A method for characterizing the lignin source in a natural degradation system

Abstract

The present invention belongs to the technical field of biological isotope detection, and relates to a method for characterizing the lignin source in a natural degradation system. The present invention realizes the accurate characterization and traceability of the lignin source by establishing an isotope response model from photosynthetic tissues and non-photosynthetic tissues to the humus soil layer. The present invention solves the technical problem in the prior art that it is difficult to distinguish different lignin sources, and provides a reliable technical means for quantifying the participation of biomass in the geochemical cycle and recycling.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological isotope detection, and relates to a method for characterizing the lignin source in a natural degradation system. Background Art

[0002] As the material basis of biomass in the agroforestry-gardening-forest ecosystem, lignocellulose has received extensive attention in the fields of biogeochemistry and biomass material applications due to its unique physical and chemical structural characteristics and renewable biomass material properties. Chemically, lignocellulose is a macromolecular biomass polymerized by cellulose, hemicellulose, and lignin through cross-linking bonds. Under natural conditions, lignocellulose has the biological resistance property of being insoluble in water, that is, the stability of the biological degradation system, which thus hinders the biogeochemical carbon cycle process and enhances the plasticity of biomass material applications.

[0003] Biodegradation, namely biological enzymatic hydrolysis, is the main way to promote the hydrolysis of cellulose and hemicellulose and the cleavage of lignin, thereby converting them into utilizable small-molecule substances. Due to the essential differences in chemical structures, lignin is relatively difficult to be degraded and utilized, and can be used as a biological degradation marker to characterize the dynamic process of biomass in the agroforestry-gardening-forest ecosystem participating in the biogeochemical cycle. The core of the chemical behavior of lignin participating in biodegradation lies in the difference in the types of its monomer composition and the site specificity of chemical structure groups. During the biosynthesis of lignin, the three hydrogen atoms of lignin methoxy all come from photosynthetic water molecules and have irreversible photolysis, while the carbon atoms come from the intermediate substances of leaf photosynthetic carbon fixation and metabolism. Therefore, once the methylation of biomass lignin is completed, the stable isotope signal carried by its methoxy group can reflect relevant information such as the biogeography and material metabolism of lignin biosynthesis. However, for the natural degradation system of biomass in the agroforestry-gardening-forest ecosystem, there is still a technical gap in the means of distinguishing lignin from different sources (such as leaves, branches, trunks, etc.), which thus hinders the quantitative characterization of lignocellulose participating in the biogeochemical carbon cycle and the traceability indication for the regeneration and utilization of biomass materials.

[0004] Currently, the application research on using the stable carbon and hydrogen isotope ratios (δ 13 C LM and δ 2 H LM ) of lignin methoxy as markers mainly focuses on the reference field of climate ecology, and the test samples are mostly tree-ring logs with high lignin content, while ignoring the characterization of leaves and branches as lignin from different sources participating in the biological degradation system and the formation of mineral soil. In existing research, there are still unknowns about how to refine the effective characterization of lignin from different tissue parts (photosynthetic tissue leaves and non-photosynthetic tissue branches, aboveground biomass materials and underground organic matter materials), and there is a lack of a method for characterizing the lignin source in a natural degradation system. Summary of the Invention

[0005] To overcome the problems such as the application gap in the existing technology, the present invention provides a method for characterizing the lignin source in a natural degradation system. The stable isotope ratios of lignin methoxy groups obtained by this method are stable and reliable, and can provide a probe for tracing the characterization of biomass lignin in the agroforestry-garden-forest ecosystem and the dynamic research of the biogeochemical material cycle process.

[0006] On the one hand, the present invention relates to a method for characterizing the lignin source in a natural degradation system, which includes:

[0007] Obtaining the δ 13 C LM values of lignin methoxy groups in plant tissues, source media, and soil in the same natural degradation system, using the δ 13 C LM value of lignin methoxy groups in the source medium as the independent variable, and the δ 13 C LM value of lignin methoxy groups in the soil as the dependent variable, constructing a regression equation model, and predicting the corresponding δ 13 C LM value of the plant tissue through the regression equation model;

[0008] Or obtaining the δ 2 H LM values of lignin methoxy groups in plant tissues, source media, and soil in the same natural degradation system, using the δ 2 H LM value of lignin methoxy groups in the source medium as the independent variable, and the δ 2 H LM value of lignin methoxy groups in the soil as the dependent variable, constructing a regression equation model, and predicting the corresponding δ 2 H LM value of the plant tissue through the regression equation model;

[0009] When the plant tissue is photosynthetic tissue, the source medium is aboveground litter, and the soil is from the 0-10 cm soil profile;

[0010] When the plant tissue is non-photosynthetic tissue, the source medium is the rootstock, and the soil is from the 10-20 cm soil profile.

[0011] Further, in the method for characterizing the lignin source in the natural degradation system provided by the present invention, the photosynthetic tissue is fresh leaves, and the non-photosynthetic tissue is branches.

[0012] Further, in the method for characterizing the lignin source in the natural degradation system provided by the present invention, the natural degradation system includes an agroforestry solid waste natural degradation system, a garden litter natural degradation system, and a forest fallen log natural degradation system.

[0013] Exemplarily, the natural degradation system of agricultural and forestry solid waste includes lignin degradation precursors, intermediates and humus products such as agricultural and forestry stems, branches, leaves and reclaimed soil organic matter; the natural degradation system of garden litter includes lignin degradation precursors, intermediates and humus products such as garden pruning waste, litter and horticultural cultivation substrates; the natural degradation system of forest fallen trees includes lignin degradation precursors, intermediates and humus products such as the stems, branches and fallen leaves of typical tree species in mountain pure forests and mixed forests that have fallen or died; the lignin degradation precursors include leaves and their litter in photosynthetic tissue parts, and stems, branches and their pruning waste in non-photosynthetic tissue parts; the lignin degradation intermediates include above-ground plant-derived substances entering the natural degradation system and degradation humus intermediates on the surface and underground; the lignin degradation humus products include degradation humus products on the surface and underground of the natural degradation system and mineralized soil organic matter.

[0014] Furthermore, in the method for characterizing lignin sources in the natural degradation system provided by the present invention, according to different natural degradation systems, the optimal regression equation model is selected, including:

[0015] In the same natural degradation system, a regression equation model is constructed with the δ 13 C LM value, and a regression equation model is constructed with the δ 2 H LM value. The regression equation model with the highest R 2 value is selected as the optimal regression equation model.

[0016] On the other hand, the present invention relates to a method for judging lignin sources in a natural degradation system, which includes:

[0017] Obtaining a regression equation model through the method for characterizing lignin sources in the natural degradation system;

[0018] The corresponding source medium of the regression equation model is above-ground litter, and the soil is a 0-10 cm soil profile. Based on the δ 13 C LM value or δ 2 H LM value of above-ground litter, a simulation data set is constructed with the regression equation model; it is judged whether there is a significant difference between the simulation data set and the data set to be judged; if there is no significant difference, it indicates that the data set to be judged is derived from photosynthetic tissue; if there is a significant difference, it indicates that the data set to be judged is not derived from photosynthetic tissue;

[0019] Or the corresponding source medium of the regression equation model is rhizomes, and the soil is a 10-20 cm soil profile. Based on the δ 13 C LM value or δ 2 HLM Construct a simulated data set with values and regression equation models; determine whether there is a significant difference between the simulated data set and the data set to be judged; if there is no significant difference ( P ≥0.05), it indicates that the data set to be judged is derived from non-photosynthetic tissues; if there is a significant difference ( P <0.05), it indicates that the data set to be judged is not derived from non-photosynthetic tissues.

[0020] Generally, when comparing the significance levels, the Student t-test is used for two-group comparisons, and ANOVA is used for multi-group comparisons.

[0021] Generally, after obtaining the sample, lignin needs to be extracted and purified from the sample, and headspace iodomethane gas is obtained through an organic chemical reaction derivative. The δ 13 C LM value or δ 2 H LM value is obtained by measuring the headspace iodomethane gas.

[0022] Preferably, according to the different interfering substances in samples such as plant tissues, source vectors, and soil, the present invention provides a preprocessing method before detecting the δ 13 C LM value or δ 2 H LM value.

[0023] Generally, after obtaining the sample, lignin needs to be extracted and purified from the sample, and headspace iodomethane gas is obtained through an organic chemical reaction derivative. The δ 13 C LM value or δ 2 H LM value is obtained by measuring the headspace iodomethane gas.

[0024] Preferably, according to the different interfering substances in samples such as plant tissues, source vectors, and soil, the present invention provides a preprocessing method before detecting the δ 13 C LM value or δ 2 H LM value.

[0025] Furthermore, in the method for characterizing the lignin source in the natural degradation system provided by the present invention, the sample to be detected contains a source donor of non-lignin methoxy, and the source donor of non-lignin methoxy is a tannic acid substance, and the sample to be detected is preprocessed;

[0026] It includes: after hydrolyzing the sample to be detected with an alkali solution to remove cellulose substances, nicotine is used to react under neutral or acidic conditions to remove the interference of tannic acid substances.

[0027] Furthermore, in the method for characterizing the lignin source in the natural degradation system provided by the present invention, the sample to be detected contains a source donor of non-lignin methoxy, and the source donor of non-lignin methoxy is pectin. The sample to be detected is pretreated, and the sample to be detected contains a cuticle;

[0028] It includes: after hydrolyzing the sample to be detected with an alkaline solution to remove cellulose substances, treating it with a mixture of the pseudo-ionic liquid zinc chloride tetrahydrate and the ionic liquid 1-ethyl-3-methylimidazolium chloride, and mixing and reacting under neutral conditions to remove the interference of pectin.

[0029] Furthermore, in the method for characterizing the lignin source in the natural degradation system provided by the present invention, the sample to be detected contains a source donor of non-lignin methoxy, and the source donor of non-lignin methoxy is pectin. The sample to be detected is pretreated, and the sample to be detected does not contain a cuticle;

[0030] It includes: after hydrolyzing the sample to be detected with an alkaline solution to remove cellulose substances, treating it with a mixture of the pseudo-ionic liquid zinc chloride tetrahydrate and the ionic liquid 1-ethyl-3-methylimidazolium chloride, mixing and reacting under acidic conditions, then adding a fuming hydrochloric acid aqueous solution as a neutralizing reagent to adjust the reaction system to neutral, and removing the interference of pectin.

[0031] Furthermore, in the method for characterizing the lignin source in the natural degradation system provided by the present invention, the sample to be detected contains a source donor of non-lignin methoxy, and the sample to be detected is soil containing humus. The sample to be detected is pretreated;

[0032] It includes: after hydrolyzing the sample to be detected with an alkaline solution to remove cellulose substances, treating it with a mixture of the pseudo-ionic liquid ammonium ferrous sulfate hexahydrate and the ionic liquid 1-ethyl-3-methylimidazolium chloride, mixing and reacting under acidic conditions, adding copper oxide as a derivatizing agent, and performing a microwave reaction under weakly alkaline conditions to selectively enrich lignin phenolic derivatives while removing pectin-like interfering substances.

[0033] Exemplarily, the sample to be detected contains a source donor of non-lignin methoxy, and the source donor of non-lignin methoxy is tannic acid substances. The specific pretreatment method is as follows:

[0034] Tannic acid is a type of ester formed by 6-hydroxybiphenyl dicarboxylic acid or phenolic carboxylic acids related to 6-hydroxybiphenyl dicarboxylic acid and polyols, mainly present in the photosynthetic leaves and litter of some specific plants. Some of its phenolic hydroxyl groups contain lignin phenolic methoxy groups due to methylation. First, the extract obtained by hydrolyzing and sieving the cellulose-like substances in the photosynthetic tissue leaves is mixed with nicotine at a mass-volume ratio of 1:2 (mg / mL); then, under medium acidic conditions, at room temperature (22 ± 1 °C), it is sealed and stirred for 20 - 30 min, allowed to stand and precipitate for 15 - 30 min, and the upper layer solution is filtered to remove tannic acid-like interfering substances containing lignin phenolic methoxy groups.

[0035] Specifically, the sample to be detected contains a source donor of non-lignin methoxy, and the source donor of non-lignin methoxy is pectin. The specific pretreatment method is as follows:

[0036] Pectin is a type of galacturonic acid polymer mainly present in the epidermal layer of plant fruits, waxy leaves, and phloem of branches. Due to the methylation and methyl esterification of its chain bond phenolic hydroxyl groups to a certain extent, it has the function of a lignin phenolic methoxy donor. For photosynthetic tissue leaves with a relatively thick cuticle, first, screening and filtration are carried out according to the pretreatment method of tannic acid-like substances above, and the upper layer filtrate obtained is used as the first extract; then, an equimolar mixture of ionic liquid zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥ 98%) and ionic liquid 1-ethyl-3-methylimidazolium chloride (Emim]FeCl4) is used as the second biphasic enrichment system for lignin material enrichment, mixed at a mass ratio (mg / mg) of 1:1 and adjusted to neutrality, and melted and reacted at 80 °C for 10 min under sealed conditions to remove pectin-like interfering substances.

[0037] For the removal pretreatment of pectin in non-photosynthetic products such as branches and rhizomes, first, an equimolar mixture of ionic liquid zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥ 98%) and ionic liquid 1-ethyl-3-methylimidazolium chloride (Emim]FeCl4) is used as the extraction system for lignin material enrichment, and medium acidic (pH ≈ 4) melting treatment (sealed microwave reaction at 80 °C for 10 min) is carried out at a mass ratio (mg / mg) of 4:1; then, 37% fuming hydrochloric acid (HCl) deionized water aqueous solution is used as a neutralizing reagent, and the neutralizing reagent and the first extract are mixed at a volume-mass ratio of 100 - 150:1 (μL / mg), and allowed to stand overnight (8 - 10 h) at room temperature (22 ± 1 °C) to adjust the pH to near neutral (6.5 - 7.5) to remove pectin-like interfering substances.

[0038] For the pretreatment of removing pectin from humus-rich soil profiles, first, an equimolar mixture of the pseudo-ionic liquid ammonium ferrous sulfate hexahydrate (NH4)2Fe(SO4)2·6H2O with a purity of ≥99% and the ionic liquid 1-ethyl-3-methylimidazolium iron(III) chloride ([Emim]FeCl4) is used as the enrichment extraction system for lignin materials, and it is mixed according to the mass ratio (mg / mg) of 15:1 with agricultural and forestry soil, 10:1 with garden soil, and 3:1 with forest soil; then, an equal mass of copper oxide is added as a derivatizing agent, and under the sealed condition of weak alkalinity with pH = 8.5, a microwave reaction is carried out at 120 °C for 70 min, so as to remove pectin-like interfering substances while selectively enriching lignin phenolic derivatives.

[0039] On the other hand, the present invention relates to the application of the method for judging the source of lignin in the natural degradation system in obtaining the source of lignin-containing samples.

[0040] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects or advantages:

[0041] The present invention utilizes the δ 13 C LM and δ 2 H LM value characteristics of lignin and their corresponding traceability response relationship models, and provides a method for characterizing the source of lignin in the photosynthetic and non-photosynthetic tissue organs of plants in agricultural, forestry, and garden ecosystems participating in the natural degradation system. The lignin source characterization method described in the present invention realizes the lignin traceability characterization of the photosynthetic tissue organs of plants - leaves participating in the biodegradation process (from leaf litter to soil organic matter), and also realizes the lignin traceability characterization of the non-photosynthetic tissue organs of plants - branches participating in the biodegradation process (from above-ground branches and stems, underground rhizomes to soil organic matter). The lignin source characterization described in the present invention uses substrates (leaf litter, garden pruning, stem branches), intermediates (humus layer degradation residues, root tissue organs), and end products (soil organic matter) participating in the natural biodegradation system such as agricultural and forestry solid wastes, garden pruning, and forest regeneration as material media, and through a one-step physicochemical two-phase extraction, chemical derivatization separation, solid-liquid extraction and other purification systems, etc., to enrich and purify lignin substances, further using a selective organic substitution derivatization reaction to selectively replace the methoxy group of lignin and obtain stable headspace iodomethane gas, and then through gas chromatography - gas stable isotope ratio mass spectrometry to measure the stable carbon and hydrogen isotope ratios, and finally using the δ 13 C LM and δ 2 H LM value characteristics of lignin in photosynthetic and non-photosynthetic tissues and their tissue organ response relationships to characterize the lignin sources of different degradation systems.

[0042] The method for lignin source characterization in the natural degradation system described in the present invention is the first to systematically elaborate on the method for source characterization of typical biomass - lignin in agricultural and forestry, gardening, and forest ecosystems during the biodegradation process, achieving the quantification of the sources of lignin in photosynthetic and non-photosynthetic tissue organs by using the δ 13 C LM and δ 2 H LM value characteristics and their traceability response relationship model, thus providing a reliable biomarker index for scientifically and effectively using the chemical structure and group specificity of lignin to characterize the dynamic research and quantitative application of biomass participation in biogeochemical cycles. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention.

[0044] Figure 1 It is a response relationship model diagram of the δ 13 C LM and δ 2 H LM values of lignin methoxy groups in relevant tissue organs of the apple tree, an agricultural and forestry plant.

[0045] Figure 2 It is a response relationship model diagram of the δ 13 C LM and δ 2 H LM values of lignin methoxy groups in relevant tissue organs of the privet, a gardening plant.

[0046] Figure 3 It is a response relationship model diagram of the δ 13 C LM and δ 2 H LM values of lignin methoxy groups in relevant tissue organs of the Quercus aliena var. acuteserrata, a forest plant.

[0047] Figure 4 It is a response relationship model diagram of the δ 13 C LM and δ 2 H LM values of lignin methoxy groups in relevant tissue organs of the Abies fargesii, a forest plant.

[0048] Figure 5 It is a response relationship model diagram of the δ 13 C LM and δ 2 H LM values of lignin methoxy groups in relevant tissue organs of the Larix chinensis, a forest plant.

[0049] Figure 6 For the comparison of the methoxyl δ of lignin before and after the pretreatment of the photosynthetic tissue leaves of garden plants 13 C LM and δ 2 H LM values.

[0050] Figure 7 For the comparison of the methoxyl δ of lignin before and after the pretreatment of the non - photosynthetic tissue branches of gymnosperms and broad - leaf plants 13 C LM and δ 2 H LM values.

[0051] Figure 8 For the comparison of the methoxyl δ of lignin before and after the lignin source - derived treatment of the humus soil layer under gymnosperms and broad - leaf plants 13 C LM and δ 2 H LM values.

[0052] Figure 9 For the lignin source characterization relationship between the photosynthetic tissue (new leaves - litter) and the surface soil, and the non - photosynthetic tissue (branches - rhizomes) and the deep soil based on the fitting relationship of the methoxyl δ 13 C LM and δ 2 H LM values. Specific implementation mode

[0053] Next, the technical solution of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments.

[0054] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments, but the embodiments cited do not limit the present invention.

[0055] Embodiment 1

[0056] This embodiment provides a method for characterizing the lignin source in a natural degradation system, specifically for the lignin source characterization of fruit tree litter, pruning materials, etc. participating in the natural biodegradation process in the agro - forestry ecosystem. It is divided into using the methoxyl δ of lignin substances in the photosynthetic tissue organs leaves - litter - soil organic matter and the non - photosynthetic tissue organs branches - rhizomes - soil organic matter 13 C LM 、δ 2 H LM value response relationship to characterize the lignin source in the natural degradation system.

[0057] The fruit tree is of the genus Malus ( MalusFor deciduous shrub apple trees (Mill.), their native mature leaves, current season's litter, branches, trunk, undisturbed profile soil (0 - 10 cm, 10 - 20 cm), and rhizomes were collected respectively. These materials were dried to a constant weight at 105 °C, crushed and ground into fine powder samples with a particle size of 80 μm for standby.

[0058] For fine powder samples of photosynthetic products such as mature leaves and current season's litter, an aqueous sodium hydroxide solution (NaOH, 1 M) was used as the first extraction system for chemical screening of non - lignin substances. The solution and the fine powder were mixed at a volume - mass ratio (mL / mg) of 1:5, stirred and dissolved at 90 °C for 4 h under sealed conditions to remove cellulose substances. Then, it was quickly quenched at 0 °C and left standing for 30 min. The lower - layer turbid precipitate was taken as the first extract. Nicotine liquid with left - and right - handed optical rotation properties and miscible in medium - acidic conditions was used as the second extraction system for lignin material enrichment. The nicotine liquid of the second extraction system was mixed with the first extract at a volume - mass ratio of 2:1 (mL / mg), stirred and reacted at room temperature (22 ± 1 °C) under sealed conditions for 20 - 30 min, left standing and precipitated for 15 - 30 min to remove tannic acid substances containing lignin phenolic methoxy groups. The upper - layer solution was filtered and taken as the second extract. After extraction with ethyl acetate at a volume - mass ratio (mL / mg) of 2:1 and rotary evaporation under reduced pressure, lignin - like substances produced by photosynthetic tissue organs were obtained.

[0059] For fine powder samples of non - photosynthetic products such as branches and rhizomes, an equimolar mixture of ionic liquid zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥ 98%) and ionic liquid 1 - ethyl - 3 - methylimidazolium chloride ferrate ([Emim]FeCl4) was used as the extraction system for lignin material enrichment. Medium - acidic (pH ≈ 4) melting treatment (sealed microwave reaction at 80 °C for 10 min) was carried out at a mass ratio (mg / mg) of 4:1. A 37% fuming hydrochloric acid (HCl) deionized water aqueous solution was used as the neutralization reagent. The neutralization reagent and the first extract were mixed at a volume - mass ratio of 100 - 150:1 (μL / mg), left standing overnight (8 - 10 h) at room temperature (22 ± 1 °C) to adjust the pH to near - neutral (6.5 - 7.5). Then, extraction with ethyl acetate at a volume - mass ratio (mL / mg) of 2:1 and rotary evaporation under reduced pressure were carried out to enrich lignin - like substances produced by non - photosynthetic tissue organs. For non - photosynthetic tissue organs such as trunk with a lignin content of 20% - 30%, their fine powder can be directly used for further determination of lignin methoxy δ 13 C LM 、δ 2 H LM value.

[0060] For the fine powder sample of organic matter in the soil profile with thin humus, an equimolar mixture of ionic liquid-like ammonium ferrous sulfate hexahydrate ((NH4)2Fe(SO4)2·6H2O, purity ≥ 99%) and ionic liquid 1-ethyl-3-methylimidazolium ferric chloride ([Emim]FeCl4) is used as the enrichment extraction system for lignin materials. It is mixed according to the mass ratio (mg / mg) of 15:1 with agricultural and forestry soil, and then an equal mass of copper oxide is added as a derivatizing agent. It is subjected to microwave reaction at 120 °C for 70 min under sealed conditions at a weak alkaline pH = 8.5, and then rapidly quenched at 0 °C. Ethyl acetate extraction is carried out at a volume-mass ratio (mL / mg) of 2:1, and lignin phenolic derivatives are obtained by concentration under reduced pressure rotary evaporation.

[0061] Weigh 8 - 10 mg of the above lignin-like substance and add it to a brown headspace liquid-phase vial containing 0.5 mL of hydroiodic acid with a mass fraction of 55%. Under sealed conditions, perform microwave displacement reaction at 110 ºC for 20 mim, and let it stand at room temperature of 22 ± 1 °C for 40 min to obtain a steady-state headspace CH3I gas with a constant isotope value. Select a Thermo Fisher TRACE 1310 model GC instrument equipped with a TG-5MS chromatographic column (30 m × 0.32 mm × 0.25 μm) for on-line separation of the headspace gas, and the injection volume is 60 - 80 μL. Settings: injection port temperature 200 °C, initial temperature of the temperature programming 40 °C, retention for 3.8 min, increase the temperature to 80 °C at 20 °C / min, retention for 1 min, then increase the temperature to 100 °C at 40 °C / min, retention for 3 min, injection split ratio 8:1, and the flow rate of high-purity helium gas as the carrier gas is 0.6 mL / min.

[0062] Under normal conditions, the CH3I reagent is in a liquid state, and it is difficult to vaporize and calibrate it. In this example, two calibrated logs, Siberian larch ( Larix gmelinii ), and Cryptomeria fortunei ( Cryptomeria fortunei ), recognized by the International Atomic Energy Agency (specifically refer to Lu Q Q, Liu X H, Treydte K, et al., Altitude-specific differences in tree-ring δ 2 H records of wood lignin methoxy in the Qinling mountains, central China. 2023, Quaternary Science Reviews) are used as calibration substances, corresponding to lignin methoxy δ 13 C LM and δ 2 H LMThe values are -35.0‰ and -29.7‰, as well as -307.5‰ and -210.1‰ respectively.

[0063] The CH3I obtained by online GC separation was further used for IRMS determination, corresponding to the δ 13 C LM value determination using the high-temperature oxidation mode. The reaction furnace temperature was set at 960 °C, and calibrated high-purity carbon dioxide was used as the reference gas. The calibrated δ 13 C LM values (-35.0‰ and -29.7‰) of the log were used as the two-point calibration standard; for the determination of the δ 2 H LM value, the high-temperature pyrolysis mode was adopted, the reaction furnace temperature was 1250 °C, high-purity hydrogen was used as the reference gas, and the H3 + value in the self-check state was 4.3 ppm / nA. The calibrated δ 2 H LM values (-307.5‰ and -210.1‰) of the log were used as the two-point calibration standard. The relative standard deviations of the lignin methoxy δ 13 C LM and δ 2 H LM values of each source sample were less than 0.4‰ and 0.7‰ respectively.

[0064] After 5 groups of repeated sample measurements, it was found that the lignin methoxy δ 13 C LM and δ 2 H LM values and the response relationship characteristics (Table 1, Figure 1 ) were stable and significantly fractionated in the photosynthetic tissue organs (leaves - litter), non-photosynthetic tissue organs (branches - rhizomes), and soil profiles (soil organic matter) of agricultural and forest plants. Among them, the lignin methoxy δ 13 C LM and δ 2 H LM values of the photosynthetic tissue, new leaves, and litter were more depleted, while the corresponding values in the soil layer were more enriched. The δ 13 C LM and δ 2 H LM values of the litter could both be used as the characterization media for the lignin sources of photosynthetic leaves and the soil surface layer. In particular, the δ 2 H LM value of the litter had a more significant response relationship with the photosynthetic leaves and the surface soil ( R 2 = 0.58 and 0.63, P <0.05), and at the same time, the δ 13 C LM and δ2 H LM values can be used as a characterization medium for the lignin sources of non - photosynthetic branches and deep - soil lignin. The δ 13 C LM and δ 2 H LM values have a more significant response relationship with deep - layer soil ( R 2 = 0.78 and 0.88, P <0.01).

[0065] Table 1: Lignin methoxy δ 13 C LM and δ 2 H LM values

[0066]

[0067] Table 2: Comparison between the simulated and measured values of lignin methoxy δ 2 H LM values of related tissue organs of Malus spectabilis in agro - forestry plants

[0068]

[0069] After Figure 1 confirmation by comparing the optimal regression equation models, the regression equation of δ 2 H LM values among photosynthetic leaves, litter, and the 0 - 10 cm soil profile is used as a prediction model. From the measured and simulated prediction results of Malus spectabilis, a plant of the same genus (Table 2), it can be seen that there is no significant difference between the predicted and measured δ 2 H LM values ( P ≥ 0.05), indicating that the lignin source of organic matter in the 0 - 10 cm soil profile comes from photosynthetic leaves through litter as a medium.

[0070] Example 2

[0071] This example provides a method for characterizing the lignin source in a natural degradation system, specifically for characterizing the lignin source involved in the natural biodegradation process of shrub litter, pruning materials, etc. in a garden ecosystem. It is divided into the response relationships of lignin - like substances methoxy δ 13 C LM and δ 2 H LM values in photosynthetic tissue organs (leaves - litter - organic matter) and non - photosynthetic tissue organs (branches - rhizomes - organic matter) to characterize the lignin source in the natural degradation system.

[0072] The large - leaf privet ( Ligustrum compactum)Evergreen broad-leaved tree species, their native mature leaves, current-season litter, branch stems, forest floor profile soil (0-10 cm, 10-20 cm), and rhizomes were collected respectively. These materials were dried to a constant weight at 105 °C, crushed and ground into fine powder samples with a particle size of 80 μm for standby.

[0073] For fine powder samples of photosynthetic products such as mature leaves and current-season litter rich in cutin, an aqueous sodium hydroxide solution (NaOH, 1 M) was used as the first extraction system for chemical screening of non-lignin substances. The solution and the fine powder were mixed at a volume-mass ratio (mL / mg) of 1:5, stirred and dissolved at 90 °C for 4 h under sealed conditions to remove cellulose substances, then quickly quenched at 0 °C and allowed to stand for 30 min. The lower-layer turbid precipitate was taken as the first extract. Nicotine liquid with left and right optical rotation characteristics and miscible in medium-acidic conditions was used as the second extraction system for lignin material enrichment. The nicotine liquid of the second extraction system was mixed with the first extract at a volume-mass ratio of 2:1 (mL / mg), stirred and reacted at room temperature (22±1 °C) under sealed conditions for 20-30 min, allowed to stand and precipitate for 15-30 min to remove tannic acid substances containing lignin phenolic methoxy groups, and the upper-layer solution was filtered and taken as the second extract. Then, an equimolar mixture of zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥98%) and ionic liquid 1-ethyl-3-methylimidazolium chloride ferric chloride ([Emim]FeCl4) was used as the second biphasic enrichment system for lignin material enrichment. They were mixed at a mass ratio (mg / mg) of 1:1 and adjusted to neutrality, melted and reacted at 80 °C for 10 min under sealed conditions to remove pectin substances, then quickly quenched at 0 °C, extracted with ethyl acetate at a volume-mass ratio (mL / mg) of 2:1, and lignin-like substances in photosynthetic tissue organs were obtained after rotary evaporation under reduced pressure.

[0074] For non - photosynthetic product fine powder samples such as branches and rhizomes, an equimolar mixture of pseudo - ionic liquid zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥ 98%) and ionic liquid 1 - ethyl - 3 - methylimidazolium chloride ferrate ([Emim]FeCl4) is used as the extraction system for lignin material enrichment. Medium - acidic (pH≈4) melting treatment is carried out according to a mass ratio (mg / mg) of 4:1 (sealed microwave reaction at 80 °C for 10 min). 37% fuming hydrochloric acid (HCl) deionized water aqueous solution is used as the neutralization reagent. The neutralization reagent and the first extract are mixed according to a volume - mass ratio of 100 - 150:1 (μL / mg), and left to stand overnight (8 - 10 h) at room temperature (22 ± 1 °C) to adjust the pH to near neutral (6.5 - 7.5). Then, ethyl acetate extraction is carried out according to a volume - mass ratio (mL / mg) of 2:1, and lignin - like substances produced by non - photosynthetic tissue organs are enriched by rotary evaporation under reduced pressure. For non - photosynthetic tissue organs such as stems with a lignin content of 20% - 30%, their fine powder can be directly used for further determination of lignin methoxy δ 13 C LM 、δ 2 H LM value determination.

[0075] For fine powder samples of soil profile organic matter with medium humus content, an equimolar mixture of pseudo - ionic liquid ammonium ferrous sulfate hexahydrate ((NH4)2Fe(SO4)2·6H2O, purity ≥ 99%) and ionic liquid 1 - ethyl - 3 - methylimidazolium chloride ferrate ([Emim]FeCl4) is used as the enrichment extraction system for lignin materials. It is mixed according to a mass ratio (mg / mg) of 10:1 with garden soil, and an equal mass of copper oxide is added as a derivatizing agent. Microwave reaction is carried out at 120 °C for 70 min under sealed conditions at weak alkaline pH = 8.5, and then rapidly quenched at 0 °C. Ethyl acetate extraction is carried out according to a volume - mass ratio (mL / mg) of 2:1, and lignin phenol derivatives are obtained by rotary evaporation under reduced pressure.

[0076] The determination method is the same as that in Example 1.

[0077] The calibrator is the same as that in Example 1.

[0078] The CH3I obtained by online GC separation is further used for IRMS determination. The determination of the corresponding lignin methoxy δ 13 C LM value adopts a high - temperature oxidation mode. The reaction furnace temperature is set at 960 °C, calibrated high - purity carbon dioxide is used as the reference gas, and the calibrated δ 13 C LM values (-35.0‰ and -29.7‰) of logwood are used as the double - point calibration standard; for the corresponding lignin methoxy δ 2 H LMThe value measurement adopts the high-temperature pyrolysis mode, the reaction furnace temperature is 1250 °C, high-purity hydrogen is used as the reference gas, and the H3 value is 4.3 ppm / nA under the self-check state. + The value is taken as the two-point calibration standard with the calibrated log δH values (-307.5‰ and -210.1‰). The relative standard deviations of the lignin methoxy δC and δH values of each provenance sample are less than 0.4‰ and 0.9‰ respectively. 2 H LM After 5 groups of repeated sample measurements, it is obtained that the lignin methoxy δC and δH values and the response relationship characteristics (Table 3) of the photosynthetic tissue organs (leaves - litter) and non-photosynthetic tissue organs (branches - stems - rhizomes) of the garden plant Ligustrum lucidum and the soil profile (soil organic matter) are all stable in tracing and have significant fractionation differences. 13 C LM and δ 2 H LM Among them, the lignin methoxy δC and δH values of the newly emerged leaves and litter of the photosynthetic tissues are more depleted. The δC and δH values of the litter can be used as the characterization media for the lignin provenance of photosynthetic leaves and the soil surface layer, while the δC and δH values of the rhizomes can also be used as the characterization media for the lignin provenance of non-photosynthetic branches and the deep soil layer. Especially, the response relationship between the δC and δH values of the rhizomes and the deep soil is more significant (r = 0.85 and 0.95, p < 0.001).

[0079] Figure 2 13 C LM and δ 2 H LM Table 3: Lignin methoxy δC and δH values of related tissue organs of the garden plant Ligustrum lucidum Figure 2 Among them, the lignin methoxy δC and δH values of the newly emerged leaves and litter of the photosynthetic tissues are more depleted. The δC and δH values of the litter can be used as the characterization media for the lignin provenance of photosynthetic leaves and the soil surface layer, while the δC and δH values of the rhizomes can also be used as the characterization media for the lignin provenance of non-photosynthetic branches and the deep soil layer. Especially, the response relationship between the δC and δH values of the rhizomes and the deep soil is more significant ( 13 C LM and δ 2 H LM The δC and δH values of the litter are more depleted. The δC and δH values of the litter can be used as the characterization media for the lignin provenance of photosynthetic leaves and the soil surface layer, while the δC and δH values of the rhizomes can also be used as the characterization media for the lignin provenance of non-photosynthetic branches and the deep soil layer. Especially, the response relationship between the δC and δH values of the rhizomes and the deep soil is more significant ( 13 C LM and δ 2 H LM The δC and δH values of the rhizomes can be used as the characterization media for the lignin provenance of non-photosynthetic branches and the deep soil layer. Especially, the response relationship between the δC and δH values of the rhizomes and the deep soil is more significant ( 13 C LM and δ 2 H LM The δC and δH values of the rhizomes can be used as the characterization media for the lignin provenance of non-photosynthetic branches and the deep soil layer. Especially, the response relationship between the δC and δH values of the rhizomes and the deep soil is more significant ( 13 C LM and δ 2 H LM The δC and δH values of the rhizomes are more significantly related to the deep soil ( R 2 r = 0.85 and 0.95, P p < 0.001).

[0080] Table 3: Lignin methoxy δC and δH values of related tissue organs of the garden plant Ligustrum lucidum 13 C LM and δ 2 H LM values

[0081]

[0082] Table 4: Lignin methoxy δC and δH values of related tissue organs of the garden plant Ligustrum lucidum13 C LM and δ 2 H LM Comparison between simulated and measured values of

[0083]

[0084] After Figure 2 confirmation by comparison with the optimal regression equation model, the regression equation of δ 2 H LM values between photosynthetic leaves, litter and 0-10 cm soil profile can be used as a prediction model. At the same time, the regression equation of δ 13 C LM values between non-photosynthetic branches, rhizomes and 10-20 cm soil profile can also be used as a prediction model. From the measured and simulated prediction results of the same plant in different regions (Table 4), it can be seen that the predicted 2 H LM values and the measured values of the corresponding related tissues have no significant difference, indicating that the lignin source of organic matter in the 0-10 cm soil profile is derived from photosynthetic leaves through litter as a medium; the predicted 13 C LM values and the measured values of the corresponding related tissues have no significant difference ( P ≥0.05), indicating that the lignin source of organic matter in the 10-20 cm soil profile is mediated by rhizomes and non-photosynthetic branches as the source.

[0085] Example 3

[0086] This example provides a method for characterizing the lignin source of a natural degradation system, specifically for characterizing the lignin source of one or several types of fallen logs, litter, etc. of deciduous broad-leaved, evergreen coniferous, and deciduous coniferous forests in a forest ecosystem participating in the natural biodegradation process. It is divided into the methoxy δ 13 C LM and δ 2 H LM value response relationship to characterize the lignin source of the natural degradation system.

[0087] Quercus aliena var. acuteserrata ( Quercus aliena ) is a deciduous broad-leaved tree species. Its native mature leaves, current-season litter, branch stems, fallen trunks, understory profile soil (0-10 cm, 10-20 cm), and rhizomes are collected respectively. After drying to a constant weight at 105 °C and crushing and grinding to a fine powder sample with a particle size of 80 μm for standby.

[0088] For photosynthetic product fine powder samples such as mature leaves rich in cutin and seasonal litter, an aqueous sodium hydroxide solution (NaOH, 1 M) is used as the first extraction system for chemical screening of non-lignin substances. The solution and the fine powder are mixed at a volume-mass ratio (mL / mg) of 1:5, and stirred and dissolved at 90 °C for 4 h under closed conditions to remove cellulose substances. Then, it is quickly quenched at 0 °C and left standing for 30 min, and the lower-layer turbid precipitate is taken as the first extract. Nicotine liquid with left and right optical rotation characteristics and miscible in medium acidic conditions is used as the second extraction system for lignin material enrichment. The nicotine liquid of the second extraction system is mixed with the first extract at a volume-mass ratio of 2:1 (mL / mg), stirred and reacted at room temperature (22 ± 1 °C) under sealed conditions for 20 - 30 min, left standing and precipitated for 15 - 30 min to remove tannic acid substances containing lignin phenolic methoxy groups, and the upper-layer solution is taken as the second extract after filtration. Then, ethyl acetate extraction is carried out at a volume-mass ratio (mL / mg) of 2:1, and lignin substances produced by photosynthetic tissue organs are obtained after rotary evaporation under reduced pressure.

[0089] For non-photosynthetic product fine powder samples such as branches and rhizomes, an equimolar mixture of ionic liquid-like zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥ 98%) and ionic liquid 1-ethyl-3-methylimidazolium chloride ferrate ([Emim]FeCl4) is used as the extraction system for lignin material enrichment. Medium acidic (pH ≈ 4) melting treatment (sealed microwave reaction at 80 °C for 10 min) is carried out at a mass ratio (mg / mg) of 4:1. 37% fuming hydrochloric acid (HCl) deionized water aqueous solution is used as the neutralization reagent. The neutralization reagent and the first extract are mixed at a volume-mass ratio of 100 - 150:1 (μL / mg), left standing overnight (8 - 10 h) at room temperature (22 ± 1 °C) to adjust the pH to near neutral (6.5 - 7.5), and then ethyl acetate extraction is carried out at a volume-mass ratio (mL / mg) of 2:1, and lignin substances produced by non-photosynthetic tissue organs are enriched by rotary evaporation under reduced pressure. For non-photosynthetic tissue organs such as fallen trunks and stems with a lignin content of 20% - 30%, their fine powder can be directly used for further determination of the δ 13 C LM 、δ 2 H LM value.

[0090] For the organic matter fine powder sample of the humus-rich soil profile, an equimolar mixture of the pseudo-ionic liquid ammonium ferrous sulfate hexahydrate (NH4)2Fe(SO4)2·6H2O, purity ≥ 99%) and the ionic liquid 1-ethyl-3-methylimidazolium iron(III) chloride ([Emim]FeCl4) is used as the enrichment extraction system for lignin materials. It is mixed according to the mass ratio (mg / mg) of 3:1 with forest soil, and then an equal mass of copper oxide is added as a derivatizing agent. It is subjected to microwave reaction at 120 °C for 70 min under sealed conditions at a weak alkaline pH of 8.5, and then rapidly quenched at 0 °C. Ethyl acetate extraction is carried out at a volume-mass ratio (mL / mg) of 2:1, and lignin phenolic derivatives are obtained by vacuum rotary evaporation enrichment.

[0091] The measurement method is the same as that in Example 1.

[0092] The calibrator is the same as that in Example 1.

[0093] The CH3I obtained by online GC separation is further used for IRMS measurement, corresponding to the lignin methoxy δ 13 C LM value measurement uses the high-temperature oxidation mode, sets the reaction furnace temperature at 960 °C, uses calibrated high-purity carbon dioxide as the reference gas, and uses the calibrated δ 13 C LM values (-35.0‰ and -29.7‰) of the log as the two-point calibration standard; for the measurement of the lignin methoxy δ 2 H LM value measurement uses the high-temperature pyrolysis mode, the reaction furnace temperature is 1250 °C, high-purity hydrogen is used as the reference gas, and the H3 + value is 4.3 ppm / nA under self-checking status. The calibrated δ 2 H LM values (-307.5‰ and -210.1‰) of the log are used as the two-point calibration standard. The relative standard deviations of the lignin methoxy δ 13 C LM and δ 2 H LM values of each source sample are less than 0.4‰ and 0.9‰, respectively.

[0094] After 5 groups of repeated sample measurements, it is found that the lignin methoxy δ 13 C LM and δ 2 H LM values and the response relationship characteristics (Table 5, Figure 3 ) are stable in tracing and significantly fractionated in the photosynthetic tissue organs (leaves - litter) and non-photosynthetic tissue organs (branches - stems - rhizomes) of the typical deciduous broad-leaved tree Quercus aliena var. acuteserrata, as well as in the soil profile (soil organic matter). Among them, the lignin methoxy δ 13 C LM and δ2 H LM values are more depleted, and the lignin methoxyl δ 13 C LM and δ 2 H LM values are more enriched. The δ 13 C LM and δ 2 H LM values of both can be used as the characterization media for the sources of photosynthetic leaves and soil surface lignin. In particular, the δ 2 H LM value of litter has a more significant source response relationship with photosynthetic leaves ( R 2 = 0.91, P <0.001). The δ 13 C LM and δ 2 H LM values of rhizomes can also be used as the characterization media for the sources of non-photosynthetic branches and soil deep lignin. In particular, the δ 2 H LM value of rhizomes has a more significant response relationship with non-photosynthetic branches ( R 2 = 0.77, P <0.01).

[0095] Table 5: Lignin methoxyl δ 13 C LM and δ 2 H LM values

[0096]

[0097] Table 6: Comparison between the simulated and measured values of lignin methoxyl δ 13 C LM and δ 2 H LM values of related tissues and organs of the forest plant Quercus aliena var. acuteserrata

[0098]

[0099] After Figure 3 confirmation by comparing the optimal regression equation models, the δ 13 C LM value regression equation between photosynthetic leaves, litter, and the 0 - 10 cm soil profile can be used as a prediction model. At the same time, the δ 2 H LM value regression equation between non-photosynthetic branches, rhizomes, and the 10 - 20 cm soil profile can also be used as a prediction model. From the measured and simulated prediction results of the same plant in different regions (Table 6), the δ of the corresponding related tissues13 C LM There was no significant difference between the predicted value and the measured value, indicating that the lignin source of soil organic matter in the 0-10 cm soil profile was derived from photosynthetic leaves through the medium of litter; the δ 2 H LM There was no significant difference between the predicted value and the measured value ( P ≥0.05), indicating that the lignin source of soil organic matter in the 10-20 cm soil profile was mediated by rhizomes and non-photosynthetic branches.

[0100] Example 4

[0101] This example provides a method for characterizing the lignin source of a natural degradation system, specifically for characterizing the lignin source of fallen logs, litter, etc. of one or more of deciduous broad-leaved, evergreen coniferous, and deciduous coniferous trees in a forest ecosystem participating in the natural biodegradation process. It is divided into the methoxy δ 13 C LM and δ 2 H LM value response relationship to characterize the lignin source of the natural degradation system.

[0102] Abies fargesii Abies fargesii is an evergreen coniferous tree species. Its native mature leaves, stem branches, fallen trunks, soil in the forest understory profile (0-10 cm, 10-20 cm), and rhizomes were collected respectively, dried to a constant weight at 105 °C, and ground into fine powder samples with a particle size of 80 μm for standby.

[0103] For photosynthetic product fine powder samples such as mature leaves rich in cutin, an aqueous sodium hydroxide solution (NaOH, 1 M) is used as the first extraction system for chemical screening of non-lignin substances. The solution and the fine powder are mixed at a volume-mass ratio (mL / mg) of 1:5, and stirred and dissolved at 90 °C for 4 h under sealed conditions to remove cellulose substances. Then, it is rapidly quenched at 0 °C and allowed to stand for 30 min, and the lower-layer turbid precipitate is taken as the first extract. Nicotine liquid with left and right circular polarization characteristics and miscible in medium acidic conditions is used as the second extraction system for lignin material enrichment. The nicotine liquid of the second extraction system is mixed with the first extract at a volume-mass ratio of 2:1 (mL / mg), and stirred and reacted at room temperature (22 ± 1 °C) under sealed conditions for 20 - 30 min, and allowed to stand and precipitate for 15 - 30 min to remove tannic acid substances containing lignin phenolic methoxy groups. The upper-layer solution is taken as the second extract by filtration. Then, an equimolar mixture of ionic liquid-like zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥ 98%) and ionic liquid 1-ethyl-3-methylimidazolium ferric chloride ([Emim]FeCl4) is used as the second biphasic enrichment system for lignin material enrichment. They are mixed at a mass ratio (mg / mg) of 1:1 and adjusted to neutrality, and melted and reacted at 80 °C for 10 min under sealed conditions to remove pectin substances. Then, it is rapidly quenched at 0 °C, extracted with ethyl acetate at a volume-mass ratio (mL / mg) of 2:1, and rotary evaporated under reduced pressure to obtain lignin-like substances produced by photosynthetic tissue organs.

[0104] For non-photosynthetic product fine powder samples such as branches and rhizomes, an equimolar mixture of ionic liquid-like zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥ 98%) and ionic liquid 1-ethyl-3-methylimidazolium ferric chloride ([Emim]FeCl4) is used as the extraction system for lignin material enrichment. Medium acidic (pH ≈ 4) melting treatment (sealed microwave reaction at 80 °C for 10 min) is carried out at a mass ratio (mg / mg) of 4:1. A 37% fuming hydrochloric acid (HCl) deionized water aqueous solution is used as the neutralizing reagent. The neutralizing reagent and the first extract are mixed at a volume-mass ratio of 100 - 150:1 (μL / mg), and allowed to stand overnight (8 - 10 h) at room temperature (22 ± 1 °C) to adjust the pH to near neutrality (6.5 - 7.5). Then, extraction with ethyl acetate is carried out at a volume-mass ratio (mL / mg) of 2:1, and rotary evaporation under reduced pressure is carried out to enrich lignin-like substances produced by non-photosynthetic tissue organs. For non-photosynthetic tissue organs such as fallen trunks and stems with a lignin content of 20% - 30%, their fine powder can be directly used for further determination of the δ 13 C LM 、δ 2 H LM value.

[0105] For the fine powder sample of organic matter in the humus-rich soil profile, an equimolar mixture of ionic liquid-like ammonium ferrous sulfate hexahydrate (NH4)2Fe(SO4)2·6H2O, purity ≥ 99%) and ionic liquid 1-ethyl-3-methylimidazolium iron chloride ([Emim]FeCl4) is used as the enrichment extraction system for lignin materials. It is mixed according to the mass ratio (mg / mg) of 3:1 with forest soil, and then an equal mass of copper oxide is added as a derivatizing agent. It is subjected to microwave reaction at 120 °C for 70 min under sealed conditions at a weak alkaline pH = 8.5, and then rapidly quenched at 0 °C. Ethyl acetate extraction is carried out at a volume-mass ratio (mL / mg) of 2:1, and lignin phenolic derivatives are obtained by rotary evaporation under reduced pressure.

[0106] The measurement method is the same as that in Example 1.

[0107] The calibrator is the same as that in Example 1.

[0108] The CH3I obtained by online separation by GC is further used for IRMS measurement, corresponding to the lignin methoxy δ 13 C LM value measurement adopts the high-temperature oxidation mode, sets the reaction furnace temperature at 960 °C, uses calibrated high-purity carbon dioxide as the reference gas, and uses the calibrated log δ 13 C LM values (-35.0‰ and -29.7‰) as the two-point calibration standard; for the measurement of the lignin methoxy δ 2 H LM value, the high-temperature pyrolysis mode is adopted, the reaction furnace temperature is 1250 °C, high-purity hydrogen is used as the reference gas, and the H3 + value under self-check state is 4.3 ppm / nA, and the calibrated log δ 2 H LM values (-307.5‰ and -210.1‰) are used as the two-point calibration standard. The relative standard deviations of the lignin methoxy δ 13 C LM and δ 2 H LM values of each source sample are less than 0.5‰ and 0.9‰ respectively.

[0109] Table 7: Lignin methoxy δ 13 C LM and δ 2 H LM values of relevant tissues and organs of the forest plant Abies fargesii

[0110]

[0111] After 5 groups of repeated measurements, stable and significantly fractionated lignin methoxy δ values and response relationship characteristics were obtained for the photosynthetic tissue organs (leaves - litter), non - photosynthetic tissue organs (branches - stems - rhizomes), and soil profiles (soil organic matter) of the typical deciduous broad - leaved tree Abies fargesii. 13 C LM and δ 2 H LM values, as well as the response relationship characteristics (Table 7, Figure 4 ). Among them, the lignin methoxy δ 13 C LM and δ 2 H LM values of the newly - grown leaves and litter in the photosynthetic tissues are more depleted, while the lignin methoxy δ 13 C LM and δ 2 H LM values in the soil layer are more enriched. The δ 13 C LM and δ 2 H LM values of the litter can be used as the characterization medium for the lignin sources of photosynthetic leaves and the soil surface layer. In particular, the response relationship between the δ 2 H LM of the litter and the lignin sources of photosynthetic leaves and the surface soil is more significant ( R 2 = 0.89 and 0.91, P <0.001). The δ 13 C LM and δ 2 H LM values of the rhizome can also be used as the characterization medium for the lignin sources of non - photosynthetic branches and the deep soil layer. In particular, the response relationship between the δ 2 H LM value of the rhizome and the non - photosynthetic branches is more significant ( R 2 = 0.83, P <0.001).

[0112] Example 5

[0113] This example provides a method for characterizing the lignin sources in a natural degradation system, specifically for characterizing the lignin sources involved in the natural biodegradation process of fallen logs, litter, etc. of one or several of deciduous broad - leaved, evergreen coniferous, and deciduous coniferous in a forest ecosystem. It is divided into the response relationships of lignin - like substances methoxy δ 13 C LM and δ 2 H LM values in the photosynthetic tissue organs (leaves - litter - organic matter) and non - photosynthetic tissue organs (branches - rhizomes - organic matter) to characterize the lignin sources in the natural degradation system.

[0114] Larix chinensis Beissn. Larch chinensis is an evergreen coniferous tree species. Its native mature leaves, current-season litter, stem branches, fallen trunks, forest floor profile soil (0 - 10 cm, 10 - 20 cm), and rhizomes were collected respectively. After being dried to a constant weight at 105 °C and crushed and ground into fine powder samples with a particle size of 80 μm, they were reserved for use.

[0115] For fine powder samples of photosynthetic products such as mature leaves and current-season litter rich in cutin, an aqueous sodium hydroxide solution (NaOH, 1 M) was used as the first extraction system for chemical screening of non-lignin substances. The solution and the fine powder were mixed according to the volume-mass ratio (mL / mg) of 1:5, and stirred and dissolved at 90 °C for 4 h under sealed conditions to remove cellulose substances. Then, it was quickly quenched at 0 °C and left standing for 30 min, and the lower layer of turbid precipitate was taken as the first extract; nicotine liquid with left and right optical rotation characteristics and miscible in medium acidic conditions was used as the second extraction system for lignin material enrichment. The nicotine liquid of the second extraction system was mixed with the first extract according to the volume-mass ratio of 2:1 (mL / mg), stirred and reacted at room temperature (22 ± 1 °C) under sealed conditions for 20 - 30 min, left standing and precipitating for 15 - 30 min to remove tannic acid substances containing lignin phenolic methoxy groups, and the upper layer solution was filtered and taken as the second extract; then, an equimolar mixture of zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥98%) and ionic liquid 1-ethyl-3-methylimidazolium chloride ferric chloride ([Emim]FeCl4) was used as the second two-phase enrichment system for lignin material enrichment. They were mixed according to the mass ratio (mg / mg) of 1:1 and adjusted to neutral, and melted and reacted at 80 °C for 10 min under sealed conditions to remove pectin substances. Then, it was quickly quenched at 0 °C, extracted with ethyl acetate according to the volume-mass ratio (mL / mg) of 2:1, and rotary evaporated under reduced pressure to obtain lignin-like substances produced by photosynthetic tissue organs.

[0116] For non - photosynthetic product fine powder samples such as branches and rhizomes, an equimolar mixture of pseudo - ionic liquid zinc chloride tetrahydrate (ZnCl2·4H2O, purity ≥ 98%) and ionic liquid 1 - ethyl - 3 - methylimidazolium ferric chloride ([Emim]FeCl4) is used as the extraction system for lignin material enrichment. Medium - acidic (pH≈4) melting treatment is carried out according to a mass ratio (mg / mg) of 4:1 (sealed microwave reaction at 80 °C for 10 min). 37% fuming hydrochloric acid (HCl) deionized water aqueous solution is used as the neutralization reagent. The neutralization reagent and the first extract are mixed according to a volume - mass ratio of 100 - 150:1 (μL / mg), and left standing overnight (8 - 10 h) at room temperature (22±1 °C) to adjust the pH to near - neutral (6.5 - 7.5). Then, extraction with ethyl acetate is carried out according to a volume - mass ratio (mL / mg) of 2:1, and lignin - like substances produced by non - photosynthetic tissue organs are enriched by rotary evaporation under reduced pressure. For non - photosynthetic tissue organs such as fallen tree trunks and stems with a lignin content of 20% - 30%, their fine powder can be directly used for further determination of lignin methoxy δ 13 C LM 、δ 2 H LM value determination.

[0117] For fine powder samples of soil profile organic matter rich in humus, an equimolar mixture of pseudo - ionic liquid ammonium ferrous sulfate hexahydrate ((NH4)2Fe(SO4)2·6H2O, purity ≥ 99%) and ionic liquid 1 - ethyl - 3 - methylimidazolium ferric chloride ([Emim]FeCl4) is used as the enrichment extraction system for lignin materials. It is mixed according to a mass ratio (mg / mg) of 3:1 with forest soil, and an equal mass of copper oxide is added as a derivatizing agent. Microwave reaction is carried out at 120 °C for 70 min under sealed conditions at weak alkaline pH = 8.5, and then rapidly quenched at 0 °C. Extraction with ethyl acetate is carried out according to a volume - mass ratio (mL / mg) of 2:1, and lignin phenolic derivatives are obtained by rotary evaporation under reduced pressure.

[0118] The determination method is the same as that in Example 1.

[0119] The calibrator is the same as that in Example 1.

[0120] The CH3I obtained by online separation through GC is further used for IRMS determination. For the determination of lignin methoxy δ 13 C LM value, a high - temperature oxidation mode is adopted. The reaction furnace temperature is set at 960 °C, calibrated high - purity carbon dioxide is used as the reference gas, and the calibrated δ 13 C LM values (- 35.0‰ and - 29.7‰) of logwood are used as the double - point calibration standard; for the corresponding lignin methoxy δ 2 H LMThe value determination adopts the high-temperature pyrolysis mode, the reaction furnace temperature is 1250 °C, high-purity hydrogen is used as the reference gas, and the H3 value in the self-check state is 4.3 ppm / nA. The calibrated log δH values (-307.5‰ and -210.1‰) are used as the double-point calibration standard. The relative standard deviations of the lignin methoxy δC and δH values of each provenance sample are less than 0.5‰ and 0.9‰ respectively. + values were used as the double-point calibration standard. The relative standard deviations of the lignin methoxy δC 2 H LM values (-307.5‰ and -210.1‰) of the calibrated log were used as the double-point calibration standard. The relative standard deviations of the lignin methoxy δC 13 C LM and δ 2 H LM values of each provenance sample are less than 0.5‰ and 0.9‰ respectively.

[0121] Table 8: Lignin methoxy δC 13 C LM and δ 2 H LM values

[0122]

[0123] After 5 groups of repeated measurements, it was found that the photosynthetic tissue organs (leaves - litter), non-photosynthetic tissue organs (branches - stems - rhizomes), and soil profiles (soil organic matter) of the typical deciduous broad-leaved tree species Quercus aliena var. acuteserrata all had stable lignin methoxy δC 13 C LM and δ 2 H LM values and response relationship characteristics (Table 8, Figure 5 ). Among them, the lignin methoxy δC 13 C LM and δ 2 H LM values of the newly emerged leaves and litter of the photosynthetic tissues are more depleted, the lignin methoxy δC 13 C LM and δ 2 H LM values of the soil layer are more enriched, and the δC 13 C LM and δ 2 H LM values of the litter can be used as the characterization medium for the lignin provenance of photosynthetic leaves and the soil surface layer. Especially, the δC 13 C LM of the litter has a more significant source response relationship with the photosynthetic leaves and the surface soil ( R 2 = 0.74, P < 0.05 and R 2 = 0.82, P < 0.02), and the δC 13 C LM and δ2 H LM values can also be used as a characterization medium for non - photosynthetic branches and deep - soil lignin sources, especially the δ 13 C LM and δ 2 H LM values have a more significant response relationship with shallow and deep - layer soils ( R 2 = 0.86 and 90, P <0.001).

[0124] Comparative Example 1

[0125] This comparative example provides a method for characterizing the lignin source of photosynthetic tissues in a natural degradation system.

[0126] For the characterization of lignin sources in photosynthetic tissues such as newly - formed leaves and current - season litter, in the lignin enrichment and pretreatment stage, considering that impurities such as pectin and tannin in lignin phenolic methoxy groups may cause interference, the extraction and purification system is the same as in Examples 1 - 5, except that an aqueous sodium hydroxide solution is used as the first screening system for non - lignin substances; nicotine solution with left - and right - handed optical rotation characteristics is used as the second removal system for substances containing lignin phenolic methoxy tannin; an equal - mixture of ionic liquid and ionic - liquid - like substances is used as the second biphasic removal system for pectin - like substances. Finally, the standard deviation of the δ 13 C LM and δ 2 H LM values is used to compare the sources of lignin - purified substances and original substances.

[0127] Taking plants with leaves rich in cutin pectin and tannin in garden applications as an example, according to the above purification method, the lignin substances are purified and the sources are compared with the original ones. Weigh 10.0 ± 0.5 mg of photosynthetic - tissue lignin substances after purification pretreatment and directly - powdered ones respectively for the determination of δ 13 C LM and δ 2 H LM values. The measurement method and calibrator are the same as in Example 1. After 15 groups of repeated measurements, the δ 13 C LM and δ 2 H LM values and standard deviations ( Figure 6 ) are obtained. The results show that for the lignin δ 13 C LM and δ 2 H LMThe standard deviations of the values are all 0.4, much lower than the corresponding standard deviations of the original sources (1.9 and 1.7).

[0128] Comparative Example 2

[0129] This comparative example provides a method for characterizing the lignin source of non - photosynthetic tissues in a natural degradation system.

[0130] For the characterization of lignin from non - photosynthetic tissue sources such as branches and rhizomes in this comparative example, in the lignin enrichment pretreatment stage, considering the interference of substances such as pectin in branches and fine roots with relatively low lignification degree, the extraction and purification system is the same as that in Examples 1 - 5, except that a biphasic mixed solution of ionic liquid and ionic liquid - like is used as the pectin - like substance removal system. For the stem with a lignin content of 20% - 30%, its fine powder can be directly used for the determination of the δ 13 C LM and δ 2 H LM values. Finally, the source comparison between the purified lignin and the original substance is carried out using the standard deviations of the δ 13 C LM and δ 2 H LM values.

[0131] Taking the branches of gymnosperms in forest ecosystems containing only meta - methoxy, i.e., G - type lignin monomers, and the rhizomes of broad - leaf plants in agro - forestry ecosystems containing both meta - and para - methoxy, i.e., G - type and S - type lignin monomers, as examples, the purification of lignin substances and the source comparison with the original are carried out according to the above purification method. Weigh 10.0 ± 0.5 mg of lignin substances from non - photosynthetic tissues after purification pretreatment and direct fine powder respectively for the determination of the δ 13 C LM and δ 2 H LM values. The measurement method and calibration substance are the same as in Example 1. After 15 groups of repeated measurements, the δ 13 C LM and δ 2 H LM values and standard deviations ( Figure 7 ) of the lignin from non - photosynthetic tissues of gymnosperms and broad - leaf plants after impurity removal pretreatment are obtained. The results show that the standard deviations of the δ 13 C LM and δ 2 H LM values of the lignin from non - photosynthetic tissues of gymnosperms and broad - leaf plants are lower than 0.4 and 0.7 respectively, much lower than the corresponding standard deviations of the original sources (2.1 and 3.4). Although the lignin of gymnosperms only contains G - type lignin with meta - methoxy donors, there are obvious δ values when measured directly without pretreatment13 C LM and δ 2 H LM value deviation.

[0132] Comparative Example 3

[0133] This comparative example provides an enrichment pretreatment method for lignin sources in the humus soil layer of a natural degradation system.

[0134] For the enrichment pretreatment of lignin sources in the humus soil profile in this comparative example, in the lignin-derived purification treatment stage, considering the differences in humus and soil types corresponding to different ecosystems under the forest, the derived purification system is the same as in Examples 1-5. The difference is that for different soil types, a biphasic mixed solution system of ionic liquid and ionic liquid-like with different mass ratios is selected. Based on the principle of maximum intensive utilization of materials, the equimolar mixing extraction system of ionic liquid and ionic liquid-like is mixed and derivatively purified with forest soil, garden soil, and agricultural and forestry soil at different mass ratios to correspond to the methoxy δ 13 C LM and δ 2 H LM values and standard deviation as the investigation indexes. Among them, for forest soil rich in humus, the standard deviations of the methoxy δ 13 C LM and δ 2 H LM values of the lignin source were investigated for the mass ratios of the biphasic extraction system to forest soil of 1:1, 3:1, 5:1, 7:1, and 9:1 respectively. For garden soil with the humus layer disturbed by garden horticultural maintenance, the standard deviations of the methoxy δ 13 C LM and δ 2 H LM values of the lignin source were investigated for the mass ratios of the biphasic extraction system to garden soil of 3:1, 5:1, 10:1, 15:1, and 20:1 respectively. For agricultural and forestry soil affected by the periodic production of agricultural and forestry fruit industries, the standard deviations of the methoxy δ 13 C LM and δ 2 H LM values of the lignin source were investigated for the mass ratios of the biphasic extraction system to agricultural and forestry soil of 5:1, 10:1, 15:1, 20:1, and 25:1 respectively.

[0135] Table 9: Comparison of the measured methoxy δ 13 C LM and δ 2 H LM values for lignin-derived purification of different types of humus soil

[0136]

[0137] After 7 groups of repeated measurements, the lignin methoxy δ of different soil types was obtained respectively 13 C LM and δ 2 H LM values and standard deviations (Table 9). It can be seen from the results that for forest soil, when the mass ratio of the biphasic extraction system is 3:1, the corresponding lignin methoxy δ 13 C LM and δ 2 H LM values are closest to the standard treatment results and the standard deviation is the smallest, indicating that the lignin-like substances in the humus soil source of the natural degradation system in the forest ecosystem are optimally derivatized and purified, the interference of the non-lignin methoxy source donors is the smallest, and the extraction system is the most economical; for garden soil, when the mass ratio of the biphasic extraction system is 10:1, the corresponding lignin methoxy δ 13 C LM and δ 2 H LM values are closest to the standard treatment results and the standard deviation is the smallest, indicating that the lignin-like substances in the humus soil source of the garden maintenance understory are optimally derivatized and purified, and the interference of the non-lignin methoxy source donors and exogenous substances is the smallest; for agricultural and forestry soil, when the mass ratio of the biphasic extraction system is 15:1, the corresponding lignin methoxy δ 13 C LM and δ 2 H LM values are closest to the standard treatment results and the standard deviation is the smallest, indicating that the lignin-like substances in the humus soil source formed by the litter in agricultural and forestry production are maximally derivatized and purified, and the interference of non-lignin methoxy source donors such as pectin and tannic acid is the smallest and the extraction system is the most economical.

[0138] Comparative Example 4

[0139] This comparative example provides a method for characterizing the lignin source of the humus soil layer in the natural degradation system.

[0140] For the characterization of the lignin source of the humus soil profile in this comparative example, in the lignin enrichment pretreatment stage, considering the differences in lignin sources and soil types corresponding to different ecosystems, its derivatization and purification system is the same as that in Examples 1-5. The difference is that for different soil types, a biphasic mixed solution system of ionic liquid and ionic liquid-like with different mass ratios is selected, and at the same time, considering the differences in lignin sources between the surface and deep soils, the derivatization and purification treatments of 0-10 cm and 10-20 cm are carried out respectively. Finally, the standard deviations of the lignin methoxy δ 13 C LM and δ 2 H LM values are used to compare the lignin sources of soil types and soil profiles.

[0141] Taking the forest soil of pure gymnosperm forests in forest ecosystems containing only meta-methoxy, i.e., G-type lignin monomers, and the farmland soil of pure broad-leaved plants in agroforestry ecosystems containing both meta- and para-methoxy, i.e., G-type and S-type lignin monomers, as examples, the purification and derivatization treatment methods of lignin substances were compared according to the above purification method. 10.0 ± 0.5 mg of the purified lignin substances were weighed respectively for the determination of the δ 13 C LM and δ 2 H LM values. The determination method was the same as that of the calibrator in Example 1. After 15 groups of repeated measurements, the δ 13 C LM and δ 2 H LM values and standard deviations ( Figure 8 ) of lignin methoxy were obtained for different soil types and soil layers. The results showed that for the soil layers under both gymnosperm and broad-leaved plants, the relevant standard deviations of the surface soil obtained after impurity removal pretreatment were all less than 0.4, and those of the deep soil were less than 0.6, while the corresponding standard deviations of the original sources were between 1.8 and 3.2.

[0142] Comparative Example 5

[0143] This comparative example provides a method for characterizing the lignin source in a natural degradation system.

[0144] For the characterization of the lignin source in the photosynthetic tissue - non-photosynthetic tissue - soil profile in this comparative example, during the analysis of the response relationship of the δ 13 C LM and δ 2 H LM values, considering the mediating role of litter as the leaves of photosynthetic tissue entering the surface soil and rhizomes as the branches of non-photosynthetic tissue entering the deep soil, the pre-purification was carried out to determine the fractionation relationship fitting of the δ 13 C LM and δ 2 H LM values respectively. The derivatization purification and determination method were the same as those in Examples 1 - 5. The difference was that a forest ecosystem with a mixture of two single life-form tree species, deciduous conifers and evergreen broad-leaved trees, was selected as an example. The lignin purification pretreatment of its photosynthetic tissue leaves and litter, non-photosynthetic tissue branches and rhizomes, and the surface and deep layers of the soil profile was carried out respectively. Finally, the lignin source was characterized by the specificity and fractionation response relationship characteristics of the δ 13 C LM and δ 2 H LM values.

[0145] As can be seen from the results in Table 10, although the forest soil in this example is the understory soil composed of two tree species with different life forms, the lignin methoxy δ 13 C LM and δ 2 H LM values of the two tree species above ground have obvious tree species attributes. At the same time, the standard deviation of the relevant values of the deciduous coniferous tree species is significantly lower than that of the evergreen broad-leaved tree species. The possible reason is that the lignin methoxy donor of the former only comes from the G-type lignin containing only meta-methoxy, while the lignin methoxy donor of the latter comes from the mixture of meta-methoxy (G-type lignin) and meta / para-methoxy (S-type lignin). The further fractionation response relationship and fitting significance also show that the lignin methoxy δ 13 C LM and δ 2 H LM values of the deciduous coniferous tree species are more stable for characterizing the lignin source. In addition, the characterization relationship of the relevant data shows that the lignin in the surface soil of the forest understory mainly comes from the litter of deciduous coniferous tree species, while the deep soil comes from the mixture of the branches of deciduous coniferous and evergreen broad-leaved tree species.

[0146] Table 10: Characterization relationship between tissue organs of mixed forests of tree species with different life forms and lignin sources in their understory humus soil

[0147]

[0148] As can be seen from Figure 9 the results, as the source medium between plant photosynthetic tissues and surface soil, although the lignin methoxy δ 13 C LM and δ 2 H LM values of the litter have significant fitting relationships with the source δ 13 C LM and δ 2 H LM of the new leaves and humus surface soil, but the fitting relationship and significance of its lignin methoxy δ 13 C LM are more significant ( Figure 9 a and 9b, P <0.001), indicating that the lignin source from photosynthetic leaves and litter can be traced by using the lignin methoxy δ 13 C LM value of the surface soil ( Figure 9 a); As the source medium between plant non-photosynthetic tissues and deep soil, although the lignin methoxy δ 13 C LM and δ 2 H LM values of the rhizome have significant fitting relationships with the source δ 13 CLM and δ 2 H LM has a significant fitting relationship ( Figure 9 c and 9d, P <0.001), but the fitting relationship data of its lignin methoxy δ 2 H LM are more uniform respectively, indicating that the use of the lignin methoxy δ 2 H LM value can be traced back to the lignin source of non-photosynthetic branches and rhizomes ( Figure 9 d).

[0149] As described above, the present invention can be preferably implemented. The above embodiments are only descriptions of the preferred implementation manners of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A method for characterizing the lignin source in a natural degradation system, characterized in that, Including: After obtaining the sample, lignin extraction and purification should be carried out on the sample. Through an organic chemical reaction, headspace iodomethane gas is derived. The δ of lignin methoxy is obtained by measuring the headspace iodomethane gas 13 C LM value or δ 2 H LM value; the sample is plant tissue, biological vector or soil; the δ 13 C LM refers to the stable carbon isotope ratio, and the δ 2 H LM refers to the stable hydrogen isotope ratio; Obtain the δ 13 C LM values of lignin methoxy in plant tissues, source media and soil in the same natural degradation system. Use the δ 13 C LM value of lignin methoxy in the source medium as the independent variable, and the δ 13 C LM value of lignin methoxy in the soil as the dependent variable to construct a regression equation model; Based on the δ 13 C LM value of lignin methoxy in the source medium, predict the corresponding δ 13 C LM value of lignin methoxy in the plant tissue through the regression equation model; Or obtain the δ 2 H LM values of lignin methoxyl in plant tissues, source media, and soil in the same natural degradation system. Use the δ 2 H LM value of lignin methoxyl in the source medium as the independent variable, and the δ 2 H LM value of lignin methoxyl in the soil as the dependent variable to construct a regression equation model; based on the δ 2 H LM value of lignin methoxyl in the source medium, predict the corresponding δ 2 H LM value of lignin methoxyl in the plant tissue through the regression equation model; When the plant tissue is photosynthetic tissue, the source medium is aboveground litter, and the soil is from the 0-10 cm soil profile; When the plant tissue is non-photosynthetic tissue, the source medium is rhizomes, and the soil is from the 10-20 cm soil profile.

2. The method for characterizing the lignin source in the natural degradation system according to claim 1, wherein The photosynthetic tissue is fresh leaves, and the non-photosynthetic tissue is branches.

3. The method for characterizing the lignin source in the natural degradation system according to claim 1, wherein The natural degradation system includes the natural degradation system of agricultural and forestry solid wastes, the natural degradation system of garden litter, and the natural degradation system of forest fallen trees.

4. The method for characterizing the lignin source in the natural degradation system according to claim 1, wherein According to different natural degradation systems, the optimal regression equation model is selected, including: In the same natural degradation system, a regression equation model is constructed with the lignin methoxy δ 13 C LM value, and a regression equation model is constructed with the lignin methoxy δ 2 H LM value. The regression equation model with the highest R 2 value is selected as the optimal regression equation model.

5. The method for characterizing the lignin source in the natural degradation system according to claim 1, characterized in that The sample to be detected contains a source donor of non-lignin methoxy, and the source donor of non-lignin methoxy is tannic acid substances. The sample to be detected is pretreated; Including: The sample fine powder is mixed with an aqueous sodium hydroxide solution at a volume-to-mass ratio of 1 mL: 5 mg, stirred and dissolved at 90 °C for 4 h under closed conditions to remove cellulose substances, then quickly quenched at 0 °C and allowed to stand for 30 min, and the lower-layer turbid precipitate is taken as the first extract; The first extract is mixed with nicotine at a mass-to-volume ratio of 1 mg: 2 mL; Then under acidic conditions, stir and react at room temperature and seal for 20-30 min, let it stand and precipitate for 15-30 min, filter and take the upper-layer solution to remove tannic acid interference substances, and extract with ethyl acetate, and lignin substances are obtained after rotary evaporation under reduced pressure.

6. The method for characterizing the lignin source in the natural degradation system according to claim 1, wherein The sample to be detected contains a source donor of non-lignin methoxy, and the source donor of non-lignin methoxy is pectin. The sample to be detected is pretreated, and the sample to be detected contains a cuticle; Including: The sample fine powder is mixed with an aqueous sodium hydroxide solution at a volume-to-mass ratio of 1 mL: 5 mg, stirred and dissolved at 90 °C for 4 h under closed conditions to remove cellulose substances, then quickly quenched at 0 °C and allowed to stand for 30 min, and the lower-layer turbid precipitate is taken as the first extract; The first extract is mixed with nicotine at a mass-to-volume ratio of 1 mg: 2 mL; Then under acidic conditions, stir and react at room temperature and seal for 20-30 min, let it stand and precipitate for 15-30 min, filter and take the upper-layer solution to remove tannic acid interference substances; Then, an equimolar mixture of zinc chloride tetrahydrate and 1-ethyl-3-methylimidazolium chloride ferric chloride, an ionic liquid, is used as the second biphasic enrichment system for lignin material enrichment, mixed at a mass ratio of 1 mg: 1 mg and adjusted to neutrality, and melted and reacted at 80 °C for 10 min under sealed conditions to remove pectin interference substances, then quickly quenched at 0 °C, extracted with ethyl acetate, and lignin substances are obtained after rotary evaporation under reduced pressure.

7. The method for characterizing the lignin source in the natural degradation system according to claim 1, characterized in that The sample to be detected contains a source donor of non-lignin methoxy, and the source donor of non-lignin methoxy is pectin. The sample to be detected does not contain a cuticle; It includes: using an equimolar mixture of zinc chloride tetrahydrate in the form of a quasi-ionic liquid and 1-ethyl-3-methylimidazolium chloride iron in the ionic liquid as the extraction system for lignin material enrichment, reacting in a sealed microwave at 80 °C for 10 min under the condition of pH = 4; then, mixing the 37% fuming hydrochloric acid deionized water aqueous solution and the extract according to a volume-mass ratio of 100 - 150 μL:1 mg, standing overnight at room temperature for 8 - 10 h to adjust the pH to 6.5 - 7.5 to remove pectin-like interfering substances, and then extracting with ethyl acetate and concentrating the lignin-like substances by rotary evaporation under reduced pressure.

8. The method for characterizing the lignin source in the natural degradation system according to claim 1, characterized in that, The sample to be detected is soil containing humus, and the sample to be detected is pretreated. It includes: First, using an equimolar mixture of ammonium ferrous sulfate hexahydrate in the form of a quasi-ionic liquid and 1-ethyl-3-methylimidazolium chloride iron in the ionic liquid as the enrichment extraction system for lignin material, mixing according to a mass ratio of 15 mg:1 mg with agricultural and forestry soil, 10 mg:1 mg with garden soil, and 3 mg:1 mg with forest soil; then, adding copper oxide as a derivatizing agent, reacting in a sealed microwave at 120 °C for 70 min under the condition of weak alkaline pH = 8.5, and then quickly quenching at 0 °C, extracting with ethyl acetate, and concentrating by rotary evaporation under reduced pressure to obtain lignin phenolic derivatives.

9. A method for determining the lignin source in a natural degradation system, characterized in that, It includes: Obtaining a regression equation model through the method for characterizing the lignin source in the natural degradation system according to any one of claims 1 - 8. The corresponding source medium of the regression equation model is the above-ground litter, and the soil is the 0-10 cm soil profile. Based on the lignin methoxy δ 13 C LM value or lignin methoxy δ 2 H LM value, a simulated data set is constructed for the regression equation model; it is judged whether there is a significant difference between the simulated data set and the data set to be judged; If there is no significant difference, it indicates that the data set to be judged is derived from photosynthetic tissues. If there is a significant difference, it indicates that the data set to be judged is not derived from photosynthetic tissues. Or the corresponding source medium of the regression equation model is the rhizome, the soil is the 10-20 cm soil profile, and based on the lignin methoxy δ 13 C LM value or lignin methoxy δ 2 H LM value to construct a simulated data set with the regression equation model; determine whether there is a significant difference between the simulated data set and the data set to be judged; If there is no significant difference, it indicates that the data set to be judged is derived from non-photosynthetic tissues. If there is a significant difference, it indicates that the data set to be judged is not derived from non-photosynthetic tissues.

10. Application of the method for judging the lignin source in the natural degradation system according to claim 9 in obtaining the source of the lignin-containing sample.

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