A method for rapidly identifying the toxicity of environmental persistent free radicals by using soil microorganisms

Abstract

The present invention discloses a method for rapidly identifying the toxicity of environmental persistent free radicals by using soil microorganisms, belonging to the field of soil microorganism research. In the present invention, the changes in the activity indexes of soil microbial biomass carbon, soil microbial biomass nitrogen, soil sucrase (S-SC), urease (S-UE), and acid phosphatase (S-ACP) are measured to judge the toxic effects of environmental persistent free radicals (EPFRs) in the soil. The results show that after adding particles containing EPFRs, the microbial biomass and the activity of microbial enzymes in black soil and brown soil are reduced, indicating that EPFRs have a toxic effect, and this toxic effect on soil microorganisms increases with the increase in the signal intensity and particle dose of EPFRs; in general, from the results of microbial biomass and microbial enzyme activity analysis, EPFRs in the soil have a toxic effect.

Description

Technical Field

[0001] The present invention belongs to the field of soil microorganism research, and through the toxicity exposure test of microorganisms in soil by EPFRs, a method for rapidly identifying the toxicity of EPFRs by using microorganisms in soil is achieved. Background Art

[0002] In recent years, a new type of environmental risk substance, environmentally persistent free radicals (EPFRs), has attracted extensive attention from researchers. Compared with ordinary short-lived free radicals (such as the lifetime of ·OH, ~10 -9 seconds), the lifetime of EPFRs can reach several days or even months. EPFRs have been widely found in environmental media such as the atmosphere and soil. Conventional risk assessment methods based on pollutant concentration are not accurate enough because they ignore the toxicity contribution of EPFRs on particles. Therefore, it is particularly important to study the environmental behavior and effects of EPFRs for assessing the environmental risks of organic pollutants.

[0003] Currently, the research on the generation mechanism and environmental effects of EPFRs mainly focuses on high-temperature combustion systems, while EPFRs can also be generated under normal temperature conditions. It has been found that metal oxides in soil (such as iron, aluminum, and copper) can interact with organic pollutants in soil to widely generate EPFRs. The toxicity effects of EPFRs in organically polluted soil have gradually attracted attention. There are many types and large quantities of microorganisms in soil (such as bacteria, fungi, and actinomycetes). Microorganisms participate in the mineralization and humification of soil organic matter, as well as the decomposition, formation, and circulation of various substances in soil. In addition, microorganisms participate in the cycling of soil nutrients such as C, N, and P, and promote the absorption of nitrogen, phosphorus, and mineral elements by plants. Microorganisms in soil are highly sensitive to environmental stress, and the widely generated EPFRs in soil will surely affect the activities of soil microorganisms. However, there is currently no research on the toxicity effects of EPFRs in soil on soil microorganisms, which is particularly important for comprehensively understanding the environmental risks of EPFRs.

[0004] Soil microbial biomass carbon (MBC) represents the total carbon in soil microorganisms, is an easily available nutrient pool in soil and the driving force for organic matter decomposition and nitrogen mineralization, and the change amount can reflect the change of soil fertility and the pollution degree of soil. Soil microbial biomass nitrogen (MBN) refers to the total nitrogen in soil microorganisms. It is one of the important links in the transformation of organic-inorganic nitrogen in soil and an important reserve pool of soil available nitrogen. Soil enzyme activity represents the ability of soil enzymes to catalyze the transformation of substances. Among many soil enzymes, ecological enzymes related to the cycling of the three major elements C, N, and P in soil have attracted extensive attention. The enzyme activities of soil sucrase (S-SC), urease (S-UE), and acid phosphatase (S-ACP) can generally reflect the overall health status of soil.

[0005] Therefore, the present invention intends to select catechol (CT) as the parent organic pollutant for generating EPFRs. CT is a typical phenolic pollutant in soil. Considering that soil contains various metal oxides and the soil composition is complex, and for the convenience of research and the rich content of aluminum oxide in soil, 1% of Al2O3 is loaded on silica (SiO2) to prepare 1%-Al2O3-SiO2 (Si-Al) particles to simulate the generation of EFPRs by organic pollutants in soil. By measuring the changes in soil microbial biomass (MBC and MBN) and related metabolic enzyme (S-ACP, S-SC, S-UE) indicators after the addition of EPERs, the toxicity of EPERs in soil can be quickly identified; the development of the present invention can expand the understanding of the environmental risks of EPERs in soil. Summary of the Invention

[0006] The purpose of the present invention is to better explore the toxicity of environmentally persistent free radical toxicity (EPFRs) widely generated in organically polluted soil, and provides a toxicity exposure test of soil EPFRs to soil microorganisms, which is a method for identifying the toxicity of EPFRs in soil by using the changes in soil microbial biomass and enzyme activity; specifically: by measuring the changes in soil microbial biomass carbon, soil microbial biomass nitrogen, soil sucrase (S-SC), urease (S-UE), and acid phosphatase (S-ACP) activity indicators, the toxic effects of EPFRs in soil can be judged.

[0007] Preferably, the present invention determines the presence of EPFRs in soil by detecting the change in soil microbial biomass carbon in soil. As the concentration of EPFRs increases, the decrease in soil microbial biomass carbon (MBC) is more significant.

[0008] Preferably, the present invention determines the presence of EPFRs in soil by detecting the change in soil microbial biomass nitrogen in soil. As the concentration of EPFRs increases, the decrease in soil microbial biomass nitrogen (MBN) is more significant.

[0009] Preferably, the present invention determines the presence of EPFRs in soil by detecting the activities of S-ACP, S-SC, and S-UE in soil. As the concentration of EPFRs increases, the decrease in the activities of S-ACP, S-SC, and S-UE in soil is more significant.

[0010] The beneficial effects of the present invention are:

[0011] The present invention relates to a toxicity exposure test of EPFRs in soil to microorganisms. EPFRs can mediate the generation of ROS (such as ·OH, ·O2 -), thereby generating oxidative stress in the organism and causing toxic effects on the organism; in the toxicity experiment of soil microorganisms, different doses and signal intensities of EPFRs particles can be added to the soil, and the changes in soil microbial biomass (MBC, MBN) and soil microbial enzyme activities (S-ACP, S-SC, S-UE) after 3-day and 7-day exposure cultures can be observed to judge the toxic effects of EPFRs in the soil. Brief Description of the Drawings

[0012] For ease of explanation, the present invention will be described in detail by the following specific embodiments and accompanying drawings.

[0013] Figure 1 Schematic diagram of the experimental procedure flow of the present invention

[0014] Figure 2 EPR signals on Si-Al-CT particles at different reaction times of the present invention.

[0015] Figure 3 EPR signals on Si-CT particles at different reaction times of the present invention.

[0016] Figure 4 Change diagram of MBC in black soil after adding different types and doses of particles of the present invention for 3 days and 7 days.

[0017] Figure 5 Change diagram of MBC in brown soil after adding different types and doses of particles of the present invention for 3 days and 7 days.

[0018] Figure 6 Change diagram of MBN in black soil after adding different types and doses of particles of the present invention for 3 days and 7 days.

[0019] Figure 7 Change diagram of MBN in brown soil after adding different types and doses of particles of the present invention for 3 days and 7 days.

[0020] Figure 8 Change diagram of S-ACP in black soil after adding different types and doses of particles of the present invention for 3 days and 7 days.

[0021] Figure 9 Change diagram of S-ACP in brown soil after adding different types and doses of particles of the present invention for 3 days and 7 days.

[0022] Figure 10 Change diagram of S-SC in black soil after adding different types and doses of particles of the present invention for 3 days and 7 days.

[0023] Figure 11 Change diagram of S-SC in brown soil after adding different types and doses of particles of the present invention for 3 days and 7 days.

[0024] Figure 12Graph of the change in S-UE of black soil after adding different types and doses of particles of the present invention for 3 days and 7 days.

[0025] Figure 13 Graph of the change in S-UE of brown soil after adding different types and doses of particles of the present invention for 3 days and 7 days. Detailed implementation manners

[0026] The present invention will be described in detail below in conjunction with embodiments, so that those skilled in the art can better understand the present invention. However, the present invention is not limited to the following embodiments.

[0027] In order to verify the feasibility of the method described in the present invention, the following experiments were conducted in the present invention, as Figure 1 shown, including experimental materials, experimental methods and data processing.

[0028] The experimental materials described in this embodiment include the tested EPFRs particles and the tested natural soil:

[0029] The tested EPFRs particles: Si-Al particles were prepared by the hydrolysis adsorption calcination method; aluminum chloride (AlCl3) was hydrolyzed to obtain an Al(OH)3 solution, which was added to SiO2, placed on a shaker and shaken, and then freeze-dried; the cold-dried particles were calcined in a muffle furnace, gradually heated from 120 °C to 450 °C to obtain particles containing 1% Al2O3 of SiO2 (abbreviated as Si-Al particles); pure SiO2 was prepared by the same method as above, except that the Al(OH)3 solution was replaced with ultrapure water (abbreviated as Si particles).

[0030] Catechol cyclohexane solution was configured as an organic precursor solution, and the organic precursor solution was dropped onto the Si-Al particles, left standing overnight, and dried with nitrogen to prepare Si-Al-CT particles, and persistent free radicals (EPFRs) were generated under natural conditions. The generation of EPFRs on the particles was monitored by an electron paramagnetic resonance spectrometer (EPR).

[0031] The tested natural soil: The tested black soil was collected from a pine forest in Yangbi County, Dali City, Yunnan Province, China, and the tested brown soil was collected from a Chinese fir forest in Liping County, Southeast Guizhou Miao and Dong Autonomous Prefecture, Guizhou Province, China; the soil was collected at a depth of 0-20 cm. After collection, the soil was wrapped in a breathable cloth bag and transported from the field to the laboratory under storage conditions of 4 °C; the soil was carefully screened through a 10-mesh sieve to remove plant residues, roots, and stones, and then naturally air-dried and stored in a 4 °C refrigerator for subsequent toxicity exposure tests of EPERs in the soil.

[0032] Among them, the experimental methods include the exposure test of EPFRs to soil microbial biomass and the exposure test of soil microbial enzyme activity.

[0033] Before the experiment, the soil was placed in a biochemical incubator and pre-incubated at 25 °C for one week. During the incubation period, ultrapure water was added to maintain the soil moisture so that the microbial activity in the soil was restored to stability. 35 g of black soil and brown soil were weighed into 250 ml wide-mouth bottles with small-hole lids respectively, and then Si-CT particles, Si-Al-CT-weak particles, and Si-Al-CT-strong particles with a mass concentration of 2% and 4% were added. A glass rod was used to stir to make the particles and the soil evenly mixed. The control group was a pure soil sample, and 3 parallels were set for each treatment group. The toxicity exposure experiment was carried out in a biochemical incubator and cultured for one week under the condition of 25 °C in the dark. During the cultivation period, deionized water was added by the weighing method every day to keep the soil water content at 40%. Soil samples were collected on the 3rd and 7th days of cultivation respectively for the determination of experimental indexes of soil microbial biomass (MBC, MBN) and soil microbial enzyme activities (S-ACP, S-SC, S-UE).

[0034] Among them, the processed data judged the toxic effects of EPFRs in the soil according to the changes in the indexes of soil microbial biomass (MBC and MBN) and soil microbial enzyme activities (S-ACP, S-SC, S-UE) after the addition of EPERs was measured.

[0035] Experimental results:

[0036] As Figure 2 , typical EPFRs signals could be detected on the Si-Al-CT particles under the condition of room temperature and darkness, and the signal intensity gradually increased with the reaction time; no EPFRs signals were detected on the Si-CT particles after 35 days of reaction ( Figure 3 ), because there was no Al2O3 on the particle surface and no EPFRs was generated. Therefore, the Si-CT particles could be used as control particles without EPFRs but containing the parent CT. The g value of EPFRs on the Si-Al-CT particles was between 2.0040 and 2.0045, indicating that the generated EPFRs were mainly oxygen-centered free radicals; in this study, the Si-Al-CT particles placed in the dark for 1 day and 35 days were used, and their EPFRs signal concentrations were 7.24×10 17 spins·g -1 (Si-Al-CT-weak) and 18.81×10 17 spins·g -1 (Si-Al-CT-strong).

[0037] After the addition of EPFRs particles, it had different effects on the MBC of black soil and brown soil ( Figure 4 and Figure 5)。Three days after the addition of EPFRs particles, there was no significant effect on the MBC of black soil, but it significantly reduced the MBC of brown soil, especially when adding 4% Si-Al-CT-strong particles. Seven days after the addition of EPFRs, it significantly reduced the MBC of black soil and brown soil, and with the increase of the particle addition dose and EPFRs concentration, the degree of reduction in MBC was more significant. Compared with the effect of EPFRs on the MBC of brown soil, the MBC of black soil was not sensitive to the addition of EPFRs particles in the short term (3 days).

[0038] As shown in ( Figure 6 and Figure 7 ), three days after the addition of EPFRs particles, the MBN of black soil and brown soil decreased. Under the condition of the same dose addition, the Si-Al-CT-strong particles had a more significant effect on reducing MBN. Seven days after the addition of EPFRs particles, the MBN in black soil and brown soil decreased, and with the increase of EPFRs concentration, the degree of reduction in MBN was more significant. The above results indicate that EPFRs reduces the MBN of black soil and brown soil, and the degree of reduction increases with the increase of EPFRs concentration.

[0039] The study found that the toxic effect of EPFRs is mainly through inducing the generation of ROS in the liquid phase environment, which attacks cell membranes, lipid proteins, DNA, etc., causing oxidative damage. Previous studies have found that the addition of AgNPs in soil significantly reduces soil microbial biomass, which is caused by the oxidative stress of the induced ROS on the microbial cell membrane. In the system of the present invention, the reduction of MBC and MBN in soil after the addition of EPFRs particles may be related to the ROS mediated by them.

[0040] As shown in ( Figures 8 - 13 ), three and seven days after the addition of EPFRs particles, there was no effect on the S-ACP of black soil, but it inhibited the activities of S-SC and S-UE in black soil, especially more significantly on the 7th day, and the inhibitory effect enhanced with the increase of the EPFRs particle dose and signal intensity; three and seven days after the addition of EPFRs particles, it significantly reduced the activities of S-ACP, S-SC, and S-UE in brown soil, and the reduction effect enhanced with the increase of the particle dose and signal intensity, especially when adding 4% Si-Al-CT-strong particles, the inhibitory effect on enzyme activity was the most significant. In addition, the inhibitory degree of EPFRs particles on the enzyme activity in brown soil was more significant than that in black soil after the addition.

[0041] Soil enzymes are derived from the secretions of soil microorganisms and plant roots, as well as the enzymes released during the decomposition of animal and plant residues. Among them, soil microorganisms are the main source of soil enzymes. Existing research has shown that the activities of S-UE, S-SC, and S-ACP are significantly positively correlated with soil microbial biomass. Therefore, the state of soil microorganisms can be indirectly indicated by changes in soil enzymes. The results show that after adding EPFRs particles to the soil, they have inhibitory effects on the activities of S-ACP, S-SC, and S-UE in black soil and brown soil, and the inhibitory effects become more significant with the increase of the signal and dose of EPFRs, especially the inhibitory effect on S-UE activity is the most significant. This result is consistent with the result of the impact of EPFRs on biomass.

[0042] Research has found that ·OH can react with polyunsaturated fatty acids (PUFAs) on the biological cell membrane to generate malondialdehyde (MDA). Malondialdehyde can inactivate proteins and change the functions of cell membrane characteristics such as enzyme activity and ion transport. ROS can also directly cause oxidative damage to proteins. ·OH and ·O2 - can damage the protein structure, change the protein function, and inhibit the activity of biological enzymes. In addition, the accumulation of ROS may be the main reason for the decrease in the activities of urease and phosphatase. Therefore, the decrease in enzyme activity in the present invention may be caused by the oxidative stress of ROS mediated by EPFRs on microorganisms. Generally, the higher the soil organic matter content, the stronger the soil buffering capacity. The more significant inhibitory effect of EPFRs particles on the enzyme activity in brown soil after addition may be due to the higher organic matter content in black soil, indicating that soil organic matter has a certain buffering capacity for the toxic effects of EPFRs.

[0043] In summary, the above results show that soil microbial biomass and enzyme activity have no toxic effects on the addition of particles without EPFRs, but soil microbial biomass and enzyme activity have toxic effects on the addition of particles containing EPFRs, and the stronger the EPFRs signal and the larger the particle dose, the greater the toxic effect; the results of soil microbial biomass (MBC, MBN) and soil microbial enzyme activity (S-ACP, S-SC, S-UE) in the present invention provide a new method for quickly identifying the toxic effects of EPFRs.

[0044] During the actual use process, add S-ACP, S-SC, and S-UE to the collected soil samples. If the activities of S-ACP, S-SC, and S-UE decrease, it proves that the soil contains EPFRs.

Claims

1. A method for rapidly identifying the toxicity of environmental persistent free radicals using soil microorganisms, characterized in that: By measuring the changes in soil microbial biomass carbon, soil microbial biomass nitrogen, soil sucrase, urease, and acid phosphatase activity indicators, the toxic effects of environmental persistent free radicals in the soil were judged; The presence of environmental persistent free radical toxicity in the soil was determined by detecting the change in soil microbial biomass carbon in the soil. As the concentration of environmental persistent free radical toxicity increased, the degree of decrease in soil microbial biomass carbon was more significant; The presence of environmental persistent free radical toxicity in the soil was determined by detecting the change in soil microbial biomass nitrogen in the soil. As the concentration of environmental persistent free radical toxicity increased, the degree of decrease in soil microbial biomass nitrogen was more significant; The presence of environmental persistent free radical toxicity in the soil was determined by detecting the activities of soil sucrase, urease, and acid phosphatase in the soil. As the concentration of environmental persistent free radical toxicity increased, the degree of decrease in the activities of soil sucrase, urease, and acid phosphatase in the soil was more significant.

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