DOM (Document Object Model) detection and reclamation influence evaluation method based on multispectrum

Abstract

The invention belongs to the field of soil dissolved organic matter analysis, and provides a multispectral-based DOM detection and reclamation influence evaluation method, which comprises the following steps: step 1, collecting dry farmland soil samples of different reclamation years, and obtaining soil column samples of a plough layer, a plow pan and subsoil; 2, extracting, centrifuging and filtering the soil sample to obtain a DOM solution; 3, detecting the DOM solution by adopting an ultraviolet-visible absorption spectrum; step 4, adopting a three-dimensional fluorescence spectrum and combining parallel factor analysis to obtain fluorescence components and source characteristics of the DOM solution; step 5, analyzing molecular composition and characteristic parameters of the DOM solution by adopting solid-phase extraction in combination with Fourier transform ion cyclotron resonance mass spectrometry; and step 6, based on the detection data in the steps 3-5, evaluating the influence of reclamation years on the composition, structure and degradation characteristics of the DOM. According to the method, multi-dimensional detection of soil DOM is realized through a multi-spectrum and mass spectrum combined technology, and DOM is comprehensively analyzed from a macrostructure to molecular composition.

Description

Technical Field

[0001] The present invention belongs to the field of soil dissolved organic matter analysis, and in particular relates to a multi-spectral based DOM detection and reclamation impact assessment method. Background Art

[0002] Soil dissolved organic matter (DOM) is a core component of soil fertility. Its molecular structure is rich in highly diverse functional groups such as hydroxyl and carboxyl groups. These active binding sites give DOM extremely high chemical reactivity, making it the most active organic matter component in the soil. The global reserve of dissolved organic matter (DOM) is about 700 PgC, mainly derived from marine DOC, supplemented by terrestrial aquatic. Although DOM only accounts for a small part of the total soil organic matter, as the largest active carbon pool in the biosphere, it is of great significance to the global carbon cycle. At the same time, DOM is an important energy source for soil microbial metabolic activities and a key driving force for soil nutrient cycling. It plays a vital role in the biological availability of soil nutrients and their transformation processes.

[0003] DOM is primarily derived from plant litter and root exudates, and once in the soil, it undergoes complex physical, chemical, and biological transformations. The structure and composition of DOM are highly complex and extremely heterogeneous, with identified chemical components accounting for less than 25% of its total organic matter. Traditionally, DOM is often characterized by DOC. However, due to the complex chemical composition and structure of DOM, only a small number of low-molecular-weight organic compounds can be directly isolated, purified, and chemically detected, while the structures of a large number of high-molecular-weight organic compounds (such as humic substances and enzymes) remain incompletely elucidated. This complexity limits our comprehensive understanding of the role of DOM in soil carbon cycling and ecological functions.

[0004] Soil microbial biomass is considered an important potential source of DOM. Fractionation and structural analysis of DOM in soil solutions revealed that microbial metabolites comprise a significant proportion of DOM. Furthermore, the chemical properties of carbohydrates in DOM reveal significant differences in composition from those in plant residues or humus. Specifically, carbohydrates in DOM exhibit a higher ratio of hexoses to deoxysugars and a lower content of pentoses, consistent with the scarcity of pentoses in microbial cells. Microorganisms profoundly influence the molecular composition of DOM through two key processes: catabolism and anabolism. Conversely, the chemical properties of DOM can drive the structure and function of soil microbial communities. Studies have shown that microbial taxa are positively correlated with DOM molecules, such as polycyclic aromatic hydrocarbons (PAHs) and phenolic polymers, and negatively correlated with unsaturated aliphatic compounds. This suggests that unsaturated aliphatic compounds are more susceptible to microbial degradation than PAHs and phenolic compounds. Furthermore, the source of DOM significantly influences the diversity of soil fungi and their functional groups. For example, fungal diversity is significantly negatively correlated with the DOM fluorescence index, indicating that plant-based DOM helps maintain high fungal diversity, while microbial-based DOM has an inhibitory effect on fungal community diversity. Therefore, DOM composition not only directly affects the structure and function of soil microbial communities but also further shapes the carbon cycle and nutrient dynamics of soil ecosystems by regulating microbial metabolic processes.

[0005] Microbial diversity plays a key role in the transformation and degradation of DOM molecular diversity. Microbial metabolic activities can significantly change the molecular composition and diversity of DOM. Degenhardt et al. revealed the relationship between microbial communities and DOM molecules through Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and 16S rRNA gene sequencing technology. Studies have shown that there are significant differences in the composition of bacterial communities in different regions, soil depths and metabolic niches, and these differences have an important impact on the degradation process of DOM. In farmland soils, the abundance of specific bacterial communities is positively correlated with the degradation rate of specific molecules in DOM (such as aromatic compounds), indicating that the composition of microbial communities directly determines the degradation efficiency of DOM. In addition, the restoration process significantly increased the content of refractory organic matter (DOM) in the soil, which not only improved the soil carbon sequestration capacity, but also had a profound impact on the soil carbon cycle and ecosystem function.

[0006] Globally, farmland soils store approximately 10% of the total global soil carbon stock (approximately 1500 PgC at a depth of 1 meter). Reclamation activities can alter the amount of DOM in soils. Studies have found that the conversion of natural forests to farmland causes a significant, short-term decline in the quantity and quality of surface soil organic matter, with a particularly pronounced decrease in dissolved organic carbon (DOC) (12%-49%). It is estimated that agricultural activities have resulted in a global loss of approximately 50 PgC or more of organic carbon in farmland soils. Reclamation activities not only exacerbate soil DOM losses but also significantly reduce the quality of soil organic matter, thereby weakening the stability of the subsoil carbon pool and its carbon sequestration function. Studies have found that the macroscopic chemical structure of soil DOM increases after natural forest conversion to farmland, with more pronounced losses of less chemically resistant carbohydrates and phenolics, indicating that reclamation activities lead to a decline in soil organic matter quality. Such changes not only affect soil carbon dynamics but may also have profound impacts on ecosystem function.

[0007] Although the impacts of land reclamation on soil DOM have been extensively studied, significant gaps remain in the in-depth characterization of soil DOM at the molecular level, particularly in the long-term monitoring and mechanistic elucidation of the molecular composition and dynamics of soil DOM. A deeper understanding of the production, transformation, and flux of soil DOM is crucial for predicting the impacts of environmental change and management practices on the soil carbon cycle. Furthermore, clarifying the importance of DOM in the environment will facilitate the development of mitigation strategies for DOM-related environmental issues, thereby optimizing soil carbon management and ecosystem services. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a multispectral DOM detection and reclamation impact assessment method to solve the problems in the prior art. The technical solution adopted by the present invention is:

[0009] A multispectral DOM detection and reclamation impact assessment method comprises the following steps:

[0010] Step 1: Collect dryland soil samples of different reclamation years and obtain soil column samples of the tillage layer, plow bottom layer and subsoil;

[0011] Step 2, extracting, centrifuging, and filtering the soil sample to obtain a DOM solution;

[0012] Step 3, detecting the DOM solution using UV-visible absorption spectroscopy;

[0013] Step 4: Using three-dimensional fluorescence spectroscopy combined with parallel factor analysis to obtain the fluorescent components and source characteristics of the DOM solution;

[0014] Step 5, using solid phase extraction combined with Fourier transform ion cyclotron resonance mass spectrometry to analyze the molecular composition and characteristic parameters of the DOM solution;

[0015] Step 6: Based on the test data from steps 3 to 5, evaluate the effect of reclamation years on the composition, structure and degradation characteristics of DOM.

[0016] Furthermore, in step 1, different reclamation periods include 10, 20, 30, 40, and 70 years. During sampling, at least three 1×1 m plots with a spacing greater than 10 m were set up in each area, and a 9.3 cm diameter soil drill was used to obtain 1 m deep soil column samples.

[0017] Furthermore, step 2 includes: passing the soil column sample through a 2mm sieve, weighing 4-25g of the soil sample, adding K2SO4 or KCl extract at a soil-water ratio of 1:5 or 1:2.5, and shaking for 30-60 minutes; then centrifuging at 4000 rpm for 20 minutes, allowing the sample to stand for stratification, collecting the supernatant, filtering it through a 0.45μm filter to remove particulate matter, and refrigerating or freezing the filtrate for future use; finally, measuring the soluble organic carbon content using a TOC analyzer, or combining it with solid-phase extraction and concentration for molecular composition analysis.

[0018] Furthermore, step 4 includes using a fluorescence spectrophotometer for measurement, using ultrapure water as a blank control, and diluting the water samples to be tested to ensure that the UV254 of all water samples to be tested is less than 0.3; the detection conditions include: excitation wavelength range of 250-600nm, emission wavelength range of 246-828nm, scanning time of 2s, wavelength interval of 2nm, and slit width of 2nm; the final spectrum is obtained after the raw data is background subtracted, inner filter effect corrected, and Raman scattering corrected;

[0019] Based on the spectral data of three-dimensional fluorescence, a mathematical model was constructed using the parallel factor analysis method. First, the raw fluorescence spectral data were preprocessed to eliminate the interference of the inner filter effect, Rayleigh scattering, and Raman scattering. Then, the DOMFluor analysis toolkit was used to perform PARAFAC modeling to decompose the three-dimensional fluorescence matrix into several independent fluorescence components, and the relative fluorescence intensity distribution of each component in the sample was quantitatively characterized.

[0020] Furthermore, step 5 includes:

[0021] The solid phase extraction method was used and the whole process was completed under nitrogen protection and fume hood conditions, including the following methods:

[0022] The PPL column was activated and cleaned with 3 mL of methanol and 6 mL of acidified ultrapure water at pH 2, respectively;

[0023] 1 L of water sample filtered through a 0.45 μm membrane was adjusted to pH = 2 and then enriched by passing it through a PPL column at a flow rate of 4 mL / min, followed by elution with 6 mL of acidified ultrapure water to remove salts;

[0024] The PPL column was dried using a nitrogen flow rate of 2 L / min, and the target component was eluted with 4 mL of methanol. The eluate was stored in the dark at 4 °C until analysis;

[0025] Based on the obtained DOM molecular formula data, the chemical properties of DOM were characterized by calculating a number of molecular characteristic parameters, including the modified aromaticity index AImod, the equivalent double bond number DBE, the nominal oxidation state of carbon NOSC, the hydrogen-carbon ratio H / C, and the oxygen-carbon ratio O / C. The calculation formulas are as follows:

[0026]

[0027]

[0028] Where C, H, N, O, P, and S represent the number of carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur atoms in the DOM molecular formula, respectively;

[0029] Each parameter in the data was normalized according to the relative abundance of the mass spectrometry peak: the average parameter of each molecular characteristic parameter was obtained by dividing the sum of the weighted parameters of each DOM molecular formula intensity by the sum of the peak intensities of each molecule. The calculation formula is as follows:

[0030]

[0031] Degradation Index I deg The calculation formula is as follows:

[0032]

[0033] Among them, NEG deg Represents relatively inert molecules in DOM; POS deg Indicates the relative activity of molecules in DOM; magnitudes indicate the mass spectrometry peak intensity of the molecules.

[0034] Furthermore, the evaluation in step 6 includes: analyzing the compound species distribution of DOM through the van Krevelen plot, evaluating the molecular diversity of DOM through the Chao1 index and the Shannon index, and evaluating the stability of DOM through the molecular instability index.

[0035] The present invention has the following beneficial effects:

[0036] This study uses multi-spectral and mass spectrometric techniques to achieve multi-dimensional detection of soil DOM. By combining UV-visible absorption spectroscopy with three-dimensional fluorescence spectroscopy, it comprehensively analyzes DOM from its macroscopic structure to its molecular composition, breaking through the limitations of traditional single indicators. Simultaneously, across different soil layers and over 10-70 years of reclamation sequences, it captures the spatiotemporal dynamics of DOM. This study provides scientific support for reclamation impact assessments, uncovering the dual impacts of reclamation on both surface and subsurface carbon pools and elucidating the evolutionary mechanisms of DOM molecular composition. The findings can guide the optimal management of soil carbon pools, inform measures to enhance carbon sequestration capacity, and provide direct evidence for assessing the impact of reclamation on soil ecological functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Flowchart of the present invention;

[0038] Figure 2 The three fluorescent components and their excitation and emission wavelengths in natural wetland and paddy field DOM identified by the PARAFAC model;

[0039] Figure 3 The van Krevelen plots of DOM molecular composition at different planting years (H1-H5) and in the tillage layer (0-20 cm), plow bottom layer (20-40 cm), and subsoil (60-80 cm) were determined based on Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) analysis;

[0040] Figure 4 The Chao 1 index and Shannon index are the soil DOM molecular diversity at different reclamation years and different soil depths. DETAILED DESCRIPTION

[0041] The following is a combination of the embodiments of the present invention Figure 1-Figure 4 , the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0042] A multispectral DOM detection and reclamation impact assessment method comprises the following steps:

[0043] Step 1: Collect dryland soil samples of different reclamation years and obtain soil column samples of the tillage layer, plow bottom layer and subsoil;

[0044] This study selected typical dryland soils from sites with varying degrees of reclamation (10, 20, 30, 40, and 70 years) for soil sampling. Within each sampling area, three 1×1 m plots were randomly selected, with each plot separated by at least 10 m to ensure spatial independence. A 9.3 cm diameter powered soil auger was used to obtain complete soil column samples approximately 1 m deep.

[0045] Step 2, extracting, centrifuging, and filtering the soil sample to obtain a DOM solution;

[0046] After the collected soil sample is passed through a 2mm sieve, 4-25g of soil sample is weighed, and distilled water, K2SO4 or KCl and other extracts are added at a soil-water ratio of 1:5 or 1:2.5, and shaken for 30-60 minutes to fully dissolve; then centrifuged at 4000 rpm for 20 minutes, allowed to stand and stratify, the supernatant is taken, filtered through a 0.45μm filter membrane to remove particulate matter, and the filtrate is refrigerated or frozen for later use; finally, the soluble organic carbon (DOC) content is determined by a TOC meter, or the molecular composition is analyzed using FT-ICR-MS and other technologies after solid-phase extraction and concentration.

[0047] Step 3, detecting the DOM solution using UV-visible absorption spectroscopy;

[0048] The process was determined using an ultraviolet-visible spectrophotometer (UV-2600, Shimadzu Instruments Suzhou Co., Ltd.). The experiment used a quartz cuvette with a 1-cm optical pathlength, ultrapure water as a blank control, and was performed at room temperature. The instrument parameters were set to: scan range 200-800 nm, wavelength interval 1 nm.

[0049] Step 4: Using three-dimensional fluorescence spectroscopy combined with parallel factor analysis to obtain the fluorescent components and source characteristics of the DOM solution;

[0050] The measurement was performed using a fluorescence spectrophotometer (Aaualog, Horiba, Japan), with ultrapure water as a blank control. The water samples to be tested were diluted to ensure that the UV254 of all water samples to be tested was less than 0.3 to reduce the interference of the inner filter effect. The detection conditions included: excitation wavelength (Ex) wavelength range 250-600nm, emission wavelength (Em) wavelength range 246-828nm, scanning time 2s, wavelength spacing 2nm, and slit width 2nm. The final spectrum was obtained after background subtraction, inner filter effect correction, and Raman scattering correction of the raw data. Based on the three-dimensional fluorescence spectral data, the present invention uses the parallel factor analysis (PARAFAC) method to construct a mathematical model. First, the raw fluorescence spectral data was preprocessed to eliminate the interference of the inner filter effect, Rayleigh scattering, and Raman scattering. Subsequently, the DOMFluor analysis toolkit was used to perform PARAFAC modeling, decompose the three-dimensional fluorescence matrix into several independent fluorescence components, and quantitatively characterize the relative fluorescence intensity distribution of each component in the sample. The spectral characteristic parameters involved in the present invention and their calculation methods are detailed in Table 1.

[0051] Table 1: Spectral characteristic parameters and their calculation methods

[0052]

[0053]

[0054] Step 5, using solid phase extraction combined with Fourier transform ion cyclotron resonance mass spectrometry to analyze the molecular composition and characteristic parameters of the DOM solution;

[0055] The solid phase extraction (PPL) column technology is used, and the entire process is completed under nitrogen protection and fume hood conditions. The specific operation process includes:

[0056] (1) Activate and clean the PPL column with 3 mL of methanol and 6 mL of acidified ultrapure water (pH = 2) in sequence;

[0057] (2) 1 L of water sample filtered through a 0.45 μm membrane was adjusted to pH = 2 and then passed through a PPL column at a flow rate of 4 mL / min for enrichment. Subsequently, 6 mL of acidified ultrapure water was used to remove salts.

[0058] (3) The PPL column was dried using a nitrogen flow rate of 2 L / min. Finally, the target component was eluted with 4 mL of methanol. The eluate was stored in the dark at 4°C until analysis. The high-resolution ion cyclotron resonance mass spectrometer used in the experiment was FT-ICRMS (Apex-Ultra 9.4T, Bruker, USA), and the ESI ionization source was Apollo II ESI.

[0059] Based on the DOM molecular formula data obtained by FT-ICRMS, the present invention systematically analyzes the molecular composition characteristics of DOM. The chemical properties of DOM are characterized by calculating multiple molecular characteristic parameters, including the modified aromaticity index (AImod), the equivalent double bond number (DBE), the nominal oxidation state of carbon (NOSC), the hydrogen-to-carbon ratio (H / C), the oxygen-to-carbon ratio (O / C), and other DOM molecular characteristic parameters. The specific calculation formula is as follows, where C, H, N, O, P, and S represent the number of carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur atoms in the DOM molecular formula, respectively:

[0060]

[0061] Each parameter in the data was normalized according to the relative abundance of the mass spectrometry peak. That is, the sum of the intensity-weighted parameters of each DOM molecular formula (I) was divided by the sum of the peak intensities of each molecule (i). The average parameters of the FT-ICRMS data, namely DBEwa, (AImod)wa, NOSCwa, H / Cwa, and O / Cwa, were obtained. The specific calculation formulas are as follows:

[0062]

[0063] Degradation index (I deg ) is a quantitative indicator for evaluating the degree of DOM molecule degradation. Its numerical range is defined between 0 and 1. A higher index indicates a higher degree of decomposition of organic molecules. The calculation formula is as follows:

[0064]

[0065] Among them, NEG deg Relatively inert molecules in DOM, including C 21 H 26 O 11 , C 17 H 20 O9, C 19 H 22 O 10 , C 20 H 22 O 10 , C 20 H 24 O 11 POS deg Indicates the relative activity of molecules in DOM, including C 13 H 18 O7, C 14H 20 O7, C 15 H 22 O7, C 15 H 22 O8, C 16 H 24 O8; magnitudes represents the mass spectrum peak intensity of the molecule.

[0066] Step 6: Based on the test data from steps 3 to 5, evaluate the effect of reclamation years on the composition, structure and degradation characteristics of DOM; specifically, analyze the distribution of DOM compound types through the van Krevelen plot, evaluate the DOM molecular diversity through the Chao1 index and Shannon index, and evaluate the stability of DOM through the molecular instability index, for example Figure 4 .

[0067] When evaluating, if Figure 3 The composition of DOM compound types is displayed through the van Krevelen diagram. Each coordinate point in the diagram represents one or more molecular formulas with specific H / C and O / C values. Different molecular formulas correspond to different H / C and O / C values. The van Krevelen diagram is divided into 7 regions according to the H / C and O / C values: lipids (H / C = 1.5-2.0; O / C = 0-0.3); proteins (H / C = 1.5-2.2; O / C = 0.3-0.67; N / C ≥ 0.05); lignin (H / C = 0.7-1.5; O / C = 0.1-0.67); carbohydrates (H / C = 1.5-2.0; O / C = 0.67-1.2); unsaturated hydrocarbons (H / C = 0.7-1.0; O / C = 0-0.1); condensed aromatic structures (H / C = 0.2-0.7; O / C = 0-0.67); and tannins (H / C = 0.5-1.5; O / C = 0.67-1.2). According to the elemental composition, the molecular formula is divided into four categories: compounds containing only CHO, nitrogen-containing compounds (CHON), sulfur-containing compounds (CHOS), and compounds containing both nitrogen and sulfur (CHONS).

[0068] In the present invention, the effects of different reclamation years on the physical and chemical properties of soil profiles are as follows:

[0069] With increasing reclamation years, soil pH in the tillage layer (0-20 cm), plow subsoil (20-40 cm), and subsoil (60-80 cm) showed a decreasing trend (p < 0.05). Silt content in all soil layers increased with reclamation years (p < 0.05). Soil SOC in the tillage layer and plow subsoil showed no significant changes with reclamation years, while in the subsoil it decreased significantly by 37.67%-66.16% after 10 years of reclamation (p < 0.01). Fe-OC content in the tillage layer increased by 45.7%-49.4% (p < 0.05), while it showed no significant changes in the plow subsoil and decreased by 30.3%-63.2% in the subsoil (p < 0.05). The DOM concentration in the tillage layer soil decreased by 28.2%-60.9% (p<0.01) with the reclamation years. Its content in the plow bottom layer had no significant change, while in the subsoil it decreased by 55.5%-62.2% (p<0.05) with the reclamation years.

[0070] like Figure 1 In the present invention, the effects of different reclamation years on the three-dimensional fluorescence spectrum characteristics of soil DOM are as follows:

[0071] The DOM fluorescence index (FI) in the cultivated layer soil showed no significant change with reclamation age, while the humification index (HIX) in the cultivated layer soil and subsoil showed opposite trends. In the cultivated layer, the HIX index increased significantly by 60% in the late reclamation period, while in the subsoil, it decreased significantly by 50.9%-69.6% after 10 years of reclamation. The autogenetic index (BIX) showed an opposite trend to the HIX index: the cultivated layer soil decreased by 7.1%-15.3% with reclamation age, while the subsoil increased by 15.1%-31.3%. No significant differences were found in the fluorescence indices of the subsoil between different reclamation years.

[0072] With increasing depth, FI and HIX in dryland soils decreased significantly (p < 0.01), while BIX and SUVA254 showed opposite trends with increasing depth. SUVA254 increased significantly with depth by 20.41%-46.45% (p < 0.01), indicating that the relative contribution of aromatic compounds to DOM increased with increasing depth.

[0073] Parallel factor analysis of the three-dimensional fluorescence spectral data of dryland DOM identified five components. Components C1 (Ex: 205 nm, Em: 380 nm), C2 (Ex: 200 nm, Em: 300 nm), C3 (Ex: 265 nm, Em: 448 nm), and C4 (Ex: 245 nm, Em: 404 nm) exhibit one excitation peak and one emission peak, while component C5 (Ex: 230 / 280 nm, Em: 330 nm) exhibits two excitation peaks and one emission peak. Components C1 and C2 are classified as protein-like substances, C3 as humic acid-like substances, C4 as fulvic acid-like substances, and C5 as protein-like substances and soluble microbial metabolites.

[0074] Protein-like substances (components C1 and C2) were the primary components of DOM in H1-H4 (10-40 years of cultivation). Components C1-C4 were more evenly distributed in H5 (70 years of cultivation), primarily including protein-like substances, humic acid-like substances, and fulvic acid-like substances. Humic acid-like substances (component C3) and fulvic acid-like substances (component C4) were significantly higher in H5 than in H1-H4, with concentrations ranging from 58.5% to 70.7%, and fulvic acid-like substances (component C4) were significantly higher in H5 than in H1-H4, with concentrations ranging from 71.1% to 81.6%. This indicates that with increasing cultivation age, the proportion of protein-like substances (components C1 and C2) decreased, while the proportions of humic acid-like substances (component C3), fulvic acid-like substances (component C4), protein-like substances, and soluble microbial metabolites (component C5) increased.

[0075] In the present study, lignin compounds dominated the DOM molecular composition, accounting for 71–84% of the total DOM, regardless of cultivation age and soil depth. Antidegradation compounds (lignin, condensed aromatic compounds, and tannins) decreased significantly with cultivation age in the tillage layer (0-20 cm), with lignin compounds in particular decreasing by 5%-7%. Similar patterns were observed for antidegradation compounds in the subsoil, but lignin compounds increased while tannins decreased significantly by 7.4%. Lignin compounds also increased by 4% in the plow bottom layer with cultivation age.

[0076] The DOM molecular diversity (Chao1 index and Shannon index) in the tillage layer first increased and then decreased with the increase of reclamation years, while the DOM molecular diversity in the plow bottom layer and subsoil did not change much. The Chao1 index and Shannon index showed a downward trend with depth.

[0077] The proportion of active DOM components showed an overall upward trend with increasing reclamation age, increasing by 29.34%-48.91% with each reclamation year, indicating that reclamation increases DOM biological activity. The DOM molecular instability index (MLB) began to increase after 10 years of reclamation, indicating that soil DOM gradually became unstable (25.46%-29.68%) after 10 years of reclamation, and this instability remained relatively stable in the later stages of reclamation. MLB gradually increased with depth, indicating that soil DOM gradually became unstable with increasing depth.

[0078] Based on the data of this invention, it is believed that long-term tillage will change the carbon stability of different soil layers, which is mainly reflected in:

[0079] 1. Long-term tillage increases the stability of topsoil carbon pool:

[0080] Long-term tillage promotes the conversion of plant residues into humic acid-based, stable organic matter, which may then bind to minerals (e.g., through adsorption of iron oxides) to form a persistent carbon pool. Our research results show that the humification index (HIX) of the tillage layer increases significantly in later stages, and the Fe-OC content of the tillage layer soil also increases significantly with age. This suggests that humification products can form stable Fe-OC complexes through coordination reactions with iron oxides, further consolidating carbon sequestration capacity. Furthermore, the formation of this mineral-bound organic carbon significantly reduces the microbial availability of organic carbon, inhibiting decomposition losses. Our finding that the biogenic index (BIX) of the tillage layer decreases with age can well explain this phenomenon.

[0081] 2. Long-term tillage reduces the stability of the bottom soil carbon pool:

[0082] Long-term tillage enhances the activity of anaerobic bacteria (such as iron-reducing bacteria) in the subsoil, increasing the activity of subsoil carbon and weakening the stability of the subsoil carbon pool. Our research found that the subsoil humification index (HIX) decreased, while the autogenous index (BIX) increased. This suggests that long-term tillage allows microorganisms to utilize humic acid as a carbon source, inhibiting the humification process. Our findings that the proportion of soluble microbial metabolites (component C5) increases with increasing cultivation age can well explain this phenomenon.

[0083] 3. Fluorescence spectral characteristics of soil DOM:

[0084] Long-term tillage drives the evolution of DOM molecular composition toward high activity and low stability by altering organic matter input and mineral-organic matter interactions. Our results show that with increasing reclamation age, CHO compounds decrease significantly, while CHON compounds and CHONS compounds increase significantly in the later stages of reclamation. This suggests that the conversion of DOM from easily degradable carbon to complex organic matter containing heteroatoms may enhance the activity of carbon available to soil microorganisms but weaken the soil's long-term carbon sequestration capacity. This may be due to the fact that long-term organic substitution (such as straw incorporation) reduces the input of low-molecular-weight easily degradable carbon and increases medium-molecular-weight carbon components (such as lignin derivatives), further weakening the accumulation of CHO compounds; long-term application of organic fertilizers or introduction of exogenous nitrogen (such as amino acids and peptides) promotes the synthesis of nitrogen-containing metabolites (such as protein-like components) by microorganisms; and minerals such as iron oxides (Fe-OC) may promote the stabilization of CHONS compounds through adsorption, thereby allowing CHONS compounds to accumulate.

[0085] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A multispectral DOM detection and reclamation impact assessment method, characterized in that: The following steps are involved: Step 1: Collect dryland soil samples of different reclamation years and obtain soil column samples of the tillage layer, plow bottom layer and subsoil; Step 2, extracting, centrifuging, and filtering the soil sample to obtain a DOM solution; Step 3, detecting the DOM solution using UV-visible absorption spectroscopy; Step 4: Using three-dimensional fluorescence spectroscopy combined with parallel factor analysis to obtain the fluorescent components and source characteristics of the DOM solution; Step 5, using solid phase extraction combined with Fourier transform ion cyclotron resonance mass spectrometry to analyze the molecular composition and characteristic parameters of the DOM solution; Step 6: Based on the test data from steps 3 to 5, evaluate the effect of reclamation years on the composition, structure and degradation characteristics of DOM.

2. The multispectral DOM detection and reclamation impact assessment method according to claim 1, characterized in that: In step 1, different reclamation periods include 10, 20, 30, 40, and 70 years. During sampling, at least three 1×1m plots with a spacing greater than 10m were set up in each area, and a 9.3cm diameter soil drill was used to obtain 1m deep soil column samples.

3. The multispectral DOM detection and reclamation impact assessment method according to claim 1, characterized in that: Step 2 includes: after passing the soil column sample through a 2mm sieve, weighing 4-25g of soil sample, adding K2SO4 or KCl extract at a soil-water ratio of 1:5 or 1:2.5, and shaking for 30-60 minutes; then centrifuging at 4000 rpm for 20 minutes, taking the supernatant after standing and stratification, filtering through a 0.45μm filter membrane to remove particulate matter, and refrigerating or freezing the filtrate for later use; finally, determining the soluble organic carbon content by a TOC instrument, or analyzing the molecular composition after solid phase extraction and concentration.

4. The multispectral DOM detection and reclamation impact assessment method according to claim 1, characterized in that: Step 4 includes measuring using a fluorescence spectrophotometer, using ultrapure water as a blank control, and diluting the water samples to be tested to ensure that the UV254 of all water samples to be tested is less than 0.3; the detection conditions include: excitation wavelength range of 250-600 nm, emission wavelength range of 246-828 nm, scanning time of 2 s, wavelength interval of 2 nm, and slit width of 2 nm; the final spectrum is obtained after the raw data is background subtracted, inner filter effect corrected, and Raman scattering corrected; Based on the spectral data of three-dimensional fluorescence, a mathematical model was constructed using the parallel factor analysis method. First, the raw fluorescence spectral data were preprocessed to eliminate the interference of the inner filter effect, Rayleigh scattering, and Raman scattering. Then, the DOMFluor analysis toolkit was used to perform PARAFAC modeling to decompose the three-dimensional fluorescence matrix into several independent fluorescence components, and the relative fluorescence intensity distribution of each component in the sample was quantitatively characterized.

5. The multispectral DOM detection and reclamation impact assessment method according to claim 1, characterized in that: Step 5 includes: The solid phase extraction method was used and the whole process was completed under nitrogen protection and fume hood conditions, including the following methods: The PPL column was activated and purified with 3 mL of methanol and 6 mL of acidified ultrapure water at pH 2, respectively; 1 L of water sample filtered through a 0.45 μm membrane was adjusted to pH = 2 and then enriched by passing it through a PPL column at a flow rate of 4 mL / min, followed by elution with 6 mL of acidified ultrapure water to remove salts; The PPL column was dried using a nitrogen flow rate of 2 L / min, and the target component was eluted with 4 mL of methanol. The eluate was stored in the dark at 4 °C until analysis; Based on the obtained DOM molecular formula data, the chemical properties of DOM were characterized by calculating a number of molecular characteristic parameters, including the modified aromaticity index AImod, the equivalent double bond number DBE, the nominal oxidation state of carbon NOSC, the hydrogen-carbon ratio H / C, and the oxygen-carbon ratio O / C. The calculation formulas are as follows: Where C, H, N, O, P, and S represent the number of carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur atoms in the DOM molecular formula, respectively; Each parameter in the data was normalized according to the relative abundance of the mass spectrometry peak: the average parameter of each molecular characteristic parameter was obtained by dividing the sum of the weighted parameters of each DOM molecular formula intensity by the sum of the peak intensities of each molecule. The calculation formula is as follows: Where X represents the abundance-weighted average of a certain molecular characteristic parameter in the entire DOM sample; X i The specific calculated value of a molecular characteristic parameter in the i-th DOM molecular formula; i Indicates the mass spectrometry peak intensity (relative abundance) corresponding to the i-th DOM molecular formula Degradation Index I deg The calculation formula is as follows: Among them, NEG deg Represents relatively inert molecules in DOM; POS deg Indicates the relative activity of molecules in DOM; magnitudes indicate the mass spectrometry peak intensity of the molecules.

6. The multispectral DOM detection and reclamation impact assessment method according to claim 1, characterized in that: The evaluation in step 6 includes: analyzing the compound species distribution of DOM through the van Krevelen plot, evaluating the molecular diversity of DOM through the Chao1 index and the Shannon index, and evaluating the stability of DOM through the molecular instability index.

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