Estuary and coastal environment ecological assessment method based on microbial indexes

Abstract

The invention relates to a microbial index-based estuary and coastal environment ecological evaluation method, which comprises the following steps of: selecting representative sampling points, collecting sediment samples, analyzing and evaluating pressure gradients generated by human activities to estuary and coastal environments through physicochemical indexes, and constructing pressure indexes according to a reference standard, the comprehensive environment pressure index of each sampling point is constructed and calculated to reflect the overall condition of the target area environment; evaluating the health condition of large invertebrate communities in estuary and coastal environments and the response of the communities to human pressure, and calculating a marine organism index AMBI, so as to display the ecological health condition of a region; a high-throughput sequencing technology is used for identifying microorganism composition, pollution indicator bacteria are defined, a microorganism index M-AMBI value is calculated according to the pollution indicator bacteria, and the ecological health condition of each sampling point is graded; and finally, verifying the reliability of an ecological evaluation result through statistical analysis. According to the method, multi-dimensional data are integrated, the ecology of the estuary and coastal environment is comprehensively evaluated, a scientific basis is provided for revealing an environment pressure source and an ecological effect thereof, formulation of a precise environment management strategy is assisted, pollution risks are effectively prevented and controlled, and sustainable development of an estuary and coastal ecological system is promoted.

Description

Technical Field

[0001] The invention relates to an estuary and coastal environmental ecological assessment method based on a microbial index, and belongs to the technical field of environmental ecological assessment. Background Art

[0002] In recent years, due to the combined effects of natural and human factors, the biodiversity of estuaries and coastal wetlands has continued to decline, and the ecosystem is facing severe challenges. Traditionally, biological indices have been widely used to monitor the health of estuaries and coastal ecosystems. These indices mostly rely on the analysis of front-end consumers of the food chain, such as large benthic invertebrates. These biological communities can respond to human disturbances in the short to medium term. However, with the sharp decline in biodiversity, the biological indicators currently used are no longer sufficient to fully and accurately reflect the overall picture of the ecological health of estuaries and coastal areas.

[0003] In this context, environmental microorganisms, as decomposers that are sensitive to environmental changes, have a rapid response and the development of high-throughput sequencing technology has made it possible to construct new ecological assessment indicators. Information on microbial communities can be converted into ecological assessment indicators to supplement the ecological information provided by macrobenthic invertebrate communities and serve as an early warning system to assess the extent of environmental impacts.

[0004] Therefore, it is particularly important to develop an ecological index based on microbial information. The application of microbial indices provides a new perspective and tool for assessing the health of estuarine and coastal ecosystems, which helps to more accurately predict and respond to ecological problems such as ecological destruction and eutrophication. In this sense, incorporating microbial indices into routine monitoring systems may be a key step in deepening the understanding of the connection between biodiversity and its ecosystem functions. In view of this, the present invention proposes an ecological assessment method for estuarine and coastal environments based on microbial indices to meet the current needs of ecosystem management for new and efficient assessment tools. Summary of the invention

[0005] In order to solve the above problems, the present invention discloses an estuarine and coastal environmental ecological assessment method based on microbial index, and its specific technical scheme is as follows:

[0006] An ecological assessment method for estuarine and coastal environments based on microbial index comprises the following steps:

[0007] Step 1: Select the study area, sample the sediment in the study area to obtain sediment samples, and measure the physical and chemical indicators of the sediment samples;

[0008] Step 2: Construct and evaluate the pressure index of anthropogenic activities on estuarine and coastal environments, and calculate the comprehensive environmental pressure index;

[0009] Step 3: Analyze macroinvertebrate species and calculate the comprehensive marine biological index (AMBI);

[0010] Step 4: Analyze the microbial community, calculate the microbial index M-AMBI, and divide the ecological health status level according to the M-AMBI value;

[0011] Step 5: Conduct statistical analysis on the microbial community data and conduct reliability tests on the ecological assessment results.

[0012] Furthermore, the sediment sample collection method in step 1 is: in the coastal tidal flat area, a nine-point sampling method is adopted, 9 sites are evenly arranged in a square unit of 5m×5m, and 100 grams of sediment sample is taken from 2-5cm below the sediment at each sampling point. All samples are fully mixed to form a comprehensive sample of about 900 grams. Subsequently, the mixed sample is placed in a sterile sealed bag, stored under low temperature conditions, and transported back to the laboratory for standby use. In addition, each sample point needs to be sampled three times, so as to obtain three replicate samples at one site.

[0013] Furthermore, the method for calculating the comprehensive environmental pressure index in step 2 is specifically as follows:

[0014] Step 2.1: Select metal elements, PCBs, PAHs, redox potential and organic matter content as additional indicators of anthropogenic pressure;

[0015] Step 2.2: Use statistical methods to standardize each variable to a range of 0 to 5 and construct an environmental stress index;

[0016] Step 2.3: Use the principal component analysis (PCA) method to calculate the weight of the environmental pressure index of each indicator and determine the weight of each indicator;

[0017] Step 2.4: Based on the weighted average, calculate the comprehensive environmental pressure index of each sampling point according to Formula 1:

[0018]

[0019] In formula 1: P i represents the comprehensive environmental pressure index of the i-th sampling point; w j is the weight of the jth physical and chemical index, obtained through PCA analysis; P {ij} is the environmental pressure index of the jth physical and chemical index at the ith sampling point; n is the total number of physical and chemical indicators.

[0020] Furthermore, the method for calculating the comprehensive marine biological index AMBI in step 3 is specifically as follows:

[0021] Step 3.1: Under a stereomicroscope, separate the macroinvertebrate specimens from each sieved replicate sample, identify and record these specimens to the genus level, and count the macroinvertebrate taxa and abundance lists;

[0022] Step 3.2: Use AMBI software to calculate the AZTI marine biological index AMBI for each macroinvertebrate species at each sampling point. AMBI is based on the response of macroinvertebrate species to pressure gradients and divides all species into five ecological groups EG: EGⅠ represents species that can only survive in very clean and unpolluted environments, EGⅡ represents species that can survive in both polluted and unpolluted environments, EGⅢ represents species that can tolerate a certain degree of environmental pressure, EGⅣ represents species that usually appear when environmental pressure is high, and EGⅤ represents species that usually only survive in severely polluted or extremely disturbed environments. The AZTI marine biological index AMBI for each macroinvertebrate species at each sampling point is calculated according to Formula 2:

[0023] AMBI=0×%EGⅠ+1×%EGⅡ+2×%EGⅢ+3×%EGⅣ+4×%EGⅤ Formula 2

[0024] In formula 2, the relative proportion of each ecological group in the sample provides an AMBI value ranging from 0 to 7, where 0 represents an undisturbed sample and 7 represents an extremely disturbed, abiotic extreme state;

[0025] Step 3.3: Use the principal component analysis (PCA) method to calculate the weight of the marine biological index of all large invertebrates at each sampling point, determine the weight of each indicator, and calculate the comprehensive marine biological index of each sampling point based on the weighted average according to Formula 3:

[0026]

[0027] Where: AMBI{ij} is the AMBI index of the jth macroinvertebrate indicator at the ith sampling site, wj is the weight of the jth indicator, calculated by the PCA method, n is the total number of macroinvertebrate indicators, and Ii is the comprehensive marine biological index at the ith sampling site.

[0028] Furthermore, the method for calculating the microbial index M-AMBI in step 4 is specifically as follows:

[0029] Step 4.1: Extract DNA from sediment samples and amplify the V4 region of the 16S rRNA gene using universal primers and Illumina adapters;

[0030] Step 4.2: Sequencing on the Illumina Miseq platform to obtain valid sequences;

[0031] Step 4.3: Use SINA aligner to classify and assign values ​​to OTU representative sequences, calculate OTU richness and Shannon index, and calculate uniformity index;

[0032] Step 4.4: Use the AMBI strategy to develop a microbial index. Classify microorganisms at the family level according to their ecological effects related to environmental pressure, and divide the microbial community into two ecological groups EG: EGⅠ represents microbial groups that are sensitive to environmental pressure, and EGⅢ represents microbial groups that are tolerant to environmental pressure; based on the relative abundance of microbial families in each ecological group in each sample, a continuous microbial index M-AMBI value is calculated. The M-AMBI value is calculated according to Formula 4.

[0033] M-AMBI=0×%EGⅠ+6×%EGⅢ Formula 4

[0034] In Formula 4: M-AMBI ranges from 0 to 6, where 0 represents 100% of the sequences are assigned to EGⅠ and 6 represents 100% of the sequences are assigned to EGⅢ.

[0035] Furthermore, step 4 divides the ecological health status into five levels according to the M-AMBI value, and each level has a specific range of M-AMBI values ​​and a corresponding ecological group contribution ratio.

[0036] Further step 5.1: convert sequence abundance data into relative abundance as input for statistical analysis;

[0037] Step 5.2: Use Bray-Curtis distance to calculate the similarity matrix and perform hierarchical clustering analysis and NMDS analysis;

[0038] Step 5.3: Use DISTLM to analyze the relationship between microbial community composition and environmental variables;

[0039] Step 5.4: Use linear model regression analysis to calculate the relationship between M-AMBI and stress index, AMBI, and putative indicator taxa and pollutant variables.

[0040] Furthermore, the metal elements in step 2.1 include mercury (Hg), cadmium (Cd), lead (Pb), copper (Cu), zinc (Zn), nickel (Ni) and arsenic (As), which are derived from industrial emissions, agricultural activities and urban sewage.

[0041] Furthermore, the organic matter in step 2.1 includes TOC and TOM.

[0042] Furthermore, the variables in step 2.2 include the marine biological index of invertebrates, microbial index, heavy metals, comprehensive environmental stress index, physical and chemical indicators, PCBs, PAHs, redox potential, TOC and TOM

[0043] The working principle of the present invention is:

[0044] The present invention provides an ecological assessment method for estuaries and coastal environments based on microbial index. The present invention selects representative sampling points and collects sediment samples, analyzes and evaluates the pressure gradient generated by human activities on estuaries and coastal environments through physical and chemical indicators, constructs a pressure index according to the reference standard, and constructs and calculates the comprehensive environmental pressure index of each sampling point to reflect the overall status of the target area environment; evaluates the health status of large invertebrate communities in estuaries and coastal environments and the response of these communities to human pressure, calculates the marine biological index AMBI, and displays the regional ecological health status; uses high-throughput sequencing technology to identify microbial composition, defines pollution indicator bacteria, and calculates the microbial index M-AMBI value to rate the ecological health status of each sampling point; finally, statistical analysis is used to verify the reliability of the ecological evaluation results. The method of the present invention integrates multi-dimensional data to comprehensively evaluate the ecology of estuaries and coastal environments, provides a scientific basis for revealing environmental stressors and their ecological effects, helps to formulate precise environmental management strategies, effectively prevents and controls pollution risks, and promotes the sustainable development of estuaries and coastal ecosystems.

[0045] The beneficial effects of the present invention are:

[0046] 1. Based on the evaluation of ecological environment by marine biological index, the present invention further uses microbial index to comprehensively evaluate estuarine and coastal environmental ecology. This method not only considers the response of large benthic invertebrates, but also incorporates the dynamic changes of microbial communities, providing more comprehensive ecological health information, which helps to more accurately identify and evaluate environmental stressors and their ecological effects.

[0047] 2. This invention introduces the microbial index (M-AMBI) as a new indicator for ecological health assessment through high-throughput sequencing technology. It provides a deeper understanding of the health of ecosystems, especially in the context of declining biodiversity, and can supplement and enhance the assessment capabilities of traditional ecological indicators, providing a new tool for early identification and management of ecology.

[0048] 3. The present invention constructs an ecological health rating system to grade ecological health conditions, providing an intuitive assessment tool for ecological management. It provides a scientific basis for environmental managers. By quantitatively assessing the impact of human activities on estuaries and coastal environments, more targeted pollution prevention and control and ecological restoration measures can be formulated to improve the effectiveness of environmental management.

[0049] 4. The present invention constructs a comprehensive environmental stress index to quantify the impact of multiple environmental parameters, providing managers with a quantitative environmental stress assessment, which helps to formulate more scientific and effective environmental management strategies and reduce the negative impact of human activities on the ecosystem.

[0050] 5. The present invention combines physical and chemical indicators, large invertebrate communities and microbial community analysis to construct a multi-dimensional ecological assessment model, which can more comprehensively assess the health of the ecosystem, avoid the limitations of single indicator assessment, and improve the accuracy and reliability of the assessment results. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0052] The present invention integrates the biological index method, divides the ecological health level based on the microbial index, evaluates the ecological health of estuaries and coastal areas, takes the coastal mudflats of Jiangsu Province as the research object, and constructs a comprehensive microbial index (Microbialbiotic-AZTI's Marine Biotic Index hereinafter referred to as M-AMBI). The ecological assessment process includes the following steps:

[0053] Step 1: Study area selection, sediment sampling and physical and chemical index determination;

[0054] Step 1.1. Select representative sampling points in the target estuaries and coastal areas. Collect four sediment samples at each sampling point using a grab sampler or other suitable sampling tools. Use a 1 mm mesh sieve to screen three sediment samples from each sampling point on site. After removing larger particles, the screened samples are preserved in formalin for morphological identification of large invertebrates. Take the surface (1 cm) sediment from the fourth sample and store it in a sterile centrifuge tube at -80°C for subsequent high-throughput analysis of microorganisms. The remaining sediment samples are used to determine physical and chemical parameters such as sediment salinity, particle size, and organic matter content.

[0055] Step 1.2, freeze-dry part of the sediment samples, and use the dry sieving method to sieve the sediment samples at a temperature of 60°C to determine their particle size distribution; burn the sediment samples at a high temperature of 450°C for 6 hours, and determine the content of organic matter (OM) by weight loss; dry the samples at 60°C, and then sieve to obtain fine-grained sediment samples (less than 63 microns), and use a mass spectrometer to determine the concentration of metal elements in the samples (such as cadmium, chromium, copper, nickel, mercury, lead and zinc, etc.); use gas chromatography-mass spectrometry to determine the concentration of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) in each sample.

[0056] Step 2: Construct and evaluate the pressure index of anthropogenic activities on estuarine and coastal environments, and calculate the comprehensive environmental pressure index;

[0057] Step 2.1, refer to relevant national and local environmental standards, such as the "National Ecological and Environmental Standards of the People's Republic of China (HJ 1300-2023) Technical Specifications for the Assessment of Seawater, Marine Sediments and Marine Organisms Quality" and "Marine Sediment Quality (GB18668-2002) Standards" to determine metal elements, polychlorinated biphenyls and polycyclic aromatic hydrocarbons, as well as redox potential and organic matter content as additional indicators of anthropogenic pressure. Using statistical methods, each variable is standardized to a range of 0 to 5 according to environmental standards to construct an environmental pressure index. Among them, 0 means that there is no pollutant pressure at all, and 5 means the maximum pressure. Concentrations exceeding the standard represent harmful effects. This limit is regarded as 2 in the pressure index (the boundary between acceptable and unacceptable pressure levels). The environmental pressure index of the physical and chemical indicators of each sampling point is calculated, and the maximum concentration recorded in the sampling area within a certain time interval (such as the past five years) is taken as the maximum pressure value;

[0058] Step 2.2: After obtaining the environmental stress index of each physical and chemical indicator, the principal component analysis (PCA) method was used to calculate the weight of the environmental stress index of all indicators at each sampling point to determine the weight of each indicator. The comprehensive environmental stress index of each sampling point was calculated based on the weighted average. Through these steps, researchers were able to evaluate the pressure gradient caused by anthropogenic activities on estuarine and coastal environments and construct a pressure index that can be used to verify other ecological indicators, such as the microbial index, to more comprehensively understand the environmental conditions and the impact of anthropogenic activities.

[0059]

[0060] Where: P i represents the comprehensive environmental pressure index of the i-th sampling point; w j is the weight of the jth physical and chemical index, obtained through PCA analysis; P {ij} is the environmental pressure index of the jth physical and chemical index at the ith sampling point; n is the total number of physical and chemical indicators.

[0061] Step 3: Macroinvertebrate species analysis and calculation of the marine biological index (AMBI);

[0062] Step 3.1. Isolate macroinvertebrate specimens from each sieved replicate under a stereomicroscope, identify and record these specimens at the lowest possible taxonomic level, and compile a list of macroinvertebrate taxa and abundance.

[0063] Step 3.2: Calculate the AZTI Marine Biota Index (AMBI) for each macroinvertebrate species at each sampling point using the AMBI software (https: / / ambi.azti.es / ). AMBI is based on the response of macroinvertebrate species to pressure gradients and divides all species into five ecological groups (EG): EGⅠ (species that can only survive in very clean and unpolluted environments), EGⅡ (species that can survive in both polluted and unpolluted environments), EGⅢ (species that can tolerate a certain degree of environmental pressure, such as pollution and habitat degradation), EGⅣ (usually appear when environmental pressure is high, such as pollution and habitat destruction), and EGⅤ (usually only survive in severely polluted or extremely disturbed environments). The specific calculation formula of AMBI is as follows:

[0064] AMBI=0×%EGⅠ+1×%EGⅡ+2×%EGⅢ+3×%EGⅣ+4×%EGⅤ Formula 2

[0065] Here, the relative proportion of each ecological group in the sample provides an AMBI value ranging from 0 (undisturbed sample) to 7 (extremely disturbed), with 7 representing an extreme state of no life.

[0066] Step 3.3: Use the principal component analysis (PCA) method to calculate the weight of the marine biological index of all large invertebrates at each sampling point and determine the weight of each indicator. The comprehensive marine biological index of each sampling point is calculated based on the weighted average value. The specific formula is as follows:

[0067]

[0068] Where: AMBI{ij} is the AMBI index of the jth macroinvertebrate indicator at the ith sampling site. wj is the weight of the jth indicator, calculated by the PCA method. n is the total number of macroinvertebrate indicators. Ii is the comprehensive marine biological index at the ith sampling site.

[0069] Specifically, through these steps, researchers were able to assess the health of macroinvertebrate communities in estuarine and coastal environments, and the response of these communities to anthropogenic pressures. Higher values ​​of the AMBI index indicate greater disturbance and poorer health of the ecosystem.

[0070] Step 4: Calculation of microbial index (M-AMBI) and classification of ecological health status;

[0071] Step 4.1. First, the sediment microbial community information of each sampling point was identified. DNA was extracted from the surface sediment samples. The bacterial / archaeal universal primers 419F (CAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) and Illumina adapters were used to amplify a 250 bp fragment of the V4 region of the 16S rRNA gene. Each sample was amplified three times in a total reaction volume of 25 μL using 12.5 μL 2×KAPA HiFi HotStart ReadyMix, 5 μL forward and reverse primers, and 2.5 μL standardized DNA (5 ng / μL). The PCR reaction conditions were as follows: preheat at 95°C for 2 min, perform 30 cycles, each cycle including 98°C for 20 s, 50°C for 60 s, and 72°C for 90 s, and finally extend at 72°C for 5 min. The three replicate PCR products were mixed and incubated with AMPure XP beads (Beckman Coulter) for purification; after mixing the dual-index PCR products of equal molar concentrations, sequencing was performed on the Illumina Miseq platform;

[0072] Step 4.2, after the quality assessment and screening steps, obtain valid sequences. Based on the 97% similarity standard, these sequences are aligned, and the OTU representative sequences are classified and assigned using SINA aligner. The standard OTU richness and Shannon index are calculated from the OTUs generated from the high-quality reads; the uniformity index is calculated based on Pielou's index; the sequences are compared with the gene library, and the sample coverage is calculated to evaluate the accuracy of the sequencing results;

[0073] Step 4.3: Use the AMBI strategy to develop a microbial index, classify microorganisms at the family level according to their ecological roles related to environmental stress, and divide the microbial community into two ecological groups (EG): EGⅠ (microbial groups sensitive to environmental stress) and EGⅢ (microbial groups tolerant to environmental stress).

[0074] Specifically, the EGⅢ ecological group is defined as microorganisms associated with environmental stress tolerance and pollution input as pollution indicator bacteria, and its ecological roles include: a. Dominant in sediments rich in organic matter; b. Response to organic pollution; c. Dominant in sediments rich in anoxic methane; d. Identified as nitrite oxidizers and related to nitrogen input; e. Found in sulfide-rich wastewater; f. Found in wastewater treatment plants; g. Degradation of alkanes during methane fermentation; h. Role in the biodegradation of aromatic compounds (including the role in petroleum product pollution); i. Potential pathogens;

[0075] Based on the relative abundance of the microbial family level in each ecological group in each sample, a continuous microbial index M-AMBI value was calculated. The specific calculation formula is as follows:

[0076] M-AMBI=0×%EGⅠ+6×%EGⅢ Formula 4

[0077] Wherein: M-AMBI ranges from 0 to 6, where 0 represents 100% of the sequences are assigned to EGⅠ, and 6 represents 100% of the sequences are assigned to EGⅢ.

[0078] Step 4.4, classify the ecological health status level of each sampling point based on the contribution of each ecological group in the microorganisms of different samples to the final M-AMBI value;

[0079] Specifically, the ecological health status is divided into five levels from "high" to "low", each with a specific range of M-AMBI values ​​and a corresponding ecological group contribution ratio. When the M-AMBI value is between 0 and 1.2, the ecological health status is considered to be "high", which means that more than 80% of the microbial families in the sample are classified as EGⅠ, that is, ecological groups not related to pollution input. As the M-AMBI value increases, the proportion of EGⅠ decreases, while the proportion of EGⅢ increases, indicating that the pressure of pollution on the environment is increasing. When the M-AMBI value is between 1.3 and 2.4, the ecological health status is "high", and the proportion of EGI and EGIII in the sample is approximately 60% and 40% respectively. With the further increase of M-AMBI value, the ecological health status gradually decreases. When the M-AMBI value is between 2.5 and 3.6, the ecological health status is "medium", and the proportion of EGⅠ and EGⅢ is approximately 40% and 60%, respectively. When the M-AMBI value is between 3.7 and 4.8, the ecological health status is rated as "low", at which point the proportion of EGⅠ drops to 20%, while the proportion of EGⅢ increases to 80%. Finally, when the M-AMBI value is between 4.9 and 6, the ecological health status is rated as "low", which means that more than 80% of the bacterial sequences in the sample are classified as EGⅢ, an ecological group associated with pollution input.

[0080] Step 5: Conduct statistical analysis on microbial community data and test the reliability of ecological assessment results.

[0081] Step 5.1. The sequence abundance data of each cluster of microbial data were converted to relative abundance (percentage of the total number of sequences in each sample) and used as input for statistical analysis using the Primer 6+ statistical software package. The similarity matrix calculated using the Bray-Curtis distance after square root transformation of the relative abundance data was used to perform hierarchical clustering based on group average (average distance between two groups, average distance between pairs of groups) and non-metric multidimensional scaling (NMDS).

[0082] Step 5.2: Perform similarity percentage (SIMPER) analysis based on the Bray-Curtis similarity matrix to determine the taxa that contribute to the differences between groups and the top 10% of similarity within each group; perform hierarchical cluster analysis based on the group average method; further analyze the samples using nonmetric multidimensional scaling (NMDS); construct an NMDS plot to explore the separation of samples based on abiotic parameters; after removing redundancy, select the main variables (such as salinity, organic matter content, oxidation potential, ∑PCB, ∑PAH, and concentrations of Zn, Pb, Cd, and Hg) to analyze the relationship between microbial community composition and environmental variables, and use a distance-based linear model (DISTLM) to examine the relationship between bacterial community composition and environmental variables.

[0083] Step 5.3: The relationships between M-AMBI and stress index, M-AMBI and AMBI, and putative indicator taxa and pollutant variables were calculated by linear model regression analysis (lm) performed in R. Forward selection was used to assess the significant response of putative indicator taxa to pollution variables. Based on the above analysis, the reliability of the results of ecological assessment using microbial indices was tested.

[0084] Although all possible combinations of technical features are not exhaustively listed in this specification, any combination of technical features and the execution order of method steps are not limited. In order to maintain the brevity of the description, only several embodiments are shown, which are specific and detailed, but should not be regarded as limiting the scope of the patent of this application. It should be clear that professionals in this field can implement various variations and improvements without departing from the core idea of ​​this application, which are also included in the protection scope of this application. Therefore, the specific protection scope of this application should be determined with reference to the attached claims.

[0085] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as those generally understood by those skilled in the art to which this application belongs. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with the meanings in the context of the prior art, and will not be interpreted with idealized or overly formal meanings unless defined as herein.

[0086] With the above-mentioned ideal embodiments of the present invention as inspiration, through the above-mentioned description content, relevant personnel can make various changes and modifications without departing from the scope of the technical idea of ​​the present invention.

Claims

1. A method for evaluating estuarine and coastal environment ecology based on microbial index, characterized in that: The following steps are involved: Step 1: Select the study area, sample the sediment in the study area to obtain sediment samples, and measure the physical and chemical indicators of the sediment samples; Step 2: Construct and evaluate the pressure index of anthropogenic activities on estuarine and coastal environments, and calculate the comprehensive environmental pressure index; Step 3: Analyze macroinvertebrate species and calculate the comprehensive marine biological index (AMBI); Step 4: Analyze the microbial community, calculate the microbial index M-AMBI, and divide the ecological health status level according to the M-AMBI value; Step 5: Conduct statistical analysis on the microbial community data and conduct reliability tests on the ecological assessment results.

2. The method for evaluating estuarine and coastal environment ecology based on microbial index according to claim 1, characterized in that: The sediment sample collection method in step 1 is as follows: in the coastal tidal flat area, a nine-point sampling method is used, 9 sites are evenly arranged in a square unit of 5m×5m, 100 grams of sediment sample is taken from 2-5cm below the sediment at each sampling point, all samples are fully mixed to form a comprehensive sample of about 900 grams, and then the mixed sample is packed into a sterile sealed bag, stored under low temperature conditions, and transported back to the laboratory for standby use. In addition, each sample point needs to be sampled three times, so as to obtain three repeated samples at one site.

3. The method for evaluating estuarine and coastal environment ecology based on microbial index according to claim 1, characterized in that: The method for calculating the comprehensive environmental pressure index in step 2 is specifically as follows: Step 2.1: Select metal elements, PCBs, PAHs, redox potential and organic matter content as additional indicators of anthropogenic pressure; Step 2.2: Use statistical methods to standardize each variable to a range of 0 to 5 and construct an environmental stress index; Step 2.3: Use the principal component analysis (PCA) method to calculate the weight of the environmental pressure index of each indicator and determine the weight of each indicator; Step 2.4: Based on the weighted average, calculate the comprehensive environmental pressure index of each sampling point according to Formula 1: In formula 1: P i represents the comprehensive environmental pressure index of the i-th sampling point; w j is the weight of the jth physical and chemical index, obtained through PCA analysis; P {ij} is the environmental pressure index of the jth physical and chemical index at the ith sampling point; n is the total number of physical and chemical indicators.

4. The method for evaluating estuarine and coastal environment ecology based on microbial index according to claim 1, characterized in that: The method for calculating the comprehensive marine biological index AMBI in step 3 is specifically as follows: Step 3.1: Under a stereomicroscope, separate the macroinvertebrate specimens from each sieved replicate sample, identify and record these specimens to the genus level, and count the macroinvertebrate taxa and abundance lists; Step 3.2: Use AMBI software to calculate the AZTI marine biological index AMBI for each macroinvertebrate species at each sampling point. AMBI is based on the response of macroinvertebrate species to pressure gradients and divides all species into five ecological groups EG: EGⅠ represents species that can only survive in very clean and unpolluted environments, EGⅡ represents species that can survive in both polluted and unpolluted environments, EGⅢ represents species that can tolerate a certain degree of environmental pressure, EGⅣ represents species that usually appear when environmental pressure is high, and EGⅤ represents species that usually only survive in severely polluted or extremely disturbed environments. The AZTI marine biological index AMBI for each macroinvertebrate species at each sampling point is calculated according to Formula 2: AMBI=0×%EGⅠ+1×%EGⅡ+2×%EGⅢ+3×%EGⅣ+4×%EGⅤ Formula 2 In formula 2, the relative proportion of each ecological group in the sample provides an AMBI value ranging from 0 to 7, where 0 represents an undisturbed sample and 7 represents an extremely disturbed, abiotic extreme state; Step 3.3: Use the principal component analysis (PCA) method to calculate the weight of the marine biological index of all large invertebrates at each sampling point, determine the weight of each indicator, and calculate the comprehensive marine biological index of each sampling point based on the weighted average according to Formula 3: Where: AMBI{ij} is the AMBI index of the jth macroinvertebrate indicator at the ith sampling site, wj is the weight of the jth indicator, calculated by the PCA method, n is the total number of macroinvertebrate indicators, and Ii is the comprehensive marine biological index at the ith sampling site.

5. The method for evaluating estuarine and coastal environment ecology based on microbial index according to claim 1, characterized in that: The method for calculating the microbial index M-AMBI in step 4 is specifically as follows: Step 4.1: Extract DNA from sediment samples and amplify the V4 region of the 16S rRNA gene using universal primers and Illumina adapters; Step 4.2: Sequencing on the Illumina Miseq platform to obtain valid sequences; Step 4.3: Use SINA aligner to classify and assign values ​​to OTU representative sequences, calculate OTU richness and Shannon index, and calculate uniformity index; Step 4.4: Use the AMBI strategy to develop a microbial index, classify microorganisms at the family level according to their ecological roles related to environmental stress, and divide the microbial community into two ecological groups EG: EGⅠ represents microbial groups that are sensitive to environmental stress, and EGⅢ represents microbial groups that are tolerant to environmental stress; Based on the relative abundance of microbial families in each ecological group in each sample, a continuous microbial index M-AMBI value was calculated. The M-AMBI value was calculated according to Formula 4: M-AMBI=0×%EGⅠ+6×%EGⅢ Formula 4 In Formula 4: M-AMBI ranges from 0 to 6, where 0 represents 100% of the sequences are assigned to EGⅠ and 6 represents 100% of the sequences are assigned to EGⅢ.

6. The method for evaluating estuarine and coastal environment ecology based on microbial index according to claim 1, characterized in that: The step 4 divides the ecological health status into five levels according to the M-AMBI value, and each level has a specific M-AMBI value range and a corresponding ecological group contribution ratio.

7. The method for evaluating estuarine and coastal environment ecology based on microbial index according to claim 1, characterized in that: The method for performing statistical analysis on the microbial community data in step 5 is specifically as follows: Step 5.1: Convert sequence abundance data to relative abundance as input for statistical analysis; Step 5.2: Use Bray-Curtis distance to calculate the similarity matrix and perform hierarchical clustering analysis and NMDS analysis; Step 5.3: Use DISTLM to analyze the relationship between microbial community composition and environmental variables; Step 5.4: Use linear model regression analysis to calculate the relationship between M-AMBI and stress index, AMBI, and putative indicator taxa and pollutant variables.

8. The method for evaluating estuarine and coastal environment ecology based on microbial index according to claim 3, characterized in that: The metal elements in step 2.1 include mercury (Hg), cadmium (Cd), lead (Pb), copper (Cu), zinc (Zn), nickel (Ni) and arsenic (As), which come from industrial emissions, agricultural activities and urban sewage.

9. The method for evaluating estuarine and coastal environment ecology based on microbial index according to claim 3, characterized in that: The organic matter in step 2.1 includes TOC and TOM.

10. The method for evaluating estuarine and coastal environment ecology based on microbial index according to claim 3, characterized in that: The variables in step 2.2 include the marine biological index of invertebrates, microbial index, heavy metals, comprehensive environmental stress index, physical and chemical indicators, PCBs, PAHs, redox potential, TOC and TOM.

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