Soil petroleum hydrocarbon pollution natural attenuation evaluation method based on fluorescence component ratio

Abstract

The invention discloses a soil petroleum hydrocarbon pollution natural attenuation evaluation method based on fluorescent components, which comprises the following steps: acquiring a three-dimensional fluorescence spectrum of soluble organic matters in petroleum hydrocarbon polluted soil to obtain fluorescence intensity of various fluorescent components; according to the difference between the fluorescence intensities of the multiple fluorescence components, whether the petroleum hydrocarbon in the polluted soil can attenuate naturally or not and the intensity of natural attenuation are evaluated; according to the method, the fluorescence spectrum technology with lower cost is adopted, and the fluorescence components of the soluble organic matters in the polluted soil are analyzed, so that the natural attenuation potential of the petroleum hydrocarbon pollution is judged, and the feasibility of the fluorescence method applied to the identification of the petroleum hydrocarbon pollution of the soil is verified; and a support can be provided for soil environment treatment of a petroleum hydrocarbon polluted site.

Description

Technical Field

[0001] This invention relates to the field of soil petroleum hydrocarbon pollution research technology, specifically to a method for assessing the natural decay of soil petroleum hydrocarbon pollution based on the ratio of fluorescent components. Background Technology

[0002] With industrial development, the demand for petroleum and its related products is constantly increasing. During petroleum exploration, extraction, transportation, and processing, environmental pollution accidents such as leaks occur frequently, leading to petroleum hydrocarbons entering the soil system. Petroleum hydrocarbons are mainly composed of volatile (e.g., benzene, toluene, ethylbenzene, and xylene) and semi-volatile (e.g., long-chain hydrocarbons and polycyclic aromatic hydrocarbons) hydrocarbon compounds. Compared to volatile petroleum hydrocarbons such as benzene series compounds (benzene, toluene, ethylbenzene, and xylene), long-chain petroleum hydrocarbons (C... 10 -C 40 Petroleum hydrocarbons exhibit significantly lower volatility and persist in soil systems for extended periods. They possess potential toxicity, mutagenicity, or carcinogenicity, posing a serious threat to ecological security and human health. Studying the migration and transformation processes of petroleum pollutants in soil is crucial for pollution control. Monitored natural attenuation (MNA) refers to the planned monitoring of contaminated sites, utilizing naturally occurring physical, chemical, and biological processes (including dilution, diffusion, volatilization, adsorption, and biodegradation) to reduce the quantity, toxicity, and mobility of pollutants in the soil to acceptable risk levels. Compared to remediation methods such as cement kiln co-processing and chemical oxidation, MNA offers advantages such as being green, low-carbon, low-disturbance, and low-cost, making it an important means of soil remediation for contaminated sites.

[0003] Since petroleum hydrocarbons are organic compounds that can be degraded by native microorganisms in the environment, an appropriate monitoring system can be established to determine whether petroleum hydrocarbons in soil exhibit natural decay and the strength of this decay trend. This allows for the development of reasonable pollution control decisions and avoids unnecessary human intervention. Traditional methods for assessing the natural decay of petroleum hydrocarbon pollution in site soil primarily rely on chromatography or mass spectrometry to test changes in petroleum hydrocarbon levels, combined with electron acceptor, microbial, or isotopic data, to comprehensively determine whether a natural decay trend exists. However, traditional identification methods suffer from drawbacks such as large sampling volumes, complex pretreatment processes, and high costs. Currently, there is an urgent need for green and low-cost methods to assess the natural decay of petroleum hydrocarbon pollution in soil. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for assessing the natural attenuation of soil petroleum hydrocarbon pollution based on the ratio of fluorescent components.

[0005] A method for assessing the natural attenuation of petroleum hydrocarbon pollution in soil based on fluorescent components includes the following steps: Obtain the three-dimensional fluorescence spectrum of dissolved organic matter (DOM) in petroleum hydrocarbon-contaminated soil; The fluorescence intensity of various fluorescent components was obtained by analyzing the three-dimensional fluorescence spectrum. The intensity of natural decay of petroleum hydrocarbons in the contaminated soil was assessed based on the differences in fluorescence intensity among various fluorescent components.

[0006] Note: The above method obtains the fluorescence intensity of multiple fluorescent components of dissolved organic matter, and then assesses the natural decay of petroleum hydrocarbons, providing an intuitive and quantifiable assessment method for monitoring the natural decay of petroleum hydrocarbon contaminated sites in soil. This method breaks through the traditional assessment method, and can more conveniently, cost-effectively and accurately determine whether petroleum hydrocarbons can decay naturally in soil and the intensity of decay, thus creating a new way to assess the natural decay of petroleum hydrocarbon pollution in soil.

[0007] Furthermore, the sources of pollution in the petroleum hydrocarbon-contaminated soil include petroleum refining enterprises, gas stations, petroleum storage / transfer stations, and timber preservation enterprises.

[0008] Note: The above-mentioned companies are involved in the generation, transportation, processing and use of petroleum hydrocarbons, which may cause soil pollution.

[0009] Furthermore, the multiple fluorescent components include tryptophan-like components (C1), petroleum-derived components (C2), and humic acid-like components (C3).

[0010] Note: The above components provide tryptophan-like signals that indicate the degree of pollution, petroleum-derived components that directly indicate the occurrence and transformation products of pollution, and humic acid-like signals that reflect the stable natural dissolved organic matter in the soil, providing key evidence for the effectiveness assessment of natural degradation processes.

[0011] Furthermore, the acquisition of the fluorescence spectrum of dissolved organic matter in petroleum hydrocarbon-contaminated soil includes: Take the sample of the petroleum hydrocarbon-contaminated soil, add the sample of the petroleum hydrocarbon-contaminated soil to water at a ratio of 1g:5~10mL, shake in the dark for 12~30h, then centrifuge at 2000~5000r / min for 15~30min to obtain a mixture, filter the mixture with a 0.22μm filter membrane to obtain a filtrate containing dissolved organic matter; The three-dimensional fluorescence spectrum of the filtrate was determined using a fluorescence spectrophotometer.

[0012] Note: The above process ensures the reliability and repeatability of fluorescence spectral data by controlling parameters such as soil-to-water ratio, shaking extraction time, centrifugation speed, and filter pore size, laying a reliable experimental foundation for accurately evaluating the natural decay process.

[0013] Furthermore, the three-dimensional fluorescence spectrum was analyzed using parallel factor analysis to establish a fluorescent component model of soluble organic matter and obtain the fluorescence intensity of each fluorescent component.

[0014] Note: Parallel factor analysis can accurately isolate and quantify the intensity of each independent fluorescent component from complex spectral signals, effectively overcoming the signal overlap interference problem commonly found in traditional analysis. This enables the precise identification and quantification of various DOM fluorescent components (such as tryptophan-like components, petroleum-derived components, and humic acid-like components). The fluorescence intensities of the tryptophan component (C1), petroleum-derived component (C2), and humic acid-like component (C3) are denoted as Fmax-C1, Fmax-C2, and Fmax-C3, respectively.

[0015] Furthermore, the difference between the various fluorescent components is the difference between the relative fluorescence intensity ratio of the petroleum-derived components (%Fmax-C2) and the relative fluorescence intensity ratio of the tryptophan-like components (%Fmax-C1). The relative fluorescence intensity ratio is the percentage of the normalized fluorescence intensity of each component to the total normalized fluorescence intensity in the petroleum hydrocarbon-contaminated soil, i.e., %Fmax-C1 = Fmax-C1 / (Fmax-C1 + Fmax-C2 + Fmax-C3). %Fmax-C2 and %Fmax-C3 are calculated similarly.

[0016] Note: The method described above, which calculates the difference in the normalized relative intensity ratios of petroleum-derived components and tryptophan-like components, transforms complex spectral information into a concise and highly sensitive indicator.

[0017] Further, the difference between the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component includes: the ratio between the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component, or the ratio between the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the petroleum-derived component, or the difference between the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component, or the ratio between the sum and difference of the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component, or the ratio between the difference and the sum of the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component.

[0018] Note: The above lists various values ​​to quantify the intensity relationship between the two key components, ensuring the reliability and accuracy of the assessment conclusions.

[0019] Furthermore, assessing the ability of microorganisms in the contaminated soil to degrade petroleum hydrocarbons (i.e., the intensity of natural decay of petroleum hydrocarbon pollution) based on the differences in fluorescence intensity among multiple fluorescent components includes: When the ratio between the relative fluorescence intensity ratio (%Fmax-C2) of the petroleum-derived component and the relative fluorescence intensity ratio (%Fmax-C1) of the tryptophan-like component is greater than 0 and less than or equal to 1, the activity of petroleum hydrocarbon-degrading microorganisms is not significant, i.e. there is no significant natural decay. When the ratio between the relative fluorescence intensity ratio (%Fmax-C2) of the petroleum-derived component and the relative fluorescence intensity ratio (%Fmax-C1) of the tryptophan-like component is greater than 1 and less than or equal to 2, the petroleum hydrocarbon degrading microorganisms begin to be active, that is, the natural decay ability of petroleum hydrocarbons is weak. When the ratio between the relative fluorescence intensity ratio (%Fmax-C2) of the petroleum-derived component and the relative fluorescence intensity ratio (%Fmax-C1) of the tryptophan-like component is greater than 2 and less than or equal to 3, the petroleum hydrocarbon degrading microorganisms are relatively active, that is, the natural decay capacity of petroleum hydrocarbons is moderate. When the ratio between the relative fluorescence intensity ratio (%Fmax-C2) of the petroleum-derived component and the relative fluorescence intensity ratio (%Fmax-C1) of the tryptophan-like component is greater than 3, the petroleum hydrocarbon degrading microorganisms are very active, that is, the petroleum hydrocarbons have a strong natural decay ability.

[0020] Note: The above method establishes a clear numerical threshold range, corresponding the proportion of fluorescence signal to the level of natural decay, which makes the evaluation process highly standardized and improves the operability and decision-making efficiency of the method in field applications.

[0021] Furthermore, it also includes: the petroleum hydrocarbon contaminated soil is identified by fluorescent component recognition; the fluorescent component recognition method includes: Target soil was collected from the area to be tested, and background soil of the same amount was collected from the background area near the area to be tested. The three-dimensional fluorescence spectra of dissolved organic matter in the target soil and the background soil were obtained respectively. The relative fluorescence intensity ratios of tryptophan-like components and petroleum hydrocarbon-derived components in the target soil and the background soil were obtained by analyzing the three-dimensional fluorescence spectra respectively. The presence of pollution in petroleum hydrocarbon-contaminated soil can be determined by the difference between the relative fluorescence intensity ratios of tryptophan-like components and petroleum hydrocarbon-derived components in the target soil and the background soil.

[0022] Note: The above method provides an efficient initial screening tool for quickly identifying and prioritizing suspected contaminated areas in actual sites.

[0023] The beneficial effects of this invention are: The method of this invention obtains the fluorescence intensity of multiple fluorescent components of dissolved organic matter, and then assesses the natural decay of petroleum hydrocarbons, providing an intuitive and quantifiable way to assess the degradation of petroleum hydrocarbon pollution in soil. This method breaks through the traditional assessment method and can more conveniently, cost-effectively and accurately determine whether petroleum hydrocarbons can decay naturally in soil and the intensity of decay, thus creating a new way to assess the natural decay of petroleum hydrocarbon pollution in soil. Attached Figure Description

[0024] Figure 1 This is a statistical distribution diagram of sampling points in the petroleum hydrocarbon contaminated soil profile in this embodiment of the invention; Figure 2 The soil contaminated with petroleum hydrocarbons (C) in the embodiments of the present invention is contaminated with petroleum hydrocarbons (C). 10 -C 40 Correlation analysis diagram of ) content, DOM fluorescence parameters and microbial community; Figure 3 This is a box plot of the diversity estimation index (Chao1) and the observed species index in different groups of petroleum hydrocarbon contaminated samples in this embodiment of the invention; Figure 4 This is a hierarchical clustering analysis (HCA) dendrogram based on fluorescence parameters in an embodiment of the present invention; Figure 5 This is a box plot of fluorescence intensity ratio (%Fmax) of different groups of petroleum hydrocarbon contaminated samples in the embodiments of the present invention. Figure 6 (a) shows the top 10 bacterial genera and petroleum hydrocarbons (C) in the soil samples of this embodiment of the invention. 10 -C 40 (a) Spearman correlation heatmap of petroleum hydrocarbon content and DOM parameters in different groups of petroleum hydrocarbon-contaminated soil samples; (b) Petroleum hydrocarbon (C) content in different groups of petroleum hydrocarbon-contaminated soil samples. 10 -C 40 A canonical correspondence analysis diagram of ) content, microbial community and DOM parameters; Figure 7 The embodiments of this invention are based on petroleum hydrocarbons (C 10 -C 40 Structural equation modeling of tryptophan content, DOM parameters, petroleum hydrocarbon-degrading bacteria, and humification index (HIX). In the DOM parameters, %Fmax-C1 and %Fmax-C2 represent the relative fluorescence intensity ratios of tryptophan-like substances and petroleum-derived components, respectively. Detailed Implementation

[0025] To further illustrate the methods and effects of this invention, the technical solution of this invention will be clearly and completely described below in conjunction with experiments.

[0026] In light of the background technology, monitoring natural degradation, as a low-cost and environmentally friendly technique, is widely used in the remediation of contaminated sites. The key to its implementation lies in verifying, through multiple sources of evidence, that pollutants in the soil exhibit natural degradation behavior (i.e., degradation of pollutants by indigenous microorganisms). Typically, evidence of natural degradation in contaminated soil includes three aspects: Primary evidence directly demonstrates that pollution is decreasing: i.e., pollutant concentration trend analysis, pollutant mass conservation and mass flux analysis, and solute transport models (analytical / numerical). Secondary evidence demonstrates that favorable environmental conditions for biodegradation have been established: such as changes in the concentrations of dissolved oxygen, nitrate, sulfate, iron ions, methane, etc. (electron acceptor / donor concentrations), and stable isotope analysis. Tertiary evidence mechanistically demonstrates the existence of microorganisms with degradation capabilities: microbiological methods such as detecting specific degradation functional genes, microbial community structure analysis (e.g., 16S rRNA sequencing), and microcosm experiments.

[0027] Dissolved organic matter (DOM) refers to organic compounds that can dissolve in water. It mainly originates from soil organic matter, plant residues, microbial activity, and human activities. Its composition is complex, primarily including humus, fulvic acid, amino acids, sugars, and proteins, and it plays a crucial role in biogeochemical cycles and the ecological environment. Studies have found that exogenous pollution inputs caused by human activities and microbial life activities can significantly alter the content and composition of soil DOM.

[0028] In the soil of petroleum hydrocarbon contaminated sites, dissolved organic matter typically comes from the following three sources: (1) Natural organic matter such as plant litter on the ground is weathered and leached into the soil system.

[0029] (2) Petroleum itself contains a large amount of organic matter, and petroleum hydrocarbons produced by petroleum refining also belong to organic matter, and there are soluble parts in both.

[0030] (3) Dissolved organic matter produced by microbial metabolism. During the process of microbial metabolism, natural dissolved organic matter and dissolved organic matter from petroleum hydrocarbons can be used to produce new microbial-derived dissolved organic matter.

[0031] This study found that although all three types of dissolved organic matter are soluble organic matter, their fluorescent components exhibit distinct characteristics. All three types of soluble organic matter are present when soil is contaminated with petroleum hydrocarbons. Detecting characteristic soluble organic matter components using fluorescence methods can help determine the degree of petroleum hydrocarbon pollution and the intensity of natural decay.

[0032] Therefore, combining the evidence of natural attenuation in the soil from the three aspects mentioned above, as well as the dissolved organic matter in the soil, this invention provides a method for assessing the natural attenuation of petroleum hydrocarbon pollution in soil based on fluorescent components, as specifically illustrated in the following embodiments: Example 1: A method for assessing the natural attenuation of petroleum hydrocarbon pollution in soil based on fluorescent components, comprising the following steps: S1. Obtain the three-dimensional fluorescence spectrum of dissolved organic matter in petroleum hydrocarbon-contaminated soil; Combination Figure 1 As shown, the sources of pollution in the petroleum hydrocarbon-contaminated soil include petroleum refining enterprises, gas stations, petroleum storage / transfer stations, and wood preservation enterprises.

[0033] The method of obtaining the fluorescence spectrum of dissolved organic matter in petroleum hydrocarbon-contaminated soil includes: Take the sample of the petroleum hydrocarbon-contaminated soil, add the sample of the petroleum hydrocarbon-contaminated soil to water at a ratio of 1g:10mL, shake in the dark for 24h, and then centrifuge at 5000r / min for 15min to obtain a mixture. Filter the mixture with a 0.22μm filter membrane to obtain a filtrate containing dissolved organic matter. The three-dimensional fluorescence spectrum of the filtrate was determined using a fluorescence spectrophotometer.

[0034] The petroleum hydrocarbon-contaminated soil was identified by fluorescent component identification; the fluorescent component identification method includes: Target soil (soil used to detect whether there is pollution in the test area) was collected from the test area, and background soil (soil that has not been contaminated by petroleum hydrocarbons) of the same amount was collected from the background area near the test area. The three-dimensional fluorescence spectra of dissolved organic matter in the target soil and the background soil were obtained respectively. The relative fluorescence intensity ratios of tryptophan-like components and petroleum hydrocarbon-derived components in the target soil and the background soil were obtained by analyzing the three-dimensional fluorescence spectra respectively. The presence of petroleum hydrocarbon contaminated soil is determined by the difference between the relative fluorescence intensity ratios of tryptophan-like components and petroleum hydrocarbon-derived components in the target soil and the background soil. Specifically, the difference indicates the presence of contamination if the percentages of tryptophan-like fluorescent components and petroleum hydrocarbon-derived components in the target soil are significantly greater than (mean background value ± 2 standard deviations) in the background soil. Furthermore, a higher ratio of tryptophan-like components to petroleum hydrocarbon-derived components indicates more severe contamination. A higher ratio of petroleum hydrocarbon-derived components to tryptophan-like fluorescent components indicates greater natural decay intensity. Generally, the longer the natural decay process, the stronger the natural decay intensity of petroleum hydrocarbons (C6N2). 10 -C 40 The higher the degree of degradation, the greater the proportion of petroleum hydrocarbon-derived components. Therefore, areas with a longer history of pollution will have a higher proportion of petroleum hydrocarbon-derived components / tryptophan-like fluorescent components compared to areas with a shorter history of pollution.

[0035] For example, this embodiment uses a southern region affected by petroleum hydrocarbons (C 10 -C 40The study area was a contaminated timber preservative plant site. Three soil profiles (DH1, 14.0 m deep; DH2 and DH3, 7.0 m deep, all above the groundwater level) were collected in the unsaturated zone, yielding a total of 31 soil samples. Petroleum hydrocarbons (C...) 10 -C 40 The concentration was determined by gas chromatography (GC-2030AF), with a detection limit of 6 mg / kg. Three-dimensional fluorescence parallel factor analysis (EEM-PARAFAC) was used to analyze soil DOM components, combined with 16S rRNA gene sequencing analysis of the microbial community. As an extension study, hierarchical cluster analysis (HCA), canonical correspondence analysis (CCA), and partial least squares structural equation modeling (PLS-SEM) were used to explore the response mechanism.

[0036] S2. Analyze the three-dimensional fluorescence spectrum to obtain the fluorescence intensity of various fluorescent components; The various fluorescent components include tryptophan-like components, petroleum-derived components, and humic acid-like components.

[0037] The three-dimensional fluorescence spectrum was analyzed using parallel factor analysis to obtain the fluorescence intensity of various fluorescent components.

[0038] For example, in this embodiment, EEM measurement is performed using a fluorescence spectrophotometer (F-4600, Hitachi). EEM correction: blank subtraction, internal filter effect correction, scattering removal, and fluorescence intensity normalized to Raman units (RU).

[0039] PARAFAC modeling: using the R language package "staRdom". Remove noise with an wavelength greater than 580 nm.

[0040] Model validation: Splitting analysis, Tucker consistency coefficient. Three fluorescent components were identified (matched with the OpenFluor database, similarity > 0.90): The various fluorescent components specifically include: C1 (Ex / Em=290 / 334nm): Tryptophan-like component.

[0041] C2 (Ex / Em=250, 290 / 360, 346nm): Components related to petroleum pollutants and their degradation products.

[0042] C3 (Ex / Em=250 / 370nm): Humic acid-like component.

[0043] Calculation parameters: Fmax represents the normalized fluorescence intensity of each component; %Fmax represents the percentage of each component's fluorescence intensity to the total fluorescence intensity; %Fmax-C2 is the percentage of petroleum-derived component Fmax to the total Fmax (i.e., relative fluorescence intensity ratio); %Fmax-C1 is the percentage of tryptophan-like component Fmax to the total Fmax (i.e., relative fluorescence intensity ratio); in the next step, the ratio (%Fmax-C2) / (%Fmax-C1) is used to assess the natural decay of petroleum hydrocarbons in the soil.

[0044] S3. Based on the differences in fluorescence intensity among various fluorescent components, assess whether petroleum hydrocarbons in the contaminated soil can decay naturally and the intensity of natural decay.

[0045] The difference between the various fluorescent components is the difference between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components. The relative fluorescence intensity ratio is the percentage of the normalized fluorescence intensity of each component to the total normalized fluorescence intensity in the petroleum hydrocarbon-contaminated soil.

[0046] The difference between the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component includes: the ratio between the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component, or the ratio between the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the petroleum-derived component, or the difference between the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component, or the ratio between the sum and difference of the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component, or the ratio between the difference and sum of the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component.

[0047] This embodiment uses the ratio between the relative fluorescence intensity ratio of the petroleum-derived component and the relative fluorescence intensity ratio of the tryptophan-like component, specifically: Furthermore, assessing the ability of microorganisms in the contaminated soil to degrade petroleum hydrocarbons (i.e., the intensity of natural decay of petroleum hydrocarbon pollution) based on the differences in fluorescence intensity among multiple fluorescent components includes: When the ratio of the relative fluorescence intensity ratio of the petroleum-derived component to the relative fluorescence intensity ratio of the tryptophan-like component is greater than 0 and less than or equal to 1, the activity of petroleum hydrocarbon-degrading microorganisms is not significant, i.e. there is no significant natural decay. When the ratio of the relative fluorescence intensity ratio of the petroleum-derived component to the relative fluorescence intensity ratio of the tryptophan-like component is greater than 1 and less than or equal to 2, the petroleum hydrocarbon degrading microorganisms begin to be active, that is, the natural decay ability of petroleum hydrocarbons is weak. When the ratio of the relative fluorescence intensity ratio of the petroleum-derived component to the relative fluorescence intensity ratio of the tryptophan-like component is greater than 2 and less than or equal to 3, the petroleum hydrocarbon degrading microorganisms are relatively active, that is, the natural decay capacity of petroleum hydrocarbons is moderate. When the ratio of the relative fluorescence intensity ratio of the petroleum-derived components to the relative fluorescence intensity ratio of the tryptophan-like components is greater than 3, the petroleum hydrocarbon-degrading microorganisms are very active, that is, the petroleum hydrocarbons have a strong natural decay ability.

[0048] The experimental basis for this invention embodiment is as follows: Taking the soil and experimental analysis results in S1 and S2 above as examples, the data processing and the resulting experimental results are as follows: 1) Data processing: Hierarchical clustering analysis (HCA): Clustering soil samples based on DOM feature parameters. Euclidean distance and Ward's method can be used.

[0049] Difference testing: Wilcoxon rank sum test to compare petroleum hydrocarbons (C) between different clusters. 10 -C 40 Differences in content, DOM parameters, and microbial diversity index.

[0050] Correlation analysis: Spearman correlation analysis and Mantel test. The R language package "corrplot" can be used for analysis.

[0051] Canonical correspondence analysis (CCA): This analyzes the relationship between environmental variables and microbial communities and DOM (microbial community and DOM). It can be performed using the R language package "vegan".

[0052] Partial Least Squares Structural Equation Modeling (PLS-SEM): Constructs a structural equation model to quantify path relationships. Analysis can be performed using SmartPLS4 software.

[0053] 2) Results Analysis: The PLS-SEM model shows: Direct effects: Petroleum hydrocarbons (C 10 -C 40 Pollution directly leads to an increase in %Fmax-C1 and %Fmax-C2, and a decrease in HIX (humification level).

[0054] Indirect effects: Petroleum hydrocarbons (C 10 -C 40Pollution indirectly leads to an increase in %Fmax-C2 and a decrease in HIX by stimulating the metabolic activity of hydrocarbon-degrading bacteria.

[0055] The ratio of (%Fmax-C2) / (%Fmax-C1) was significantly positively correlated with the abundance of petroleum hydrocarbon-degrading bacteria, but not with the abundance of petroleum hydrocarbons (C1). 10 -C 40 There was no direct and significant correlation between the concentration and the concentration.

[0056] The Biogenicity Index (BIX): The ratio of fluorescence intensity at an emission wavelength (Em) of 380 nm to that at 430 nm when the excitation wavelength (Ex) is 310 nm. A BIX > 1 indicates that the DOM (Dissolved Oxygenate) is primarily derived from microbial activity. The Humus Index (HIX): The ratio of fluorescence intensity area in the emission wavelength (Em) region of 435-480 nm to that in the region of 300-345 nm when the excitation wavelength (Ex) is 254 nm. A high HIX indicates a high degree of humification and more stable DOM properties.

[0057] ① Petroleum hydrocarbons (C 10 -C 40 The correlation between microbial communities and soil DOM (e.g.) Figure 2 (as shown) With petroleum hydrocarbons (C 10 -C 40 The concentrations of Fmax-C1, Fmax-C2, Fmax-C3, %Fmax-C1, %Fmax-C2, and BIX showed a significant positive correlation (indicating that pollution enhances fluorescence intensity and increases the contribution of microbial sources).

[0058] With petroleum hydrocarbons (C 10 -C 40 The concentrations showed a significant negative correlation: %Fmax-C3,HIX (indicating that pollution reduced the proportion of humus and the degree of humification).

[0059] BIX is positively correlated with %Fmax-C1 and %Fmax-C2, and negatively correlated with %Fmax-C3 and HIX (indicating that %Fmax-C1 and %Fmax-C2 are associated with microbial activity).

[0060] Mantel test: Microbial community composition was significantly correlated with Fmax-C1, Fmax-C2, %Fmax-C1, %Fmax-C2, %Fmax-C3, BIX, and HIX.

[0061] ② HCA-based petroleum hydrocarbons (C 10 -C 40 Content distribution (combined with) Figure 4 (as shown) HCA divided the 31 samples into three clusters with different levels of contamination: Cluster A (Low Pollution): Petroleum hydrocarbons (C 10 -C 40 The median concentration was 57 mg / kg (range 21-240 mg / kg), with depths ranging from 2.0 to 3.6 m. No samples exceeded the limit.

[0062] Cluster B (Moderate Pollution): Petroleum Hydrocarbons (C 10 -C 40 The median concentration was 404 mg / kg (range 18-631 mg / kg), with a depth of 2.5-4.5 m. No samples exceeded the standard.

[0063] Cluster C (High Pollution): Petroleum hydrocarbons (C 10 -C 40 The median concentration was 765 mg / kg (range 84-2490 mg / kg) at depths of 4.5-14.0 m. 45% of the samples exceeded the soil screening value for Class I construction land in GB 36600 (>826 mg / kg).

[0064] ③ The influence of microbial community composition on petroleum hydrocarbons (C 10 -C 40 ) Pollution response (e.g. Figure 3 (as shown) Diversity: The diversity estimate index (Chao1) and observed species index of Cluster C (high pollution) were significantly lower than those of Cluster A (low pollution), indicating that pollution reduced microbial abundance. Cluster B was in a transitional state. Phylum level: Proteobacteria was the most abundant phylum in Cluster C (mean 49.0%, Cluster A: 7.5%, Cluster B: 17.7%), and is the major potential hydrocarbon-degrading phylum. Genus level: The key hydrocarbon-degrading genera *Pseudomonas* and *Sphingomonas* were the most abundant in Cluster C (mean 22.7% and 5.5%, Cluster A: 0.9% and 0.2%, Cluster B: 6.1% and 1.4%), indicating that pollution stimulated their growth and natural decline potential. *Acinetobacter* was also associated with petroleum hydrocarbons (C... 10 -C 40 Positively correlated with pollution.

[0065] ④ Characteristics of soil DOM fluorescence components (e.g.) Figure 5 (as shown) Component ratio (%Fmax): Cluster A (Low Pollution): High %Fmax-C3 (humic acid-like components, median 89.3%), low %Fmax-C1 (tryptophan-like components, 7.9%), very low %Fmax-C2 (petroleum-derived components, 0.0%). High HIX (high degree of humification), low BIX.

[0066] Cluster B (moderate contamination): %Fmax-C3 decreased (33.2%), while %Fmax-C1 (25.1%) and %Fmax-C2 (41.0%) increased. HIX decreased, and BIX increased.

[0067] Cluster C (high contamination): lowest %Fmax-C3 (16.0%), highest %Fmax-C1 (28.1%) and %Fmax-C2 (55.1%). HIX is the lowest, BIX is the highest (indicating that the DOM is mainly from microbial sources and has active metabolism).

[0068] (%Fmax-C2) / (%Fmax-C1) ratio: Clusters B and C are significantly higher than Cluster A. Although Cluster B has higher petroleum hydrocarbon (C) ratios... 10 -C 40 The concentration of C1 was lower than that of Cluster C, but its %Fmax-C2 and (%Fmax-C2) / (%Fmax-C1) ratios were significantly higher than those of Cluster A, and the abundance of petroleum hydrocarbon degrading bacteria was also higher, indicating that Cluster B had experienced high pollution and initiated natural decay (manifested as an increase in the proportion of C2 component).

[0069] ⑤ Soil DOM on petroleum hydrocarbons (C 10 -C 40 ) Pollution response mechanisms (such as Figure 6 , Figure 7 (as shown) Spearman correlation and canonical correspondence analysis ( Figure 6 Petroleum hydrocarbons (C) 10 -C 40 The concentration of hydrocarbon-degrading bacteria (Pseudomonas, Sphingomonas, Acinetobacter) was positively correlated with the concentration of hydrocarbon-degrading bacteria. Hydrocarbon-degrading bacteria were positively correlated with %Fmax-C1, %Fmax-C2, (%Fmax-C2) / (%Fmax-C1), and BIX, and negatively correlated with %Fmax-C3 and HIX. HIX clustered with %Fmax-C2 and (%Fmax-C2) / (%Fmax-C1), indicating a strong correlation with microbial metabolic activity. CCA showed that petroleum hydrocarbons (C... 10 -C 40), %Fmax-C1, and %Fmax-C2 are positively correlated; %Fmax-C2 and ((%Fmax-C2) / (%Fmax-C1)) are positively correlated with Pseudomonas abundance.

[0070] PLS-SEM model ( Figure 7 ): Quantified the direct and indirect effect pathways. Petroleum hydrocarbons (C 10 -C 40 Direct effects of pollution (path coefficients, p<0.001): Significantly increased %Fmax-C1 (+0.498) and %Fmax-C2 (+0.416). Significantly decreased HIX (-0.509) (i.e., reduced humification). Significantly increased abundance of petroleum hydrocarbon-degrading bacteria (+0.327, p<0.05).

[0071] Direct effects of petroleum hydrocarbon-degrading bacteria activity: Significantly increased %Fmax-C2 (+0.348, p<0.001) and (%Fmax-C2) / (%Fmax-C1) (+0.423, p<0.001). Significantly decreased HIX (-0.224, p<0.01). No significant direct effect on %Fmax-C1 (+0.027, p>0.05). The (%Fmax-C2) / (%Fmax-C1) ratio was primarily driven by the direct effect of petroleum hydrocarbon-degrading bacteria activity (+0.423), which was far greater than the effect of petroleum hydrocarbon (C) activity. 10 -C 40 The direct effect of concentration (+0.224, not significant p>0.05).

[0072] The following mechanism can be summarized from the above content: Direct effects (physical mixing): petroleum hydrocarbons (C 10 -C 40 It enters the soil and directly alters the DOM composition, increasing the fluorescence intensity of tryptophan-like and petroleum hydrocarbon-derived components, and reducing the degree of humification (HIX).

[0073] Indirect effects (microbial-mediated): Petroleum hydrocarbons (C 10 -C 40 This stimulates the growth of petroleum hydrocarbon-degrading bacteria. The activity of these bacteria produces more petroleum hydrocarbon-derived components associated with petroleum degradation. It enhances microbial metabolism, consumes small molecule precursors, and inhibits their conversion to humic acid-like substances, further reducing the degree of humification (HIX). This leads to a significant increase in the (%Fmax-C2) / (%Fmax-C1) ratio, which mainly reflects the degradation of petroleum hydrocarbons by microorganisms (C... 10 -C 40 (Activities)

[0074] This invention provides a method for rapidly identifying petroleum hydrocarbons (C). 10-C 40 The significance of pollution and its natural decay is as follows: A two-stage assessment framework based on DOM fluorescence characteristics is proposed: 1. Physical stage (recent pollution): After petroleum hydrocarbon pollution enters the soil, it rapidly alters the DOM through processes such as dissolution / emulsification, leading to a significant increase in %Fmax-C1. This is a general indicator of pollution presence. 2. Biological response stage (days / weeks after pollution to historical pollution): Indigenous microorganisms are activated and begin to degrade petroleum hydrocarbon pollution. Their metabolites alter DOM fluorescence, resulting in a continuous increase in the (%Fmax-C2) / (%Fmax-C1) ratio. This ratio reflects the intensity of microbial degradation activity and the initiation / sustainability of the natural decay process.

[0075] The above experiments show that petroleum hydrocarbons (C 10 -C 40 Petroleum hydrocarbon pollution significantly reduced microbial diversity (e.g., the Chao1 index) but increased the abundance of potential petroleum hydrocarbon-degrading bacteria (e.g., *Pseudomonas* and *Sphingomonas*). 10 -C 40 The concentration of DOM was significantly positively correlated with total fluorescence intensity, %Fmax-C1, %Fmax-C2, and biogenic index (BIX), and significantly negatively correlated with %Fmax-C3 and humification index (HIX).

[0076] Soil DOM for petroleum hydrocarbons (C 10 -C 40 Pollution exhibits both direct (physical mixing) and indirect (microbial-mediated) responses. %Fmax-C1 (the proportion of tryptophan-like components) can serve as a potential indicator for rapid screening of soil petroleum hydrocarbon pollution (the higher the proportion, the more severe the pollution). The (%Fmax-C2) / (%Fmax-C1) ratio (the ratio of petroleum-derived components to tryptophan-like components) can serve as a potential indicator for monitoring the intensity of natural petroleum hydrocarbon degradation (a higher ratio indicates more active microbial degradation activity). These findings provide new and rapid spectroscopic-based methods for monitoring and assessing in-situ microbial remediation.

[0077] Example 2 differs from Example 1 in that a sample of the petroleum hydrocarbon-contaminated soil was taken and added to water at a ratio of 1g:5mL. The mixture was shaken in the dark for 12 hours and then centrifuged at 4000r / min for 20 minutes to obtain a mixed solution.

[0078] Example 3 differs from Example 1 in that a sample of the petroleum hydrocarbon-contaminated soil was taken and added to water at a ratio of 1g:8mL. The mixture was shaken in the dark for 30 hours and then centrifuged at 2000r / min for 30 minutes to obtain a mixed solution.

Claims

1. A method for assessing natural attenuation of soil petroleum hydrocarbon pollution based on fluorescent components, characterized in that, The method comprises the following steps: acquiring a three-dimensional fluorescence spectrum of dissolved organic matter in the oil-contaminated soil; analyzing the three-dimensional fluorescence spectrum to obtain fluorescence intensities of multiple fluorescence components; evaluating the strength of natural attenuation of the oil in the oil-contaminated soil according to the difference between the fluorescence intensities of the multiple fluorescence components.

2. A method for assessing natural attenuation of soil petroleum hydrocarbon pollution based on fluorescent components according to claim 1, characterized in that, The oil-contaminated soil is from petrochemical enterprises, wood preservation enterprises, and oil storage / transshipment stations.

3. A method for assessing natural attenuation of soil petroleum hydrocarbon pollution based on fluorescent components according to claim 1, characterized in that, The method for acquiring the fluorescence spectrum of the dissolved organic matter in the oil-contaminated soil comprises: taking a sample of the oil-contaminated soil, adding the sample into water at a ratio of 1 g:5-10 mL, oscillating in the dark for 12-30 hours, then centrifuging at a speed of 2000-5000 r / min for 15-30 minutes to obtain a mixed solution, filtering the mixed solution with a 0.22 μm filter membrane to obtain a filtrate containing the dissolved organic matter; measuring the three-dimensional fluorescence spectrum of the filtrate with a fluorescence spectrophotometer.

4. A method for assessing natural attenuation of soil petroleum hydrocarbon pollution based on fluorescent components according to claim 1, characterized in that, analyzing the three-dimensional fluorescence spectrum with a parallel factor analysis method to obtain the fluorescence intensities of the multiple fluorescence components.

5. A method for assessing natural attenuation of soil petroleum hydrocarbon pollution based on fluorescent components according to claim 1, characterized in that, The multiple fluorescence components include tryptophan-like components, petroleum-derived components, and humic acid-like components.

6. A method for assessing natural attenuation of soil petroleum hydrocarbon pollution based on fluorescent components according to claim 5, characterized in that, The difference between the multiple fluorescence components is the difference between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components, wherein the relative fluorescence intensity ratio is the percentage of the normalized fluorescence intensity of each component in the total normalized fluorescence intensity of the oil-contaminated soil.

7. A method for assessing natural attenuation of soil petroleum hydrocarbon pollution based on fluorescent components according to claim 6, characterized in that, The difference between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components includes the ratio between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components, or the ratio between the relative fluorescence intensity ratio of the tryptophan-like components and the relative fluorescence intensity ratio of the petroleum-derived components, or the difference between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components, or the ratio of the sum and the difference between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components, or the ratio of the difference and the sum between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components.

8. A method for assessing natural attenuation of soil petroleum hydrocarbon pollution based on fluorescent components according to claim 7, characterized in that, The evaluation of the strength of natural attenuation of the oil in the oil-contaminated soil according to the difference between the fluorescence intensities of the multiple fluorescence components comprises: when the ratio between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components is greater than 0 and less than or equal to 1, the oil has no significant natural attenuation; when the ratio between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components is greater than 1 and less than or equal to 2, the oil has weak natural attenuation ability; when the ratio between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components is greater than 2 and less than or equal to 3, the oil has moderate natural attenuation ability; when the ratio between the relative fluorescence intensity ratio of the petroleum-derived components and the relative fluorescence intensity ratio of the tryptophan-like components is greater than 3, the oil has strong natural attenuation ability.

9. A method for assessing natural attenuation of soil petroleum hydrocarbon pollution based on fluorescence components according to claim 7, characterized in that, The method further comprises: The petroleum hydrocarbon contaminated soil is identified by a fluorescence component; The fluorescence component identification method comprises: Taking equal amounts of target soil and background soil, three-dimensional fluorescence spectra of dissolved organic matters in the target soil and the background soil are obtained respectively; The three-dimensional fluorescence spectra are analyzed to obtain relative fluorescence intensity ratios of tryptophan-like components and petroleum hydrocarbon derived components in the target soil and the background soil respectively; Whether the target soil is contaminated by petroleum hydrocarbon is determined according to the relative fluorescence intensity ratios of the tryptophan-like components in the target soil and the background soil.

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