Analysis of functional bacterial activity in organically contaminated sites based on quinone fingerprint characteristics

Abstract

The present invention relates to a method for analyzing the activity of functional bacteria in organic contaminated sites based on quinone fingerprint characteristic maps, which specifically includes the following steps: Step 1, site pollution background investigation, where the indicator to be investigated is the organic pollutant concentration value, and the site focus points and characteristic pollutants are determined; Step 2, sampling and monitoring the pollutant concentration, pH value, and ORP value of the sample on a time scale to determine the biodegradation characteristics of the pollutants; Step 3, drawing the quinone fingerprint map of the focus points to obtain the dominant quinone type of the characteristic pollutant; Step 4, comparing with the microbial quinone spectrum library to screen the pollutant characteristic quinone type with the functional bacteria community indication function; Step 5, evaluating the strength of the functional bacteria community activity at each point and at each time by the total concentration of the pollutant characteristic quinone. The method of the present invention is simple, fast, universal, and highly accurate. Relying on data, it focuses on solving the problem that traditional microbial detection technology cannot quickly and accurately reflect the activity of functional microorganisms.

Description

Technical Field

[0001] The present invention belongs to the technical field of pollution control, and in particular relates to a method for analyzing the activity of functional bacteria in organically contaminated sites based on a quinone fingerprint characteristic spectrum. Background Art

[0002] The quinone profile method is a method for analyzing microbial population composition using microbial isoprenoid quinones (quinones) as markers. The presence of different quinone types is measured to provide fingerprints of different microorganisms in a sample. Quinones are diverse in species, primarily comprising ubiquinone (UQ) and menaquinone (MK). The way microorganisms synthesize quinones depends on their characteristics and electron transfer requirements. A microorganism typically has only one dominant quinone type. By using the correspondence between different quinone types and microbial flora, quinones can be used as an effective biochemical taxonomic indicator to indicate microbial community structure. Quinones are ubiquitous electron transporters and proton carriers in the electron transport chain. During cellular biochemical metabolic reactions, microorganisms utilize redox reactions to maintain electron transfer and material gradients across the cytoplasmic membrane. Therefore, quinones can serve as markers reflecting microbial activity.

[0003] Compared with traditional soil microbial enumeration methods, the quinone fingerprint method has the advantages of simple operation and no need for microbial culture. Compared with the carbon source utilization analysis method, the quinone fingerprint method does not require culture in a carbon source microplate, is not restricted by carbon sources and indicators, and can detect a wider range of microorganisms. Compared with the phospholipid fatty acid method, the quinone fingerprint method is not only more stable in the extraction of markers and less affected by site environmental factors, but also can detect archaea due to its polar lipid structure. Compared with the most commonly used modern molecular biology methods, studies have shown that the results of the quinone fingerprint method in characterizing microbial diversity are basically consistent with the results of deformed gradient gel electrophoresis, fluorescence in situ hybridization, and 16S rRNA cloning and sequencing. The extraction and detection of quinone is cheaper than these methods and has strong engineering applicability. In addition, as an active substance in cellular respiration, the concentration of quinone itself can directly reflect the activity of microorganisms, which is simpler and more intuitive than extracting nucleic acids. In addition, since it is difficult to quantitatively extract nucleic acids from some microorganisms, such as actinomycetes, the quinone fingerprint method shows a better indicator effect in the characterization of these microorganisms.

[0004] The quinone fingerprint method has attracted widespread attention and has been applied in the field of microbial activity detection in contaminated sites due to its advantages of simplicity, convenience, high sensitivity, stable marker extraction, and little influence from environmental factors. However, most existing methods analyze the activity of various microorganisms based on the absolute concentration of each quinone type separately, without combining the degradation characteristics of specific pollutants to analyze and summarize the quinone types that indicate the activity of functional bacterial communities. There are practical problems such as insufficient site-specific accuracy and poor universality of the analysis results, and the inability to reflect the activity of functional microorganisms.

[0005] Therefore, a method for analyzing the activity of functional bacteria in organic contaminated sites based on quinone fingerprint characteristic maps is proposed, in order to screen out pollutant characteristic quinone types that indicate the activity of functional bacteria in organic contaminated sites, so as to achieve the purpose of quickly and accurately indicating the activity of functional bacteria through the concentration of pollutant characteristic quinone types. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to propose a universal and rapid indication method for indicating the degradation activity of functional bacteria in polluted sites that can solve the above problems.

[0007] The technical solution for solving the technical problem of the present invention is: the present invention discloses a method for analyzing the activity of functional bacteria in organic contaminated sites based on a quinone fingerprint characteristic spectrum, comprising performing activity analysis on the functional bacteria in the organic contaminated site based on the quinone fingerprint characteristic spectrum, and evaluating the activity of the functional bacteria at each point and time in the organic contaminated site according to the total concentration of the characteristic quinones of the pollutants.

[0008] Further, the following steps are included:

[0009] Step 1: Site pollution background investigation, the indicators that need to be investigated are organic pollutant concentration values, and the focus points and characteristic pollutants of the site are determined;

[0010] Step 2: Sampling and monitoring the pollutant concentration, pH value, and redox potential value of the sample on a time scale to determine the biodegradation characteristics of the pollutants;

[0011] Step 3: Draw the quinone fingerprint of the focus point to obtain the dominant quinone type of the characteristic pollutant;

[0012] Step 4: Match the dominant quinone type with the microbial quinone spectrum library to screen the pollutant characteristic quinone type that has the function of indicating the degradation functional bacteria;

[0013] Step 5: Evaluate the activity of functional bacteria at each location at different times based on the total concentration of characteristic quinones of pollutants.

[0014] Furthermore, the activity of functional bacteria at each location at different times is evaluated by the total concentration of characteristic quinones of pollutants, including the following steps:

[0015] S51. Characteristic quinone concentration detection of characteristic quinone type of pollutants Based on the characteristic quinone concentration detection results, the absolute concentrations of each characteristic quinone screened out are summed to obtain the total concentration of characteristic quinone;

[0016] S52. Based on the difference in the absolute value of the total concentration of characteristic quinones in the samples, the relative strength of the corresponding functional microbial activity in the samples can be qualitatively determined;

[0017] S53. A linear regression equation was constructed by establishing the total concentration of characteristic quinones and the corresponding degradation functional enzyme activity parameters to semi-quantitatively determine the absolute intensity of functional microbial activity.

[0018] S54. By comparing the ratio of the total concentration of characteristic quinones to the total quinone concentration in soil samples collected from different locations and at different times in the target site, the activity of the functional bacterial community at each location at different times can be evaluated.

[0019] Furthermore, the step 4 further includes the following steps:

[0020] S41. After determining the dominant quinone type, match it with the corresponding relationship between quinone type and microbial type in the microbial quinone spectrum library;

[0021] S42. Obtain the microbial type information corresponding to the dominant quinone type, and further confirm whether it contains functional microbial populations related to pollutant degradation based on the microbial classification standards. If the microorganisms corresponding to the dominant quinone type include microbial populations with pollutant degradation functions, then the microorganisms corresponding to the dominant quinone type are screened as pollutant characteristic quinone types with functional flora activity indicators, and the characteristic quinone concentration of the pollutant characteristic quinone type is detected.

[0022] Furthermore, in step 1, the selection basis of the site focus points is relatively high pollutant concentration; the selection basis of the characteristic pollutants is the pollutant with the highest concentration in the environment.

[0023] Furthermore, in the step 2, sampling is performed by drilling the surface soil of the original soil layer through a drilling device, collecting and mixing the soil samples evenly, and removing debris such as plant residues and hard rocks.

[0024] Furthermore, in the step 4, the method for determining the dominant quinone type is that the abundance in the quinone fingerprint is greater than 10%.

[0025] Beneficial effects:

[0026] Compared with the existing technology, the present invention proposes to analyze the activity of functional bacteria in organic contaminated sites based on the total concentration of characteristic quinones. By comparing the ratio of the total concentration of characteristic quinones to the total quinone concentration in soil samples collected from different points and at different times in the target site, the activity of the functional bacteria that degrade the target pollutants at each point at different times is evaluated.

[0027] The present invention focuses on the correlation between characteristic quinone types and the activity of functional bacterial communities, clarifies the method for constructing the indication relationship between characteristic quinone types of pollutants and functional bacterial communities, and proposes a universal rapid indication method for indicating the degradation activity of functional bacterial communities that degrade target pollutants in contaminated sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1Graph showing changes in pH, ORP, and pollutant concentration over time in an embodiment of the present invention;

[0029] Figure 2 This is the fingerprint of quinone contamination by toluene in the embodiment of the present invention;

[0030] Figure 3 Graph showing the change of total concentration of characteristic quinones over time in an embodiment of the present invention;

[0031] Figure 4 The results of fitting the total concentration of characteristic quinones and polyphenol oxidase activity in the embodiment of the present invention are shown in FIG.

[0032] Figure 5 Graph showing changes in pH, ORP, and pollutant concentration over time in an embodiment of the present invention;

[0033] Figure 6 This is the fingerprint of trichloroethylene contamination quinone in the embodiment of the present invention;

[0034] Figure 7 Graph showing the change of total concentration of characteristic quinones over time in an embodiment of the present invention;

[0035] Figure 8 This is the fitting result of the total concentration of characteristic quinones and dehydrogenase activity in the examples of the present invention. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0037] The present invention discloses a method for analyzing the activity of functional bacteria in organically contaminated sites based on a quinone fingerprint characteristic spectrum, which specifically comprises the following steps:

[0038] Step 1: Site pollution background investigation, the indicators that need to be investigated are organic pollutant concentration values, and the focus points and characteristic pollutants of the site are determined;

[0039] Step 2: Sampling and monitoring the pollutant concentration, pH value, and ORP value of the samples over a time scale to determine the biodegradation characteristics of the pollutants;

[0040] Step 3: Draw the quinone fingerprint of the focus point to obtain the dominant quinone type of the characteristic pollutant;

[0041] Step 4: Match the dominant quinone type with the microbial quinone library to screen for pollutant-characteristic quinone types that indicate degradation of functional bacteria. The specific steps are as follows:

[0042] S41. After determining the dominant quinone type, match it with the corresponding relationship between quinone type and microbial type in the microbial quinone spectrum library;

[0043] S42. Obtain the microbial type information corresponding to the dominant quinone type, and further confirm whether it contains functional microbial populations related to pollutant degradation based on the microbial classification standards. If the microorganisms corresponding to the dominant quinone type include microbial populations with pollutant degradation functions, the microorganisms corresponding to the dominant quinone type will be screened as pollutant characteristic quinone types with functional flora activity indicators, and the characteristic quinone concentration of the pollutant characteristic quinone type will be detected.

[0044] Step 5: Evaluate the activity of functional bacteria at each location at different times based on the total concentration of characteristic quinones of pollutants. The specific operation is as follows:

[0045] S51. Based on the characteristic quinone concentration detection results, the absolute concentrations of the selected characteristic quinones are summed to obtain the characteristic quinone total concentration;

[0046] S52. Based on the difference in the absolute value of the total concentration of characteristic quinones in the samples, the relative strength of the corresponding functional microbial activity in the samples can be qualitatively determined;

[0047] S53. A linear regression equation was constructed by establishing the total concentration of characteristic quinones and the corresponding degradation functional enzyme activity parameters to semi-quantitatively determine the absolute intensity of functional microbial activity.

[0048] S54. By comparing the ratio of the total concentration of characteristic quinones to the total quinone concentration in soil samples collected from different locations and at different times in the target site, the activity of the functional bacterial community at each location at different times can be evaluated.

[0049] In this method, step three is to screen the dominant quinone type through the drawn quinone fingerprint map, and step four is to realize the screening of the characteristic quinone type corresponding to the target pollutant based on the dominant quinone type. The two are in a progressive relationship. Constructing a linear regression equation is the content of step five. The purpose is to construct an indicative relationship (regression equation) between the total concentration of characteristic quinones and the total activity of microorganisms through data from several points of the target site. A unified indicative relationship can be shared in the same site. Therefore, the microbial activity of the remaining points in the same site can be directly calculated by obtaining the total characteristic quinone concentration through the regression equation. Among them, the regression equation varies according to different sites.

[0050] Example 1

[0051] This example introduces a method for analyzing the activity of functional bacteria in organic contaminated sites based on quinone fingerprint characteristic patterns, which specifically includes the following steps:

[0052] Step 1: Select a retired pesticide site in Jiangyin as the sampling source, and measure the background concentration of organic pollutants in soil samples from eight locations. The background values ​​of pollutant concentrations are shown in Table 1, where W1 and W2 are sites with relatively high pollution concentrations, and the characteristic pollutant of the site is toluene;

[0053] Table 1 Background values ​​of organic pollutants in the underground at various points on the site

[0054]

[0055] Step 2: Using the soil from the site as the sample source, a detailed site survey and multi-point soil sampling were conducted on the organic contaminated site. The sampling was done by drilling 1.5m deep into the original soil layer with drilling equipment. The soil samples were collected and mixed evenly to remove plant residues, hard rocks and other debris. The pollutant concentration, pH value and ORP value of the samples were sampled and monitored on a time scale. The changes of various indicators over time were as follows: Figure 1 As shown in the figure, there was a degradation process of toluene at two points during the monitoring period. The degradation of pollutants occurred in an aerobic environment, and the pH showed a downward trend. Acidic substances such as benzoic acid were produced during the aerobic degradation of toluene, while no acidic substances were produced during the anaerobic degradation process. The biodegradation pathway of toluene in this case was aerobic degradation.

[0056] Step 3: Draw a fingerprint of quinone contamination with toluene based on the abundance detection value of quinone. The fingerprint of quinone in this embodiment is as follows: Figure 2 As shown, UQ-8, UQ-9, MK-7 and MK-8(H2) have an abundance greater than 10% in the quinone fingerprint, which are the dominant quinone types in the toluene contamination quinone fingerprint;

[0057] Step 4: Compare the dominant quinone types related to pollutant degradation with the microbial quinone library. The three quinone types, UQ-8, UQ-9, and MK-8 (H2), are the characteristic quinone types of pollutants in toluene-contaminated sites.

[0058] Step 5: Draw a graph showing the change of the total concentration of characteristic quinones at each point over time to evaluate the activity of the functional bacteria at each point at each time. Figure 3 As shown, in this embodiment, the activity of the functional flora at point W1 was significantly higher than that at point W2 (P < 0.05), and the activity of the functional flora showed an increasing trend over time;

[0059] In this case, the correlation between the toluene degradation microbial activity and the total concentration of characteristic quinones is as follows: Figure 4 As shown in the results, there was a linear relationship between the polyphenol oxidase activity and the total concentration of characteristic quinones in toluene-contaminated soil.

[0060] Example 2

[0061] This example introduces a method for analyzing the activity of functional bacteria in organic contaminated sites based on quinone fingerprint characteristic patterns, which specifically includes the following steps:

[0062] Step 1: Select a retired pesticide site in Nantong as the sampling source and measure the background concentration of organic pollutants in soil samples from eight locations. The background concentration values ​​of pollutants are shown in Table 2. Among them, W3 and W4 are sites with relatively high pollution concentrations, and the characteristic pollutant of the site is trichloroethylene;

[0063] Table 2 Background values ​​of organic pollutants in the underground at various points on the site

[0064]

[0065] Step 2: Using the soil from the site as the sample source, a detailed site survey and multi-point soil sampling were conducted on the organic contaminated site. The sampling was done by drilling 1.5m deep into the original soil layer with drilling equipment. The soil samples were collected and mixed evenly to remove plant residues, hard rocks and other debris. The pollutant concentration, pH value and ORP value of the samples were sampled and monitored on a time scale. The changes of various indicators over time were as follows: Figure 5 As shown in the figure, there was a degradation process of trichloroethylene at the two points during the monitoring period. The degradation of pollutants occurred in an aerobic environment, and the pH showed a downward trend. Organic acids such as formic acid were produced during the aerobic degradation of trichloroethylene, while no acidic substances were produced during the anaerobic degradation process. The biodegradation pathway of trichloroethylene in this case was aerobic degradation.

[0066] Step 3: Draw a fingerprint of quinone contamination with toluene based on the abundance detection value of quinone. The fingerprint of quinone in this embodiment is as follows: Figure 6 As shown, the abundance of UQ-9, MK-7, MK-8, and MK-9 in the quinone fingerprint is greater than 10%, which is the dominant quinone type in the quinone fingerprint of toluene contamination;

[0067] Step 4: Compare the dominant quinone types related to pollutant degradation with the microbial quinone library. The three quinone types, UQ-9, MK-7, and MK-9, are the characteristic quinone types of pollutants in toluene-contaminated sites.

[0068] Step 5: By plotting the total concentration of characteristic quinones at each point over time, the activity of the functional bacteria at each point at each time can be evaluated. Figure 7 As shown, in this embodiment, the activity of the functional flora at W3 was significantly higher than that at W4 (P < 0.05), and the activity of the functional flora at W3 showed an upward trend with time.

[0069] In this case, the semi-quantitative relationship between the activity of trichloroethylene-degrading microorganisms and the total concentration of characteristic quinones is as follows: Figure 8 As shown in the figure, it can be seen that the linear regression model can be used to fit the correlation between dehydrogenase activity and total concentration of characteristic quinones in trichloroethylene contaminated soil.

[0070] As can be seen from the above examples, the method of the present invention is simple, fast, universal, and highly accurate. Based on data, it focuses on solving the problem that traditional microbial detection technologies cannot quickly and accurately reflect the activity of functional microorganisms.

[0071] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for analyzing the activity of functional bacteria in organically contaminated sites based on quinone fingerprint characteristics, characterized by: Based on the quinone fingerprint characteristic spectrum, the activity of the functional bacteria in the organic contaminated site is analyzed, and the total concentration of the characteristic quinones of the pollutants is used to evaluate the activity of the functional bacteria at each point and time in the organic contaminated site; The following steps are included: Step 1: Site pollution background investigation, the indicators that need to be investigated are organic pollutant concentration values, and the focus points and characteristic pollutants of the site are determined; Step 2: Sampling and monitoring the pollutant concentration, pH value, and redox potential value of the sample on a time scale to determine the biodegradation characteristics of the pollutants; Step 3: Draw the quinone fingerprint of the focus point to obtain the dominant quinone type of the characteristic pollutant; Step 4: Match the dominant quinone type with the microbial quinone spectrum library to screen the pollutant characteristic quinone type that has the function of indicating the degradation functional bacteria; The step 4 further comprises the following steps: S41. After determining the dominant quinone type, match it with the corresponding relationship between quinone type and microbial type in the microbial quinone spectrum library; S42. Obtain information on the microbial type corresponding to the dominant quinone type, and further confirm whether it contains functional microbial populations related to pollutant degradation according to microbial classification standards. If the microbial populations corresponding to the dominant quinone type contain microbial populations capable of pollutant degradation, screen the microbial populations corresponding to the dominant quinone type as pollutant-characteristic quinone types that indicate functional microbial activity, and perform characteristic quinone concentration testing on the pollutant-characteristic quinone type. Step 5: Evaluate the activity of functional bacteria at each location at different times based on the total concentration of characteristic quinones of pollutants; The activity of functional bacteria at different points at different times is evaluated by the total concentration of characteristic quinones of pollutants, including the following steps: S51 characteristic quinone concentration detection of pollutants based on the characteristic quinone concentration detection results, the absolute concentration of each characteristic quinone screened out is summed to obtain the characteristic quinone total concentration; S52. Based on the difference in the absolute value of the total concentration of characteristic quinones in the samples, the relative strength of the corresponding functional microbial activity in the samples can be qualitatively determined; S53. A linear regression equation was constructed by establishing the total concentration of characteristic quinones and the corresponding degradation functional enzyme activity parameters to semi-quantitatively determine the absolute intensity of functional microbial activity; S54. By comparing the ratio of the total concentration of characteristic quinones to the total quinone concentration in soil samples collected from different locations and at different times in the target site, the activity of the functional bacterial community at each location at different times can be evaluated.

2. The method for analyzing the activity of functional bacteria in organic contaminated sites based on quinone fingerprint characteristic patterns according to claim 1, characterized in that: In the step 1, the focus points of the site are selected based on relatively high pollutant concentrations; and the characteristic pollutants are selected based on the pollutants with the highest concentrations in the environment.

3. The method for analyzing the activity of functional bacteria in organic contaminated sites based on quinone fingerprint characteristic patterns according to claim 1, characterized in that: In the second step, sampling is performed by drilling the surface soil of the original soil layer through a drilling device, collecting and mixing the soil samples, and removing plant residues and hard rocks.

4. The method for analyzing the activity of functional bacteria in organic contaminated sites based on quinone fingerprint characteristic patterns according to claim 1, characterized in that: In step 4, the method for determining the dominant quinone type is that the abundance in the quinone fingerprint is greater than 10%.

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