Method for evaluating heavy metal pollution risk in high geological background soil by using biological indexes

Abstract

The invention discloses a method for evaluating heavy metal pollution risk in high geological background soil by using biological indexes, and belongs to the field of microbiology and environmental science. The method comprises the following steps: firstly, screening by adopting propionium bromide azide to obtain active microorganisms in soil, extracting DNA of the active microorganisms, carrying out gene high-throughput sequencing on the extracted DNA, screening obtained common microorganisms, and establishing a relationship between relative abundance of the common microorganisms and heavy metals; the heavy metal pollution risk of the soil can be judged according to the relative abundance of the microorganisms in the later period. The method provided by the invention not only improves the microbiological analysis accuracy of the heavy metal contaminated soil with the high geological background, but also provides a new method for heavy metal pollution risk assessment of the heavy metal contaminated soil.

Description

Technical Field

[0001] The invention relates to a method for evaluating the heavy metal pollution risk in soil with high geological background by using biological indicators, and belongs to the fields of microbiology and environmental science. Background Art

[0002] In recent years, heavy metal pollution in soil has attracted much attention. The content of heavy metals in soil with high geological background is significantly higher than that in other areas. The heavy metal content in this soil is mainly caused by the natural weathering of geological materials, the long-term weathering and migration of rocks, and the natural enrichment of heavy metal elements in specific rock types (such as basalt, black shale, etc.). Heavy metals in these soils, such as cadmium (Cd), arsenic (As), chromium (Cr), nickel (Ni), etc., pose a threat to environmental quality, food safety and ecological security. Risk assessment of heavy metal pollution in soil with high geological background has always been an important issue in the field of environmental science.

[0003] Traditional risk assessment methods mainly rely on the determination of heavy metal content, including the analysis of total and effective content, and cannot directly evaluate ecological risks. Therefore, the development of effective biological indicators is crucial for the assessment and monitoring of soil heavy metal pollution. The importance of detecting and monitoring heavy metal content in soils in high geological background areas is self-evident, but this method cannot fully reflect the ecological risks of heavy metals. In addition, morphological analysis methods, such as the risk assessment coding method (RAC) and the secondary phase to primary phase ratio method (RSP), although they can provide more information on the behavioral characteristics and ecological activity of heavy metals, have certain complexities and uncertainties in actual operations. For example, Chen Jiangjun et al. (Chen Jiangjun, Liu Bo, Cai Liegang, et al. Comparison of soil heavy metal pollution risk assessment based on multiple methods - taking a typical site in Jianghan Plain as an example [J]. Hydrogeology and Engineering Geology, 2018, 45(6):9.) took a typical site in Jianghan Plain as an example and pointed out that the potential ecological risk index method based on total amount and the RAC method based on morphology each have their own advantages and disadvantages. The former has direct indicative significance, and the latter can use the morphology of heavy metals to explain their behavioral characteristics and ecological activity in the soil environment. However, neither the potential ecological risk index method based on total amount nor the RAC method based on morphology takes into account the role of soil microorganisms. Therefore, although the heavy metal content and morphology analysis methods have made certain progress in risk assessment, there are still defects, especially in assessing the actual impact of heavy metals on the ecosystem. Therefore, the development of effective biological indicators is crucial for evaluating and monitoring heavy metal pollution in soils in high geological background study areas. These biological indicators can not only reflect the biological effects of soil pollution, but also reveal the adaptability and ecological functions of microorganisms in polluted environments.

[0004] Active microorganisms are an important component of soil ecosystems, and their response to soil heavy metal pollution in high geological background study areas can provide a more direct indication of ecological risks. Compared with single chemical analysis methods, active microbial methods have many advantages. First, active microorganisms can reflect the overall health of soil ecosystems because they are highly sensitive to environmental changes. Second, changes in microbial community structure can be used as a biological indicator of the impact of heavy metal pollution, because different microbial populations have different tolerance and responses to heavy metals. Finally, research techniques based on all microbial DNA can easily lead to an overestimation of microbial abundance and community diversity. Active microorganisms provide a more accurate evaluation indicator that can reflect the immediate response and adaptability of soil ecosystems to heavy metal pollution.

[0005] Among all the methods for separating active and dead microorganisms, the PMA method combined with sequencing methods has received the most attention and recognition. However, current research has not elaborated on the specific operating steps for extracting soil active microbial DNA using PMA, nor has it optimized its experimental conditions. A review (Hu Xiaojing, Bai Xin, Qu Juanjuan, et al. Principles and applications of azidopropidium bromide for screening environmental active microorganisms [J]. Soil and Crops, 2021, 10(03): 306-313.) mentioned the use of PMA to extract soil active microorganisms and proposed that the extraction method faces many problems, but the document did not elaborate on its specific operating steps. If the specific experimental operating steps of this method are further clarified, and the experimental conditions are improved on the basis of traditional methods to find the experimental conditions and methods for the best extraction effect, the responsiveness research of heavy metals and microbial communities will be more accurate.

[0006] In summary, screening common microorganisms and exploring their pollution response laws are of great significance for monitoring and evaluating heavy metal pollution in high geological background research areas. The screened common microorganisms can be used as biomarkers. By monitoring the population changes, diversity and activity of these microorganisms, the extent of heavy metal pollution and potential ecological risks can be assessed. For example, the increased abundance of certain metal-tolerant bacteria in heavy metal-contaminated environments may indicate a high level of pollution, while the reduction of sensitive species may indicate an increase in ecological risks. Secondly, common microorganisms play a role in the biogeochemical cycle of heavy metals, and they affect the migration and transformation of heavy metals through metabolic activities. For example, some microorganisms are able to change the valence state of heavy metals through redox reactions, or fix heavy metals through adsorption and complexation, reducing their bioavailability in the environment. The metabolic activities and interactions of these microorganisms can be studied through molecular ecological network analysis, thereby revealing their ecological functions and network positions in response to heavy metal pollution. Finally, the ecological network analysis of common microorganisms can reveal their competitive and cooperative relationships under heavy metal pollution, as well as their adaptability to environmental changes. Network analysis can identify key species and functional modules, which may play an indicative and early warning role in the ecological risk assessment of heavy metal pollution. By integrating the ecological functions and network structure information of these microorganisms, the impact of heavy metal pollution on the ecosystem can be more comprehensively assessed, providing a scientific basis for pollution remediation and management. Summary of the invention

[0007] The object of the present invention is to provide a method for evaluating the risk of heavy metal pollution in soil with high geological background using biological indicators. The method comprises: screening out common microorganisms based on the α diversity of active microorganisms, and evaluating the risk of heavy metal pollution in soil with high geological background through the relationship between the relative abundance of common microorganisms and heavy metals.

[0008] Preferably, the heavy metal includes one or more of arsenic, chromium or mercury.

[0009] Preferably, when the heavy metal pollution is arsenic, the common microorganisms screened are Aquicella, Acidibacter and Panacagrimonas, among which the relative abundance of Aquicella is positively correlated with the arsenic content in the soil with high geological background, the relative abundance of Acidibacter is negatively correlated with the arsenic content in the soil with high geological background, and the relative abundance of Panacagrimonas is positively correlated with the arsenic content in the soil with high geological background.

[0010] Preferably, the method for evaluating the risk of heavy metal pollution in soil with high geological background using biological indicators comprises the following specific steps:

[0011] (1) Take the high geological background soil to be tested and use propidium azidobromide to screen the active microorganisms in the high geological background soil.

[0012] (2) Extract DNA of active microorganisms in soil with high geological background.

[0013] (3) The extracted DNA is subjected to high-throughput gene sequencing, and common microorganisms are obtained based on α-diversity screening.

[0014] (4) Establish the relationship between the relative abundance of common microorganisms and heavy metals.

[0015] Preferably, the specific operation steps of step (1) are:

[0016] (a) Take fresh soil, add PBST buffer solution and then shake culture.

[0017] (b) After the shaking is completed, low-speed centrifugation is performed, the supernatant is collected, and the supernatant is centrifuged again at high speed. After the centrifugation is completed, 0.9 to 1.2 mL of liquid is retained.

[0018] (c) The retained liquid is mixed with the bacterial plaque to obtain a bacterial solution, and the bacterial solution is filtered using a filter membrane.

[0019] (d) Add propidium azidobromide solution to the filtered bacterial solution and perform shaking reaction in the dark.

[0020] (e) After the light-proof oscillation reaction is completed, a light reaction is carried out under ice bath conditions. After the light reaction is completed, the reaction is allowed to stand in a dark environment and then centrifuged to retain the precipitate. The obtained precipitate is the active microorganism.

[0021] Preferably, in step (a), the solid-liquid ratio of the fresh soil to the PBST buffer solution is 3:(5-20), in units of g:mL.

[0022] Preferably, the low-speed centrifugation in step (b) is performed at a speed of 1500 to 2000 r / min for 5 to 6 min; the high-speed centrifugation is performed at a speed of 7500 to 8500 r / min for 3 to 4 min.

[0023] Preferably, the pore size of the filter membrane in step (c) is 40 um.

[0024] Preferably, in step (d), the volume ratio of the bacterial liquid to the propidium bromide solution is (900-1200):(5-20), and the molar concentration of the propidium bromide solution is 1 mmol / L.

[0025] Preferably, in step (e), the illumination reaction time is 5 to 15 minutes, and the standing time in a dark environment is 30 to 40 minutes; the high-speed centrifugation conditions are: centrifugation at 12000 to 13000 r / min for 3 to 4 minutes.

[0026] Beneficial Effects of the Invention

[0027] (1) The present invention can separate active microorganisms from dead microorganisms in soil samples, which is beneficial for accurately evaluating the activity of microorganisms in soil samples.

[0028] (2) The present invention is applicable and extensive, especially in areas with high geological backgrounds. By screening common microorganisms, it can provide universal biological indicators for ecological risk assessment of heavy metal pollution and is suitable for soil samples in different regions.

[0029] (3) The present invention can promote ecological risk management by identifying and screening microorganisms that have a common response to heavy metal pollution. This method helps to develop targeted bioremediation strategies and provides a scientific basis for the management and risk assessment of soil heavy metal pollution in high geological background study areas.

[0030] (4) The process of extracting active microbial DNA in the present invention is simple and easy to standardize, and is suitable for promotion and application in different laboratories.

[0031] (5) The present invention further optimizes the extraction effect by comparing the extraction effects of different amounts of PMA (propidium azide bromide) additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The electrophoresis diagrams are obtained by PCR and gel processing of the soil active microbial DNA extracted in Examples 1, 2 and 3, with dead microorganisms used for comparison.

[0033] Figure 2 This is a correlation analysis chart of the abundance of active microorganisms and As concentration in soil samples from Example 4 (sampling areas in Lanping, Yunnan, Tongren, Guizhou, and Zhanjiang, Guangdong). DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0035] Preparation of PBST buffer: 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, and 0.2 mL / L Tween-20.

[0036] The bacterial genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., model: YDP302.

[0037] Unless otherwise specified, the methods described in the embodiments are routine operations well known to those skilled in the art.

[0038] Example 1

[0039] A method for screening active microbial DNA in soil, specifically comprising the following steps:

[0040] (1) Weigh 3 g of fresh soil sample, add 5 mL of prepared PBST buffer solution, put into a sterile centrifuge tube, and place it on a micro-oscillator at a frequency of 300 r / min for 15 min.

[0041] (2) After the shaking is completed, low-speed centrifugation is performed at a speed of 1500 r / min for 6 min, and then all the supernatant is transferred to another centrifuge tube for high-speed centrifugation at a speed of 7500 r / min for 4 min.

[0042] (3) After centrifugation, remove the supernatant and discard it, leaving 0.9 mL of liquid in the centrifuge tube. Gently blow the bacterial plaque, filter it with a 40 μm filter membrane, transfer the remaining 0.9 mL of bacterial liquid to a 12-well plate, then add 5 μL of 1 mmol / L propidium azidobromide solution, and place it on a microplate oscillator in the dark and oscillate at a frequency of 200 r / min for 25 minutes.

[0043] (4) After the shaking is completed, place an ice pack under the well plate and a halogen tungsten lamp 20 cm above it for illumination for 10 min. After the shaking is completed, the well plate is left in the dark for 40 min, then transferred into a sterile centrifuge tube and centrifuged at high speed at 12,000 r / min for 4 min, and the supernatant is discarded.

[0044] (5) The bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (centrifugal column type, TIANGEN). The operation steps refer to the official instructions of the kit.

[0045] (6) The extracted active microbial DNA is amplified by PCR.

[0046] Example 2

[0047] A method for screening active microbial DNA in soil, specifically comprising the following steps:

[0048] (1) Weigh 3 g of fresh soil sample, add 15 mL of prepared PBST buffer solution, put into a sterile centrifuge tube, and place it on a micro-oscillator at a frequency of 200 r / min for 25 min.

[0049] (2) After the shaking is completed, low-speed centrifugation is performed at a speed of 2000 r / min for 5 min, and then all the supernatant is transferred to another centrifuge tube and high-speed centrifugation is performed at a speed of 8500 r / min for 3 min.

[0050] (3) After centrifugation, remove the supernatant and discard it, leaving 1 mL of liquid in the centrifuge tube. Gently blow the bacterial plaque, filter it using a 40 μm filter membrane, transfer the remaining 1 mL of bacterial liquid to a 12-well plate, then add 10 μL of 1 mmol / L propidium azidobromide solution, and place it on a microplate oscillator in the dark and oscillate at a frequency of 300 r / min for 15 minutes.

[0051] (4) After the shaking is completed, place an ice pack under the well plate and a halogen tungsten lamp 20 cm above it for illumination for 5 min. After the shaking is completed, the well plate is left in the dark for 30 min, then transferred into a sterile centrifuge tube and centrifuged at high speed at 13,000 r / min for 3 min, and the supernatant is discarded.

[0052] (5) The bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (centrifugal column type, TIANGEN). The operation steps refer to the official instructions of the kit.

[0053] (6) The extracted active microbial DNA is amplified by PCR.

[0054] Example 3

[0055] A method for screening active microbial DNA in soil, specifically comprising the following steps:

[0056] (1) Weigh 3 g of fresh soil sample, add 20 mL of prepared PBST buffer solution, put into a sterile centrifuge tube, and place it on a micro-oscillator at a frequency of 300 r / min for 15 min.

[0057] (2) After the shaking is completed, low-speed centrifugation is performed at a speed of 1500 r / min for 6 min, and then all the supernatant is transferred to another centrifuge tube. After the liquid is balanced with PBS solution, high-speed centrifugation is performed again at a speed of 7500 r / min for 4 min.

[0058] (3) After centrifugation, remove the supernatant and discard it, leaving 1.2 mL of liquid in the centrifuge tube. Gently blow the bacterial plaque, filter it with a 40 μm filter membrane, transfer the remaining 1.2 mL of bacterial liquid to a 12-well plate, then add 20 μL of 1 mmol / L propidium azidobromide solution, and place it on a microplate oscillator in the dark and oscillate at a frequency of 200 r / min for 25 minutes.

[0059] (4) After the shaking is completed, place an ice pack under the well plate and a halogen tungsten lamp 20 cm above it for illumination for 15 min. After the shaking is completed, the well plate is left in the dark for 40 min, then transferred into a sterile centrifuge tube and centrifuged at high speed at 12,000 r / min for 4 min, and the supernatant is discarded.

[0060] (5) The bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (centrifugal column type, TIANGEN). The operation steps refer to the official instructions of the kit.

[0061] (6) The extracted active microbial DNA is amplified by PCR.

[0062] The PCR products of Examples 1 to 3 and Comparative Example 1 were subjected to gel electrophoresis. The results are as follows: Figure 1 As shown, it can be seen from the figure that the method of the present invention can screen out active microbial DNA in the soil, 5 is the PCR result of the DNA obtained in Example 1, 10 is the PCR result of the DNA obtained in Example 2, 20 is the PCR result of the DNA obtained in Example 3, and dead bacteria are used as a control. It can be seen that the DNA of dead bacteria cannot be extracted using the methods of Examples 1 to 2.

[0063] Example 4

[0064] The soil samples in this embodiment were taken from three different places, namely, the surface soil near a certain industrial zone in Donghai Island, Zhanjiang, a high geological background area, the surface soil in Lanping, Yunnan, a high geological background area, and the surface soil in Tongren, Guizhou, a high geological background area.

[0065] A method for assessing the risk of heavy metal contamination in soil with high geological background using biological indicators, comprising the following steps:

[0066] (1) Weigh 3 g of fresh soil samples from different places, add 20 mL of prepared PBST buffer solution, put into a sterile centrifuge tube, and place it on a micro-oscillator at a frequency of 300 r / min for 15 min.

[0067] (2) After the shaking is completed, low-speed centrifugation is performed at a speed of 1500 r / min for 6 min, and then all the supernatant is transferred to another centrifuge tube. After the liquid is balanced with PBS solution, high-speed centrifugation is performed again at a speed of 7500 r / min for 4 min.

[0068] (3) After centrifugation, remove the supernatant and discard it, leaving 1.2 mL of liquid in the centrifuge tube. Gently blow the bacterial plaque, filter it with a 40 μm filter membrane, transfer the remaining 1.2 mL of bacterial liquid to a 12-well plate, then add 20 μL of 1 mmol / L propidium azidobromide solution, and place it on a microplate oscillator in the dark and oscillate at a frequency of 200 r / min for 25 minutes.

[0069] (4) After the shaking is completed, place an ice pack under the well plate and a halogen tungsten lamp 20 cm above it for illumination for 15 min. After the shaking is completed, the well plate is left in the dark for 40 min, then transferred into a sterile centrifuge tube and centrifuged at high speed at 12,000 r / min for 4 min, and the supernatant is discarded.

[0070] (5) The bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (centrifugal column type, TIANGEN). The operation steps refer to the official instructions of the kit.

[0071] (6) The extracted active microbial DNA was used for high-throughput gene sequencing to obtain the relative abundance and α diversity of active microorganisms. The three microorganisms Aquicella, Acidibacter and Panacagrimonas were screened and found to have the highest correlation with As concentration. The relative abundance of these three microorganisms was linearly regressed with the arsenic (As) concentration in the soil. The results are as follows: Figure 2 As shown, it can be seen from the figure that the relative abundance of Aquicella is positively correlated with the arsenic content in the soil with high geological background, the relative abundance of Acidibacter is negatively correlated with the arsenic content in the soil with high geological background, and the relative abundance of Panacagrimonas is positively correlated with the arsenic content in the soil with high geological background.

Claims

1. A method for assessing the risk of heavy metal pollution in soil with high geological background using biological indicators, characterized in that: Based on the α-diversity of active microorganisms, common microorganisms were screened out, and the risk of heavy metal pollution in soils with high geological background was evaluated by the relationship between the relative abundance of common microorganisms and heavy metals.

2. The method for assessing the risk of heavy metal pollution in soil with high geological background using biological indicators according to claim 1, characterized in that: The heavy metals include one or more of arsenic, chromium or mercury.

3. The method for assessing the risk of heavy metal pollution in soil with high geological background using biological indicators according to claim 1, characterized in that: When the heavy metal pollution is arsenic, the common microorganisms screened are Aquicella, Acidibacter and Panacagrimonas. The relative abundance of Aquicella is positively correlated with the arsenic content in the soil with high geological background, the relative abundance of Acidibacter is negatively correlated with the arsenic content in the soil with high geological background, and the relative abundance of Panacagrimonas is positively correlated with the arsenic content in the soil with high geological background.

4. The method for evaluating the risk of heavy metal pollution in soil with high geological background using biological indicators according to claim 1, characterized in that: The specific steps are as follows: (1) taking soil with a high geological background to be tested, and screening active microorganisms in the soil with a high geological background using propidium azidobromide; (2) Extract DNA of active microorganisms in soil with high geological background; (3) performing high-throughput gene sequencing on the extracted DNA and screening the common microorganisms based on α-diversity; (4) Establish the relationship between the relative abundance of common microorganisms and heavy metals.

5. The method for evaluating the risk of heavy metal pollution in soil with high geological background using biological indicators according to claim 4, characterized in that: The specific steps of step (1) are as follows: (a) Take fresh soil, add PBST buffer solution and then shake culture; (b) After the shaking is completed, low-speed centrifugation is performed, the supernatant is collected, and the supernatant is then subjected to high-speed centrifugation, leaving 0.9 to 1.2 mL of liquid after the centrifugation; (c) mixing the retained liquid with the bacterial plaque to obtain a bacterial solution, and filtering the bacterial solution using a filter membrane; (d) adding propidium azidobromide solution to the filtered bacterial solution and performing a shaking reaction in the dark; (e) After the light-proof oscillation reaction is completed, a light reaction is carried out under ice bath conditions. After the light reaction is completed, the reaction is allowed to stand in a dark environment and then centrifuged to retain the precipitate. The obtained precipitate is the active microorganism.

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