficient or iron-free, thereby increasing the content of one or more of the elements iron, calcium, copper, and zinc in the root and stem parts of rice plants.

[0019] In a sixth aspect, the present invention provides a use of the fertilizer additive as described in the second aspect above, specifically: adding it to irrigation water throughout the entire growth cycle of rice to promote earlier flowering of rice or increase the number of rice tillers or increase the number of rice grains.

[0020] Compared with existing technologies such as lignin pyrolysis, photocatalysis, metal catalysis, and hydrogen peroxide oxidation for high-value conversion of lignin, this invention modifies sulfated lignin (a highly condensed industrial lignin, insoluble in water and organic solvents) through a short and low-cost hydrothermal reaction. The water-soluble lignin-based material (HSAL) obtained by hydrothermal alkali-cutting modification of sulfated lignin (SAL) not only cleaves monomer bonds, causing lignin depolymerization, but also leads to a decrease in methoxy groups and an increase in phenolic hydroxyl groups. This structural change makes HSAL an environmentally friendly metal ion chelating agent, improving the bioavailability of trace metals in the growth medium and apoplast space of rice and Arabidopsis thaliana by chelating metal ions, thereby promoting plant growth. The modified hydrothermal sulfated lignin has good water solubility and, after hydroponics and 1 / 2 MS medium, shows a significant effect on restoring root environment metal element deficiency in model plants such as rice and Arabidopsis thaliana, and promoting root growth under normal metal element conditions. Taking iron as an example, the mechanism by which HSAL promotes iron content in different model plants is different. In strategy I plants (Arabidopsis thaliana), HSAL chelates Fe. 3+ Reduced to Fe by FRO2 iron reductase 2+ Then, it is transported into plant cells via the IRT1 transporter protein. In Strategy II plants (rice), Fe... 3+ It may be released from the HSAL chelate and transported to the root cells in the form of an iron-carrier chelate via the YSL transporter protein. Attached Figure Description

[0021] Figure 1 The changes in methoxyl content of modified lignin sulfate (HSAL) in Example 1 under different hydrothermal reaction conditions include: a) changes in HSAL methoxyl content under a temperature gradient (200–300 °C) for 2 hours of hydrothermal treatment; and b) changes in HSAL methoxyl content under a time gradient (1–6 h) for a reaction temperature of 275 °C.

[0022] Figure 2 This is the effect of whether or not HSAL was added on the growth phenotype of Arabidopsis thaliana (wild type Col-0) in 1 / 2 MS medium with a single deficiency of each nutrient element (from top to bottom: normal 1 / 2 MS as control and single deficiency of iron, calcium, magnesium, zinc, copper, nitrogen, and carbon).

[0023] Figure 3The data in Example 3 are the root and stem length (a,b), root and stem magnesium content (c,d), root and stem iron content (e,f), root and stem copper content (g,h), root and stem calcium content (i,j), and root and stem zinc content (k,l) of rice seedlings after 10 days of planting under hydroponic conditions with 0.05% HSAL and iron deficiency and normal iron (36μMEDTA-Fe(II)).

[0024] Figure 4 This is a comparison of the effects of applying 0.05% HSAL on the entire growth cycle of rice under soil cultivation conditions in Example 4. a) Applying 0.05% HSAL accelerated the flowering period of soil-cultivated rice; b) Applying 0.05% HSAL effectively promoted tillering of soil-cultivated rice; c) Applying 0.05% HSAL effectively increased the final number of rice grains per plant in soil-cultivated rice.

[0025] Figure 5 The differences in total chlorophyll content (a) and root length (b) of Arabidopsis thaliana (wild-type Col-0 and mutants opt3-2, irt1-1 and fro2) in 1 / 2 MS medium (36 μM EDTA-Fe(II)) and iron-deficient medium (-Fe) in Example 5 are shown in Example 5.

[0026] Figure 6 The comparison of total chlorophyll content (a / b) and root length (c / d) of Arabidopsis thaliana (wild-type Col-0 and mutants opt3-2, irt1-1 and fro2) with gradient concentrations of EDTA-Na2 (0-1300 μM) in Example 6 under 1 / 2 MS medium (36 μM EDTA-Fe(II)) and iron-deficient 1 / 2 MS medium (-Fe) was made.

[0027] Figure 7 These are the chelation products of HSAL(a) and HSAL with FeSO4(b), FeCl3(c) and CaCl2(d) respectively in Example 7;

[0028] Figure 8 Example 7 compares the metal chelating abilities of HSAL and EDTA using complexometric titration analysis.

[0029] Figure 9The results are X-ray photoelectron spectroscopy (XPS) analysis of HSAL and the chelate products of HSAL with FeSO4 / FeCl3 (a) and CaCl2 (b) in Example 7. The results show that HSAL formed stable chelate structures with FeSO4 / FeCl3 (a) and CaCl2 (b), respectively (the positions of the Fe(2p1 / 2) / Fe(2p3 / 2) peaks in the chelate products in Figure a are 723.3-723.9 eV and 709.7-710.6 eV, respectively; the positions of the Ca(2p1 / 2) / Ca(2p3 / 2) peaks in the chelate products in Figure b are 346.5-347.0 eV and 349.5-350.0 eV, respectively). Detailed Implementation

[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Technical features in the various embodiments of the present invention can be combined accordingly without mutual conflict.

[0031] This invention prepares a fertilizer additive based on sulfated lignin modification. Specifically, sulfated lignin (SAL, which is the type of lignin discharged from the delignified lignin fiber biomass refining industry by concentrated sulfuric acid hydrolysis) is subjected to hydrothermal alkaline ablation treatment (treated in a strong alkaline solution at 200-300°C for 1-6 hours), and then subjected to impurity removal treatment to obtain water-soluble lignin-based material (HSAL).

[0032] It should be noted that the aforementioned sulfated lignin, also known as Classen lignin, can be obtained by grinding coniferous wood (such as Chinese fir) into powder, then processing the powder using the Classen process to separate the acid-insoluble portion, and finally washing it to neutral and drying it. The above impurity removal process mainly removes some low-molecular-weight inorganic salts and organic molecular fragments produced after hydrothermal alkaline cutting, while retaining high-molecular-weight lignin. A feasible impurity removal method is to continuously wash the hydrothermal alkaline-cut lignin in running water and then thoroughly filter and replace it using a semi-permeable membrane with a molecular weight cutoff of 3500 to remove inorganic salts and organic molecular fragments that can pass through the membrane.

[0033] In addition, after hydrothermal alkali cutting and desiccation, the above-mentioned sulfated lignin can be further freeze-dried to obtain fertilizer additives in the form of dry powder for subsequent formulation and use.

[0034] The water-soluble lignin-based substance prepared by this invention can be used as a fertilizer additive. When added to various culture media (hydroponics and 1 / 2 MS) lacking a single element, it can restore and promote root growth in plants (rice and Arabidopsis thaliana), and even under conditions of sufficient elements, it can significantly promote plant growth. Compared with other current explorations of high-value lignin development, the HSAL conversion process is simple, the reaction conditions are relatively mild (280℃-2h), the main reactant (NaOH solution) is inexpensive, and it can be recycled multiple times. Furthermore, this discovery innovatively links the high-value conversion and development of lignin waste emitted from the biomass refining industry with green agricultural production. The potential lignin consumption in agricultural production is sufficient to absorb the increasing industrial lignin emissions, and it points to a positive cycle that promotes plant biomass synthesis. The large-scale application of this lignin conversion process not only reduces the difficulty of disposing of solid lignin waste emitted from the biomass refining industry but also increases ecological carbon sequestration, plant biomass, and even crop yield. Furthermore, RNA-Seq analysis was performed on the root tips (0-0.5 cm) of hydroponically grown rice treated with HSAL. The results showed that ion-binding proteins, especially iron-binding proteins, were significantly enriched in the significantly altered genes in the HSAL-treated root tips, and the expression of genes related to iron absorption and transport was also significantly upregulated. Additionally, HSAL significantly increased the content of iron, calcium, and zinc in rice roots and aboveground parts, and significantly improved the iron deficiency tolerance of rice. Therefore, HSAL, as a novel modified material of industrial lignin, has the potential to be developed into an environmentally friendly fertilizer additive.

[0035] Example 1: Preparation of HSAL

[0036] In this embodiment, a method for preparing a fertilizer additive based on sulfated lignin modification is provided: Residual wood chips are ground into powder (60-100 mesh), treated with classen lignin (acid hydrolysis with 72% sulfuric acid, diluted with water to a 3-4% sulfuric acid concentration, and treated in an oil bath at 90°C for 90 minutes), filtered to obtain the acid-insoluble portion, rinsed until neutral, and then placed in a NaOH solution for hydrothermal alkaline ablation (temperature 280°C, time 2 hours). The powder is then thoroughly filtered and replaced with running water using a BioDesign-Mw3500 membrane to remove low molecular weight inorganic salts and organic fragments. The powder is then freeze-dried to obtain HSAL powder (denoted as HSAL280-2), which can be used as a fertilizer additive. The molecular weight (MW) of HSAL 280-2 is determined to be 7200±200 g / mol, and the methoxy content is 2±0.05 mmol / g.

[0037] It should be noted that the HSAL powder was obtained by freeze-drying under the experimental conditions of this embodiment in order to better control the purity and application concentration of the fertilizer additive. In the actual production process, freeze-drying can be omitted, and the HSAL concentration can be directly calculated and adjusted for application.

[0038] Furthermore, although the hydrothermal alkaline ablation treatment in this embodiment was performed at a temperature of 280°C for 2 hours, these parameters can be adjusted within a treatment temperature range of 200–300°C and a treatment time range of 1–6 hours. The molecular weight (as shown in Table 1) and methoxy content (as shown in Table 2) of the final HSAL product obtained under different treatment times and temperatures are also discussed. Figure 1 (As shown) There may be differences, but they all have similar performance to HSAL 280-2.

[0039] Table 1. Changes in molecular weight of products from the SAL hydrothermal reaction under time and temperature gradients.

[0040]

[0041] Therefore, based on the HSAL280-2 prepared in this embodiment, its effect as a fertilizer additive is demonstrated in Examples 2 to 7. The demonstration effect is specifically divided into the following aspects:

[0042] a. HSAL recovery experiment on single nutrient deficiency in culture medium: With Arabidopsis thaliana 1 / 2MS (agar medium) as a control, the growth recovery of Arabidopsis thaliana under single nutrient deficiency (iron, calcium, magnesium, zinc, copper, nitrogen, carbon) culture medium was set up with and without HSAL addition.

[0043] b. Restoration of Rice under Hydroponic Conditions and Elemental Analysis by HSAL: In a rice hydroponic system, two conditions were set up: an iron-sufficient control group without HSAL and an experimental group with HSAL. The effects of HSAL on rice root and stem growth and changes in the content of iron, magnesium, copper, zinc, and calcium were observed. Elemental analysis of HSAL itself and the hydroponic system was performed to determine the direct source of non-iron elements from HSAL, which can be used to restore rice growth under iron-deficient conditions.

[0044] c. Effects of HSAL on root and stem and chloroplast content of Arabidopsis iron transporter-reduction mutants: Two groups were set up under iron-sufficient and iron-deficient conditions, with a control group without HSAL and an experimental group with HSAL. The effects of HSAL on root and stem length and leaf chlorophyll content of Arabidopsis wild-type (Col-0) and iron transporter-reduction (irt1-1, fro2 and opt3-2) mutant materials were observed.

[0045] d. Experiment on the full cycle of rice cultivation in soil: Rice was grown in soil in pots, and 0.05% HSAL was added during the growth process. Compared with the control soil-grown rice, the growth differences of rice under soil cultivation with HSAL addition were observed throughout the entire cycle (tiller number, stem height, flowering period and final number of rice grains per plant, etc.).

[0046] e. Comparison of iron chelating capacity of HSAL and EDTA: Under the conditions of Arabidopsis thaliana 1 / 2MS (agar medium), two groups were set up: one with sufficient iron and one with deficient iron. The control group without EDTA-Na2 and the experimental group with gradient concentrations of EDTA-Na2 were observed to have different concentrations of EDTA-Na2 on the root and stem length and leaf chlorophyll content of Arabidopsis thaliana wild type (Col-0), iron-absorbing mutants (irt1-1 and fro2), and iron-transporting mutant opt3-2.

[0047] f. Morphological analysis of HSAL-metal chelates: HSAL was prepared by reacting it with FeSO4, FeCl3, and CaCl2 solutions, respectively. The resulting HSAL-metal chelates were then analyzed by X-ray electron spectroscopy (XPS). Thorough morphological description and characterization analysis of HSAL and its metal chelates showed that HSAL possesses excellent chelating properties, forming a relatively stable HSAL-metal chelate.

[0048] It should be noted that the "single nutrient deficiency" in the culture medium of this invention means that the culture medium is lacking only one element required for plant growth, while the other elements are sufficient.

[0049] The specific methods and results of the above-mentioned effect demonstration will be shown below through specific examples 2 to 7.

[0050] Example 2: HSAL experiment on the recovery of a single nutrient deficiency in culture medium

[0051] Using Arabidopsis thaliana as the culture plant, the growth recovery of Arabidopsis thaliana under single-deficiency (iron, calcium, magnesium, zinc, copper, nitrogen, and carbon) media was investigated with 1 / 2 MS medium (agar medium) as the control. (0.05% HSAL addition refers to the addition of HSAL at a mass percentage of 0.05 wt.% of the medium.)

[0052] The results of the HSAL experiment on the recovery of a single nutrient deficiency in the culture medium showed that ( Figure 2 HSAL did not show significant differences in response to single nitrogen or single carbon deficiencies, but exhibited significant recovery and growth-promoting phenotypes (leaf color depth, leaf size, root and stem length, etc.) in response to deficiencies of micronutrients (iron, calcium, magnesium, zinc, and copper). This result indicates that HSAL does not promote plant growth and recovery as an organic carbon or nitrogen source, but is highly correlated with responding to micronutrient deficiencies in plants.

[0053] Based on this conclusion, the HSAL prepared in this invention can be applied as a fertilizer additive that chelates metal elements to plant culture media lacking a single trace metal element, restoring the growth phenotypic defects caused by nutrient deficiency; or it can be applied as a fertilizer additive that chelates metal elements to plant culture media with normal nutrient supply, increasing the content of trace metal elements in plants and promoting plant growth.

[0054] It should be noted that the specific growth phenotypic defects caused by nutrient deficiency in plants in this invention vary depending on the element. Taking a 1 / 2 MS medium with only iron deficiency as an example, the growth phenotypic defect caused by iron deficiency is yellowing of leaves. Figure 2 The 1 / 2MS-Fe group showed that applying HSAL 280-2 as a fertilizer additive that chelates iron ions to the mono-iron-deficient Arabidopsis culture medium can promote the absorption of iron by Arabidopsis, increase the chlorophyll content of Arabidopsis leaves that are yellowing due to iron deficiency, and restore the leaf growth phenotype of Arabidopsis.

[0055] It should also be noted that the plant culture medium in this invention is not limited to 1 / 2MS medium, but can also be other soil culture medium or hydroponic substrate.

[0056] Example 3: Growth Regulation of Hydroponic Rice Based on HSAL Addition

[0057] First, rice seeds were immersed twice in a 10% hydrogen peroxide solution for 10 minutes each time, and then treated with 10% sodium hypochlorite for 15 minutes. These rice seeds were then vernalized at 28°C for 3 days, and then transferred to an iron-deficient (without EDTA-Fe(II)) or iron-sufficient (36 μM EDTA-Fe(II)) IRRI hydroponic solution (hydroponic nutrient solution based on the International Rice Research Institute's formula), with or without 0.05% HSAL added, in an opaque plastic container, and grown in a growth chamber under long-day conditions (16 hours light at 16000 lux at 28°C / 8 hours darkness at 25°C) for 10 days. Root and stem lengths of rice seedlings were measured with a ruler. Root / stem samples were taken and dried for elemental analysis. The effects of HSAL on rice root and stem growth and changes in iron content were observed. Among the findings, the phytonutrient content of HSAL itself was measured, and the hydroponic formula developed by the International Rice Research Institute revealed that the phytonutrient content of HSAL itself was extremely low compared to the corresponding phytonutrient content in the hydroponic solution, far below the requirements for restoring plant growth. These results fully demonstrate that HSAL does not act as a direct source of plant nutrients, but rather promotes plant growth phenotypes by regulating and facilitating the transport and absorption of plant nutrients.

[0058] like Figure 3As shown, HSAL addition not only promotes rice root and stem growth under iron-deficient conditions but also has a significant promoting effect under normal iron conditions. Furthermore, HSAL addition increases the content of iron, calcium, copper, and zinc in the rice root and stem. The increases in iron and calcium content are the most significant. We further precisely quantified the content of each major nutrient element based on the International Rice Research Institute's hydroponic solution formulation (IRRI) and the effect of 0.05% HSAL addition on each major nutrient element content (Table 2). Comparing this result with the total increase in each element in hydroponically grown rice caused by 0.05% HSAL addition (Table 3), it can be further confirmed that the increase in iron content in rice seedlings due to 0.05% HSAL does not originate from iron containing HSAL.

[0059] Table 2 Comparison of major nutrients in 0.05% HSAL and IRRI media

[0060]

[0061] Note: IRRI medium is based on the hydroponic nutrient solution formula of the International Rice Research Institute.

[0062] Table 3 shows that the iron content in rice seedlings increased by 0.05%. HSAL did not originate from iron containing HSAL.

[0063]

[0064]

[0065] Note: The control group (IRRI-Fe) used the hydroponic nutrient solution formula from the International Rice Research Institute (IRRI) without the addition of Fe-EDTA, while the experimental group (IRRI-Fe + 0.05% HSAL) used the same formula but without Fe-EDTA, but with the addition of 0.05% HSAL. Furthermore, each 1L hydroponic container held 12 rice seedlings.

[0066] Based on this conclusion, the HSAL prepared in this invention can be applied as a fertilizer additive that chelates metal ions to rice culture media that are either iron-deficient or iron-free, thereby increasing the content of one or more of the elements iron, calcium, copper, and zinc in the roots and stems of rice plants.

[0067] It should also be noted that the rice culture medium in this invention is not limited to hydroponic substrate, but can also be other soil culture medium or hydroponic substrate.

[0068] Example 4: HSAL Experiment on the Full Cycle of Rice Cultivation in Soil

[0069] Rice seedlings (Nipponbare) were transplanted into soil pots for soil cultivation. Throughout the entire growth cycle, a control group using purified water and a group using 0.05% HSAL (irrigation water) were set up. Compared with the soil-grown rice control, the effects of HSAL addition on the growth of rice in soil cultivation (tiller number, stem height, flowering period, and final number of grains per plant, etc.) were observed throughout the entire growth cycle.

[0070] The results are as follows Figure 4 As shown, the soil-grown rice in the 0.05% HSAL group flowered about 5 days earlier and the number of tillers more than doubled, resulting in a significant increase in the number of rice grains per plant.

[0071] Based on this conclusion, the HSAL prepared in this invention can be added to irrigation water throughout the entire growth cycle of rice to promote earlier flowering, increase the number of tillers, or increase the number of rice grains.

[0072] Example 5: Regulating the growth of Arabidopsis thaliana in 1 / 2 MS medium based on HSAL addition

[0073] Arabidopsis seeds (in this example, Arabidopsis thaliana was used with the addition of Colombian wild-type (Col-0) and its iron-absorbing mutants (irt1-1 and fro2), and the iron-transporting mutant opt3-2) were sterilized with 1% sodium hypochlorite and then vernalized in a dark room at 4°C for 2 days. Two conditions were established in 1 / 2 MS medium: iron-sufficient (50 μM EDTA-Fe(II)) and iron-deficient conditions. A control group without HSAL and an experimental group with 0.05% (m / m) HSAL were established. The plates were placed vertically in a growth chamber at 22°C (GXZ-380C-4, Jiangnan, China), with a 16-hour light / 8-hour dark cycle. After culturing in the growth chamber for ten days, the root and stem length and leaf chlorophyll content of wild-type and each mutant material were measured under iron-deficient and normal iron conditions, with and without HSAL.

[0074] The results are as follows Figure 5 As shown, 0.05% HSAL significantly restored chlorophyll content and root length of opt3-2 to wild-type levels, but did not restore chlorophyll content and root length of irt1-1 and fro2. Figure 5(a, b). These results indicate that FRO2- and IRT1-mediated iron uptake are crucial for the uptake of HSAL-chelated iron into plants. Membrane-localized FRO2 reduces extracellular iron chelates to ferrous iron, which is then transported into cells via the plasma membrane by IRT1. Furthermore, we precisely quantified the content of each major nutrient in 1 / 2 MS medium and the effect of 0.05% HSAL addition on each major nutrient (Table 4). Comparing this result with the total increase in elemental content in Arabidopsis thaliana planted in 1 / 2 MS medium due to 0.05% HSAL addition (Table 5) further confirms that the increase in iron content in Arabidopsis thaliana seedlings under iron-deficient conditions due to 0.05% HSAL does not originate from HSAL-containing iron.

[0075] Table 4 Comparison of major nutrients in 0.05% HSAL and 1 / 2MS media

[0076]

[0077] Table 5 shows that the iron content of Arabidopsis seedlings increased by 0.05% under iron deficiency conditions. HSAL does not originate from iron containing HSAL.

[0078]

[0079]

[0080] Note: The control group (1 / 2 MS-Fe) was prepared by removing Fe-EDTA under 1 / 2 MS conditions. The experimental group (1 / 2 MS-Fe + 0.05% HSAL) was prepared by removing Fe-EDTA under 1 / 2 MS conditions and adding 0.05% HSAL. Additionally, 15 Arabidopsis seedlings were planted on growth plates containing 50 mL of agar medium.

[0081] Example 6: Regulation of Arabidopsis growth in 1 / 2 MS medium based on EDTA-Na2 supplementation

[0082] Arabidopsis seeds (in this example, Arabidopsis Columbia wild-type (Col-0) and its iron-absorbing mutants (irt1-1 and fro2), and iron-transporting mutant opt3-2) were sterilized with 1% sodium hypochlorite and then vernalized in a dark room at 4°C for 2 days. Gradient molar concentrations (0, 65, 130, 1300 μm) of EDTA-Na2 were added to 1 / 2 MS medium under both iron-sufficient (50 μM EDTA-Fe(II)) and iron-deficient conditions. The plates were placed vertically in a growth chamber at 22°C (GXZ-380C-4, Jiangnan, China) with a 16-hour light / 8-hour dark cycle. After culturing in the growth chamber for ten days, the root and stem length and leaf chlorophyll content of wild-type and mutant materials were measured under iron-deficient and normal iron conditions with and without HSAL addition.

[0083] In addition, to quantify the iron chelation values ​​of HSAL and EDTA, 0.1 g of HSAL or EDTA was dissolved in 10 ml of distilled water with an indicator (5% sulfosalicylic acid) and titrated with a 0.01 M NH4Fe(SO4)2 standard solution. The titration endpoint was indicated by a change in solution color from yellow to light red. The Fe values ​​of HSAL and EDTA were calculated based on the titration volume. 3+ Chelation value.

[0084] like Figure 6 As shown, the effects of the iron chelator EDTA were compared in wild-type Arabidopsis and its iron-absorbing mutants (irt1-1 and fro2), and the iron-transporting mutant opt3-2. The recovery pattern of iron-deficient phenotypes in these iron-uptake-deficient mutants by EDTA was very similar to that of HSAL. This result further supports that, like EDTA, HSAL can serve as a model for trivalent iron chelators to improve iron bioavailability and restore plant growth under iron-deficient conditions.

[0085] Example 7: Speciation analysis of HSAL chelates with FeSO4, FeCl3 and CaCl2

[0086] The preparation of HSAL-metal chelates using the aforementioned HSAL and metal compounds is carried out according to the following steps:

[0087] a. Chelation reaction: Weigh an appropriate amount (0.2 g) of HSAL and dissolve it in 20 mL of ultrapure water. Add 0.1 M FeSO4, FeCl3 and CaCl2 respectively and stir thoroughly at room temperature for 6 hours.

[0088] b. Purification: Place the chelation reaction mixture into a molecular sieve (BioDesign-Mw3500) membrane and allow water to fully displace any residual small molecules;

[0089] c. Freeze-drying: The HSAL-metal chelate solution obtained by filtering the chelation reaction mixture through a molecular sieve is then filtered through simple filter paper to remove insoluble substances, and finally freeze-dried to obtain HSAL-metal chelate powder.

[0090] The obtained HSAL metal chelate was then subjected to thorough morphological description and characterization analysis (X-ray photoelectron spectroscopy (XPS) analysis). The results showed that HSAL has good chelating properties and can form a relatively stable HSAL-metal chelate.

[0091] Morphological observation of HSAL and HSAL-metal chelates as follows: Figure 7 As shown, unlike the porous and loose structure of HSAL, the chelate complexes of HSAL with FeSO4 exhibit a fine, sandy texture, while the chelate complexes of HSAL with FeCl3 and CaCl2 show a more fluffy appearance. These morphological changes in the chelates indicate the successful chelation of HSAL with metal compounds.

[0092] To quantify the chelating ability of HSAL, we used chelation titration analysis to measure the ability of HSAL chelated with FeCl3 and CaCl2 and compared it with the well-known chelating agent EDTA. The results showed that HSAL chelates Fe... 3+ The capacity is 101.9 mg / g. -1 Slightly lower than EDTA (138.8 mg / g). -1 ). HSAL chelates Ca 2+ The capacity is 69.7 mg / g. -1 The chelating capacity of EDTA is 173.8 mg / g. -1 ( Figure 8 Since the average molecular weight of HSAL (7.5 kDa) is 25.6 times that of EDTA, an equimolar amount of HSAL can chelate more metals than EDTA. In summary, these results demonstrate that HSAL possesses metal-chelating capabilities.

[0093] X-ray photoelectron spectroscopy (XPS) was used to analyze the binding ratio and binding energy, further investigating the elemental composition, valence states, and chelate complexes of HSAL with different metal compounds. Figure 9 FeSO4 on Fe(2p) 3 / 2 The binding energy of FeCl3 is between 711.9 and 712.3 eV, while that of FeCl3 with Fe(2p) is between 711.9 and 712.3 eV. 3 / 2 The binding potential of ) is between 711.0 and 711.5 eV. For example... Figure 9 As shown in figure a, the Fe2p peaks of the HSAL chelate complexes with FeSO4 and FeCl3 shifted to 709.7-710.6 eV and 723.3-723.9 eV, respectively, corresponding to Fe(2p) peaks.3 / 2 ) and Fe(2p 1 / 2 While HSAL did not detect the corresponding peaks, these results indicate that FeCl3 and FeSO4 were successfully chelated by HSAL.

[0094] In addition, Ca(2p) in CaCl2 3 / 2 The binding energy of Ca2p to the chelate complex of HSAL and CaCl2 is between 347.9 and 348.6 eV. The binding energies of Ca2p to the chelate complex of HSAL and CaCl2 decompose into two main peaks at 346.5–347.0 eV and 349.5–350.0 eV, respectively, corresponding to Ca2p... 3 / 2 ) and Ca(2p 1 / 2 ()( Figure 9 b). This result shows that HSAL can also successfully chelate with CaCl2.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. The use of a fertilizer additive based on sulfate-modified lignin, characterized in that, It is applied as a fertilizer additive that chelates metal elements to plant culture media lacking a single micronutrient to restore the plant's growth phenotypic defects caused by nutrient deficiency; the plant culture media lacking a single micronutrient is a culture medium lacking iron, calcium, magnesium, zinc, or copper; the plant is rice or Arabidopsis thaliana. The preparation method of this fertilizer additive is as follows: lignin sulfate is subjected to hydrothermal alkaline ablation treatment in a strong alkaline solution at 200~300 ℃ for 1~6 h. After treatment, impurity removal treatment is performed to obtain the fertilizer additive. The lignin sulfate is obtained by grinding Chinese fir slices into powder, then treating the powder with the Klassen process to separate the acid-insoluble part, and finally washing it to neutral and drying it. The impurity removal treatment involves continuously rinsing the hydrothermal alkaline ablation treatment lignin in running water and fully filtering and replacing it with a semi-permeable membrane with a molecular weight cutoff of 3500 to remove inorganic salts and organic molecular fragments that can pass through the membrane.

2. The use of a fertilizer additive based on sulfate-modified lignin, characterized in that, It was applied as a fertilizer additive that chelates iron ions to the culture medium of Arabidopsis thaliana with iron deficiency, which promoted the absorption of iron by Arabidopsis thaliana, increased the chlorophyll content of Arabidopsis thaliana leaves that turned yellow due to iron deficiency, and restored the leaf growth phenotype of Arabidopsis thaliana. The preparation method of this fertilizer additive is as follows: lignin sulfate is subjected to hydrothermal alkaline ablation treatment in a strong alkaline solution at 200~300 ℃ for 1~6 h. After treatment, impurity removal treatment is performed to obtain the fertilizer additive. The lignin sulfate is obtained by grinding Chinese fir slices into powder, then treating the powder with the Klassen process to separate the acid-insoluble part, and finally washing it to neutral and drying it. The impurity removal treatment involves continuously rinsing the hydrothermal alkaline ablation treatment lignin in running water and fully filtering and replacing it with a semi-permeable membrane with a molecular weight cutoff of 3500 to remove inorganic salts and organic molecular fragments that can pass through the membrane.

3. The use of a fertilizer additive based on sulfate-modified lignin, characterized in that, It was applied as a fertilizer additive that chelates metal ions to a single iron-deficient rice culture medium to increase the iron content in the roots and stems of rice plants. The preparation method of this fertilizer additive is as follows: lignin sulfate is subjected to hydrothermal alkaline ablation treatment in a strong alkaline solution at 200~300 ℃ for 1~6 h. After treatment, impurity removal treatment is performed to obtain the fertilizer additive. The lignin sulfate is obtained by grinding Chinese fir slices into powder, then treating the powder with the Klassen process to separate the acid-insoluble part, and finally washing it to neutral and drying it. The impurity removal treatment involves continuously rinsing the hydrothermal alkaline ablation treatment lignin in running water and fully filtering and replacing it with a semi-permeable membrane with a molecular weight cutoff of 3500 to remove inorganic salts and organic molecular fragments that can pass through the membrane.

4. The use as described in any one of claims 1 to 3, characterized in that, After hydrothermal alkali cutting and impurity removal, sulfated lignin needs to be freeze-dried to obtain a fertilizer additive in the form of dry powder.

5. The use as described in claim 1, characterized in that, The culture medium was 1 / 2 MS medium, and the plant was Arabidopsis thaliana.

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