A method for adjusting soil microenvironment to improve the degradation rate of phthalate

Abstract

The application discloses a soil microenvironment adjusting method for improving the degradation speed of phthalate esters, and comprises the following steps: step S1, soil sampling; step S2, crushing one of the samples in step S1 into small particles; step S3, detecting the content of phthalate esters in the small particle sample; step S4, grouping the other samples in step S2; step S5, respectively placing each group of samples in step S4 in different environments; step S6, crushing the samples in different environments into small particles; step S7, detecting the content of phthalate esters in the soil crushed into small particles in step S6, and comparing the content with the content of phthalate esters in the sample in step S3; and step S8, constructing the soil environment according to the environment of the sample with the lowest content. The application can take multiple samples from the land to be adjusted, make targeted adjustment schemes according to different land plots, and further improve the treatment level of phthalate esters.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental protection, and particularly relates to a soil microenvironment adjustment method for improving the degradation speed of phthalate esters. BACKGROUND

[0002] Phthalate esters (PAEs) are a kind of environmental hormones. They can be used as pesticide carriers, insect repellents, cosmetics, lubricants and raw materials for detergents, among which the largest amount is plastic plasticizer. With the development of the plastic industry, the production and use of PAEs have increased linearly, and a large amount of PAEs have entered the environment. PAEs are difficult to degrade in the environment, leading to residues in soil, water and air. Phthalate esters have carcinogenicity and can cause damage to the endocrine system and nervous system of the human body. The United States Environmental Protection Agency (EPA) has listed six phthalate esters in the list of 129 priority pollutants, and three phthalate esters are included in the blacklist of environmental priority pollutants in China.

[0003] Phthalate esters (PAEs) are also typical environmental endocrine disruptors. They are stable in the environment, difficult to degrade and have a long residence time. PAEs mainly enter the environment through factory emissions, plastic waste incineration, automobile exhaust, agricultural films and coatings. PAEs have become one of the important pollutants that threaten the safety of the ecological environment. In addition, long-distance migration and deposition of the atmosphere significantly promote the accumulation of PAEs in soil, so soil becomes the final sink of PAEs, which seriously affects the soil environment.

[0004] Studies have shown that microbial degradation has become the main way to reduce PAEs in the environment. Bacteria have good PAEs tolerance and degradation capacity, and can participate in the degradation process of PAEs alone or as a bacterial community. The current PAEs degrading bacterial community is mainly divided into two types. One is a bacterial community enriched from environmental samples using PAES as a substrate. The other is an artificial constructed bacterial community. According to the source of the bacterial strains, the bacterial community can be divided into a specific bacterial community composed of single degrading bacteria isolated from the same natural bacterial community and a bacterial community composed of single degrading bacteria from different bacterial communities. Therefore, adjusting the soil environment and ensuring the growth environment of the bacterial community can effectively degrade phthalate esters. SUMMARY

[0005] The present application aims to provide a soil microenvironment adjustment method for improving the degradation speed of phthalate esters, so as to solve the problem that the in-situ soil is not suitable for the survival of microorganisms, resulting in slow degradation of phthalate esters in the prior art.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is:

[0007] A soil microenvironment adjustment method for improving the degradation speed of phthalate esters, comprising the following steps:

[0008] Step S1: sampling the target soil, and measuring the phthalate ester content, pH, total organic matter content, and other physicochemical properties of the soil;

[0009] Step S2: extracting total DNA from the soil sample, detecting soil microbial community structure, diversity, and functional gene information using high-throughput sequencing technology, and identifying microorganisms with phthalate ester degradation function;

[0010] Step S3: constructing a microbial community interspecies co-occurrence network, and screening collaborative bacteria that are strongly positively correlated with phthalate ester degrading bacteria using network analysis;

[0011] Step S4: determining key environmental factors that are strongly positively correlated with the relative abundance of phthalate ester degrading bacteria and their collaborative bacteria using Spearman correlation analysis;

[0012] Step S5: dividing the soil sample into multiple portions; crushing one of the portions into small particles, and detecting the content of phthalate esters;

[0013] Step S6: grouping the other samples in step S5;

[0014] Step S7: designing an orthogonal test for the other sample groups in step S6 according to the key environmental factors screened, adjusting the soil microenvironment conditions by adding exogenous substances, placing each group of samples in step S6 in different environments for 1-7 days to allow the soil microbial community to evolve in a direction conducive to phthalate ester degradation;

[0015] Step S8: crushing the samples in different environments into small particles;

[0016] Step S9: detecting the content of phthalate esters in the soil crushed into small particles in step S8, and comparing it with the content of phthalate esters in the sample in step S1;

[0017] Step S10: constructing a soil environment according to the environment of the sample with the lowest content, and adjusting the environmental conditions suitable for the growth of soil microbial communities with phthalate ester degradation function.

[0018] Further, in step S1, there is at least one target sampling site. When the number of sampling sites is greater than 1, the samples from multiple sampling sites are detected according to steps S1-S9, and the soil environment is constructed according to the environment of the sample with the lowest content from multiple sampling sites, and the environmental conditions suitable for microbial growth are adjusted to find the soil environment with the highest microbial content.

[0019] Further, the environment for the growth of the microorganism includes temperature, humidity, pH value, organic matter content and other physical and chemical factors of the soil.

[0020] The application discloses a method for adjusting a soil microenvironment to improve the degradation speed of phthalate esters, and a detection method for the content of the phthalate esters.

[0021] Step A1: dry, grind and screen small granular soil to obtain a sample;

[0022] Step A2: mix the measured sample with an extraction agent to a saturated state, and then remove solid impurities by centrifugation or filtration to obtain an extraction liquid;

[0023] Step A3: concentrate the extraction liquid to 1%-10% of the volume of the extraction liquid to obtain a concentrated solution of the target compound;

[0024] Step A4: purify the concentrated solution;

[0025] Step A5: analyze the purified sample by GC-MS or LC-MS, qualitatively analyze the specific phthalate ester contained in the sample based on the comparison of the retention time and the mass spectrum, and quantitatively obtain the content of the phthalate ester by comparing the area of the chromatographic peak with the known concentration of the standard sample.

[0026] Further, the extraction agent in step A2 is ethyl acetate or dichloromethane.

[0027] Further, the purification method of step A4 includes solid phase extraction (SPE) and liquid-liquid extraction.

[0028] Further, the method for obtaining the small granular soil sample is as follows: after the collected soil is naturally dried, stones and animal and plant residue foreign matters in the soil are removed, the soil is rolled by a mortar, uniformly mixed by passing through a 20-mesh screen, and then the soil passing through the 20-mesh screen is baked at 8-10 DEG C for 8-10 h, and then rolled by a mortar and passed through a 100-mesh screen, and then uniformly mixed and placed in a desiccator for standby.

[0029] Further, the temperature of the adjusted soil in step S7 is not more than 40 DEG C, the humidity is not more than 7%, the pH value is between 4.5-8.5, and the organic matter content is not more than 10%.

[0030] Compared with the prior art, the application has the advantages and positive effects that:

[0031] This invention analyzes samples from the site to be adjusted to identify suitable growth environment data for microorganisms in the site. Based on the suitable growth environment data, the environmental data of the local soil, including temperature, humidity, pH value, and organic matter content, are adjusted to improve the treatment level of phthalates in the site and protect the soil environment.

[0032] The present invention can also perform multi-site sampling on the site to be adjusted, and make targeted adjustment plans according to different plots, thereby further improving the treatment level of phthalates. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a flowchart of the soil microenvironment adjustment method of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1:

[0037] like Figure 1 As shown, a method for adjusting the soil microenvironment to improve the degradation rate of phthalates includes the following steps:

[0038] Step S1: Soil samples were taken from a facility vegetable field in a certain area. There was one sampling site. The phthalate content of the soil was measured to be ND-4987.6 μg·kg-1, with an average value of 658.7 μg·kg-1, an average pH of 6.8, and a total organic matter content of 1.8%.

[0039] Step S2: Extract total DNA from soil samples and use high-throughput sequencing technology to detect information such as soil microbial community structure, diversity, and functional genes to identify microorganisms with phthalate degradation function;

[0040] Step S3: Construct a co-occurrence network among microbial community species, and use network analysis to screen for cooperative bacteria that are strongly positively correlated with phthalate-degrading bacteria;

[0041] Step S4: Determine the key environmental factors that are strongly positively correlated with the relative abundance of phthalate-degrading bacteria and their collaborative bacteria using Spearman correlation analysis;

[0042] Step S5: Divide the soil sample into multiple portions; crush one of the portions into small particles and detect the content of phthalates;

[0043] Step S6: Group the other samples in step S5;

[0044] Step S7: Design an orthogonal test based on the key environmental factors screened out for the other samples in step S6, adjust the soil microenvironment conditions by adding exogenous substances, the temperature of the adjusted soil is 38℃, the humidity is not more than 6%, the pH value is between 5.8-6.5, and the organic matter content is 2%; then place each group of samples in step S6 in different environments for 1-7 days, so that the soil microbial community evolves in the direction conducive to phthalate degradation;

[0045] Step S8: Crush the samples in different environments into small particles;

[0046] Step S9: Detect the content of phthalates in the soil crushed into small particles in step S8 and compare it with the content of phthalates in the sample in step S1;

[0047] Step S10: Construct the soil environment according to the environment of the sample with the lowest content, and adjust the environmental conditions suitable for the growth of soil microbial communities with phthalate-degrading function.

[0048] A soil microenvironment adjustment method for improving the degradation speed of phthalates, the detection method of the content of phthalates comprising the following steps:

[0049] Step A1: Dry, grind and screen the small particle soil to obtain a sample;

[0050] Step A2: Mix the measured sample with an extraction agent to a saturated state, then remove the solid impurities by centrifugation or filtration method to obtain an extraction liquid;

[0051] Step A3: Concentrate the extraction liquid to a volume of 3% of the extraction liquid to obtain a concentrated liquid of the target compound;

[0052] Step A4: Purify the concentrated liquid;

[0053] Step A5: The purified sample is analyzed by GC-MS or LC-MS, and the specific phthalate ester contained in the sample is qualitatively analyzed based on the comparison of the retention time and the mass spectrum. The content of the phthalate ester is quantitatively obtained by comparing the area of the chromatographic peak with the known concentration of the standard sample.

[0054] In this embodiment, the extraction agent in step A2 is ethyl acetate or dichloromethane.

[0055] In this embodiment, the purification method of step A4 includes solid phase extraction (SPE) and liquid-liquid extraction.

[0056] In this embodiment, the method for obtaining small particle soil samples is as follows: after the collected soil is naturally dried, stones and animal and plant residues are removed, the soil is crushed with a mortar, mixed and passed through a 20-mesh sieve, the soil passing through the 20-mesh sieve is baked at 8-10°C for 8-10h, then crushed with a mortar and passed through a 100-mesh sieve, mixed and placed in a desiccator for use.

[0057] Example 2:

[0058] As shown in Figure 1 A method for adjusting the soil microenvironment to improve the degradation rate of phthalate esters, comprising the following steps:

[0059] Step S1: Soil sampling in a certain facility vegetable field, there are 8 sampling sites, the content of phthalate esters in the soil is measured to be ND-3864 μg·kg-1, the average value is 634.5 μg·kg-1, the average pH is 7.2, and the total organic matter content is 3.5%;

[0060] Step S2: Extracting total DNA from the soil sample, using high-throughput sequencing technology to detect soil microbial community structure, diversity and functional gene information, and identifying microorganisms with phthalate ester degradation function;

[0061] Step S3: Constructing a microbial community species co-occurrence network, and using network analysis to screen for collaborative bacteria that are strongly positively correlated with phthalate ester degrading bacteria;

[0062] Step S4: Using Spearman correlation analysis to determine the key environmental factors that are strongly positively correlated with the relative abundance of phthalate ester degrading bacteria and their collaborative bacteria;

[0063] Step S5: Dividing the soil sample into multiple portions; crushing one of the portions into small particles and detecting the content of phthalate esters;

[0064] Step S6: Grouping the other samples in step S5;

[0065] Step S7: Grouping the other samples of step S6 according to the key environmental factors screened, adjusting the soil microenvironment conditions by adding exogenous substances, adjusting the temperature of the soil to 40℃, the humidity to not more than 4%, the pH value to 7, and the organic matter content to not more than 2.5%, and then placing each group of samples of step S6 in different environments for 1-7 days to make the soil microbial community evolve in a direction conducive to the degradation of phthalate esters;

[0066] Step S8: Crushing the samples in different environments into small particles;

[0067] Step S9: Detecting the content of phthalate esters in the soil crushed into small particles in step S8 and comparing it with the content of phthalate esters in the sample in step S1;

[0068] Step S10: Constructing a soil environment according to the environment of the sample with the lowest content and adjusting the environmental conditions suitable for the growth of soil microbial communities with phthalate ester degradation function.

[0069] In this embodiment, the samples of 8 sampling sites are detected according to steps S1-S9, and the soil environment of the sample with the lowest content of multiple sampling sites is constructed, the environmental conditions suitable for microbial growth are adjusted, and the soil environment with the highest microbial content is found out.

[0070] A soil microenvironment adjustment method for improving the degradation speed of phthalate esters, the detection method of the content of phthalate esters comprising the following steps:

[0071] Step A1: Drying, grinding and screening the small particle soil to obtain a sample;

[0072] Step A2: Mixing the measured sample with an extraction agent to a saturated state, and then removing solid impurities by centrifugation or filtration to obtain an extraction liquid;

[0073] Step A3: Concentrating the extraction liquid to a volume of 4% of the extraction liquid to obtain a concentrated liquid of target compounds;

[0074] Step A4: Purifying the concentrated liquid;

[0075] Step A5: Analyzing the purified sample by GC-MS or LC-MS, qualitatively analyzing the specific types of phthalate esters contained in the sample based on the comparison of retention time and mass spectrum, and quantitatively obtaining the content of phthalate esters by comparing the area of the chromatographic peak with the known concentration of the standard sample.

[0076] In this embodiment, the extraction agent in step A2 is ethyl acetate or dichloromethane.

[0077] In the embodiment, the purification method of step A4 comprises solid phase extraction (SPE) and liquid-liquid extraction.

[0078] In the embodiment, the method for obtaining the small granular soil sample comprises the following steps: after the collected soil is naturally dried, stones and animal and plant residues in the soil are removed, the soil is rolled by a mortar, uniformly mixed by passing through a 20-mesh sieve, and then the soil passing through the 20-mesh sieve is baked at 8-10 ℃ for 8-10 h, and then the soil is rolled by the mortar, passed through a 100-mesh sieve, uniformly mixed, and then placed in a desiccator for standby.

[0079] Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor, any modification, equivalent replacement, improvement, etc., should be included in the protection scope of the present application.

Claims

1. A method for adjusting the microenvironment of soil to increase the degradation rate of phthalate esters, characterized by, The method comprises the following steps: Step S1: soil sampling, determining the content of phthalate esters, pH, and total organic matter content of the soil; Step S2: extracting total DNA from the soil sample, detecting the structure, diversity, and functional gene information of the soil microbial community by using high-throughput sequencing technology, and identifying microorganisms with phthalate ester degradation function; Step S3: constructing a microbial community interspecies co-occurrence network, and screening collaborative bacteria that are strongly positively correlated with phthalate ester degrading bacteria by using network analysis; Step S4: determining key environmental factors that are strongly positively correlated with the relative abundance of phthalate ester degrading bacteria and their collaborative bacteria by using Spearman correlation analysis; Step S5: dividing the soil sample into multiple parts; crushing one of the parts into small particles, and detecting the content of phthalate esters; Step S6: grouping the other samples in step S5; Step S7: designing an orthogonal test according to the key environmental factors screened, adjusting the soil microenvironment conditions by adding exogenous substances, placing each group of samples in step S6 in different environments for 1-7 days, so that the soil microbial community evolves in a direction conducive to phthalate ester degradation; Step S8: crushing the samples in different environments into small particles; Step S9: detecting the content of phthalate esters in the small particle soil in step S8, and comparing it with the content of phthalate esters in the sample in step S1; Step S10: constructing a soil environment according to the environment of the sample with the lowest content, and adjusting the environmental conditions suitable for the growth of soil microbial communities with phthalate ester degradation function; In step S1, there is at least one sampling site, when the number of sampling sites is greater than 1, the samples of multiple sampling sites are detected respectively according to steps S1-S9, then the soil environment is constructed according to the environment of the sample with the lowest content in multiple sampling sites, and the environmental conditions suitable for microbial growth are adjusted to find the soil environment with the highest microbial content; The microbial growth environment includes the temperature, humidity, pH value, and organic matter content of the soil; The adjusted soil in step S7 has a temperature not higher than 40℃, a humidity not greater than 7%, a pH value between 4.5 and 8.5, and an organic matter content not higher than 10%; The method for obtaining small particle soil samples comprises the following steps: The detection method of the content of phthalate esters comprises the following steps: Step A1: drying, grinding, and screening the small particle soil to obtain a sample; Step A2: mixing the measured sample with an extraction agent to a saturated state, and then removing solid impurities by centrifugation or filtration to obtain an extraction liquid; Step A3: concentrating the extraction liquid to a volume of 1%-10% of the extraction liquid to obtain a concentrated solution of target compounds; Step A4: purifying the concentrated solution; Step A5: The purified sample is analyzed by GC-MS or LC-MS, based on the comparison of retention time and mass spectrum, the specific phthalate ester contained in the sample is qualitatively analyzed, and the content of phthalate ester is quantitatively obtained by comparing the area of the chromatographic peak with the known concentration of the standard sample.

2. The method for adjusting the micro-environment of soil for increasing the degradation rate of phthalic acid esters according to claim 1, characterized by: The extraction agent in step A2 is ethyl acetate or dichloromethane.

3. The method for adjusting the micro-environment of soil for increasing the degradation rate of phthalic acid esters according to claim 1, characterized by: The purification method of step A4 includes solid phase extraction (SPE), liquid-liquid extraction.

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