Method for detecting influence of microplastics and triclosan on structure and function of sediment microbial community

Abstract

The invention provides a method for detecting the influence of microplastics and triclosan on the structure and function of a sediment microbial community. The method comprises the following specific steps: firstly, collecting a sediment sample in a specified sampling area; then adding the micro-plastic particles and a triclosan solution into the sediment sample, and incubating; dNA of the microorganisms is extracted, and 16S rRNA sequencing is carried out; and finally, analyzing the diversity of the microbial community and evaluating the structure and function of the microbial community according to the sequencing data. The comprehensive influence of micro-plastic and triclosan combined pollution on the structure and function of the sediment microbial community is systematically analyzed for the first time, the efficiency and accuracy of sediment microbial community detection are effectively improved by adopting a 16S rRNA detection technology, and a reliable basis is provided for formulating an environment protection strategy, optimizing pollution control and promoting ecological restoration engineering.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of environmental protection, and particularly relates to a method for detecting the effects of microplastics and triclosan on the structure and function of sediment microbial communities. BACKGROUND

[0002] In recent years, with the rapid development of global economy, the large-scale use of plastic products and personal care products has caused increasingly serious environmental pollution problems. As emerging pollutants, microplastics and the antibacterial agent triclosan have attracted widespread attention due to their accumulation and spread in the environment. Microplastics have become a global environmental pollution problem that deserves attention due to their small size, abundant sources, large specific surface area, strong hydrophobicity, easy migration and transformation in the environment, and difficulty in degradation. Microplastics not only exist widely in soil and water environments, but also pose potential threats to ecosystems and even human health through the food chain and biological accumulation. On the other hand, as a compound of pharmaceutical and personal care products, triclosan is a multifunctional broad-spectrum antibacterial agent widely used in consumer products and exhibits strong microbial toxicity and difficulty in degradation, and is easily spread into the natural environment through pollution discharge and further spread and accumulate in the natural environment, affecting environmental microorganisms.

[0003] Sediments are not only an important sink for pollutants, but also an important habitat for microbial communities, and are an important part of global ecosystems and a key node of the biochemical cycle, affecting element cycling and energy flow. Microorganisms in sediments play an important role in maintaining ecological balance, promoting material cycling and energy flow. However, the combined pollution of microplastics and triclosan can have a significant impact on the structure and function of sediment microbial communities, thereby disrupting the balance and normal function of the ecosystem.

[0004] Currently, there has been some progress in the study of the effects of microplastics and triclosan on sediment microbial communities and functional genes, but there are still many challenges and deficiencies. First, conventional microbial technology methods, such as culture method and microscopic observation method, can characterize the basic information of microbial surface structure, but cannot comprehensively reflect the diversity and complexity of microbial communities. Second, the interaction mechanism of microplastics and triclosan in sediments and how their combined pollution affects microbial communities and functions are still lacking in-depth understanding and research.

[0005] In addition, existing evaluation methods often focus on the effects of a single pollutant, ignoring the comprehensive effects under the combined action of multiple pollutants. In actual environments, microplastics and triclosan often coexist, and their interaction can have more complex and unpredictable effects on microbial communities. Therefore, there is an urgent need to develop a new method that can comprehensively consider the effects of microplastics and triclosan combined pollution and fully evaluate the changes in the structure and function of sediment microbial communities. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the shortcomings of the existing technology and provide a novel, accurate and comprehensive evaluation system that combines modern molecular biology techniques (such as 16S rRNA sequencing) and ecological methods to deeply reveal the impact mechanism of microplastics and triclosan on the structure and function of sediment microbial communities, and provide scientific basis and technical support for environmental protection, pollution control and ecological restoration.

[0007] To address the aforementioned technical problems, this invention provides a method for detecting the effects of microplastics and triclosan on the structure and function of sediment microbial communities. The method includes the following steps:

[0008] S1. Obtain sediment samples at the sampling point;

[0009] S2. Add microplastic particles and triclosan solution to the sediment sample and incubate.

[0010] S3. Extract microbial DNA and perform 16S rRNA sequencing.

[0011] S4. Based on the sequencing results, conduct an assessment of microbial community diversity and ecological function.

[0012] The above method, further, includes S1 comprising:

[0013] Sediment samples were taken at a depth of 25m in the selected study area. After sampling, the sediment samples were thoroughly mixed, air-dried, impurities removed, and sieved before being stored in a 4°C refrigerator for later use.

[0014] The above method, further, includes S2 comprising:

[0015] S2-1. Add 1% by mass of microplastic particles and 10 mg / kg of triclosan solution to the pretreated sediment sample; add deionized water so that the water level is about 2 mm above the sediment surface, and replenish it every 3 days to maintain its condition.

[0016] S2-2. Incubate for 60 days in the dark at 25°C to simulate the long-term interaction between microplastics and triclosan in sediments.

[0017] The above method, further, includes S3 comprising:

[0018] DNA from microorganisms was extracted from sediments and amplified by PCR using 338F and 806R primers, followed by high-throughput sequencing to specifically target the 16S rRNA V3-V4 region.

[0019] The gene sequence of the 338F is shown as SEQ ID NO. 1, and the gene sequence of the 806R is shown as SEQ ID NO. 2.

[0020] The method, further, in the S4, adopts the Bray-Curtis distance combined with the principal coordinate analysis method to analyze the community diversity of the sequencing results, and evaluates the differences in the microbial community composition among different treatments.

[0021] The method, further, in the S4, adopts the community bar graph to represent the community structure composition at the microbial door level among different treatments.

[0022] The method, further, in the S4, adopts the community heatmap graph to represent the community structure composition at the microbial genus level among different treatments.

[0023] The method, further, in the S4, adopts the FAPROTAX ecological function prediction method to predict the ecological function of the microorganism in the sediment according to the microbial community structure information.

[0024] The method, further, in the S4, constructs a co-occurrence network graph to analyze the correlation between microbial species and the interaction relationship of key species.

[0025] Compared with the prior art, the advantages of the present application are:

[0026] (1) The present application provides a method for detecting the effects of microplastics and triclosan on the structure and function of sediment microbial communities, overcoming the challenges of complex pollution research, and comprehensively analyzing the comprehensive effects of microplastics and triclosan complex pollution on the structure and function of microbial communities in sediments for the first time, filling the research gap and providing a new perspective for understanding the environmental impact of complex pollutants.

[0027] (2) The present application provides a method for detecting the effects of microplastics and triclosan on the structure and function of sediment microbial communities, which uses 16S rRNA detection technology to improve the detection speed and accuracy of sediment microbial communities, and enhances the understanding of microbial dynamics under the influence of microplastics and triclosan.

[0028] (3) The present application provides a method for detecting the effects of microplastics and triclosan on the structure and function of sediment microbial communities, which provides strong support for evaluating the actual impact of complex pollution on ecosystem function through FAPROTAX tools.

[0029] (4) The present application provides a method for detecting the effects of microplastics and triclosan on the structure and function of sediment microbial communities, which provides solid support for formulating environmental protection strategies, optimizing pollution control and implementing ecological restoration projects. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application.

[0031] Figure 1 is a flow chart of the method of the present application embodiment 1;

[0032] Figure 2 is the PCoA analysis chart of each group of the present application embodiment at the ASV level, the door level column chart and the genus level heat map;

[0033] Figure 3 is the sediment microbial co-occurrence network diagram of the present application embodiment;

[0034] Figure 4 is the variation chart of the relative abundance of genes in the FAPROTAX database of the present application embodiment. DETAILED DESCRIPTION

[0035] The present application will be further described below in combination with specific preferred embodiments, but the protection scope of the present application is not limited thereby.

[0036] The materials, reagents and instruments used in the following examples can be obtained from commercial channels. The experimental methods in the following examples are conventional methods in the art, unless otherwise specified.

[0037] Example 1

[0038] A method for detecting and evaluating the effects of microplastics and triclosan on the structure and function of sediment microbial communities according to the present application, the flow chart is as shown in Figure 1 , which specifically comprises the following steps:

[0039] S1, obtaining sediment samples at the sampling point. Sediment sampling is carried out at a depth of 25 m from the river bank, and the sampling is immediately transferred to the laboratory. The sediment is air-dried under good ventilation conditions, and then plant residues, gravel and other impurities are removed by crushing and manual sorting with tweezers. The sample is ground and sieved through a 1 mm sieve, and stored in a 4℃ refrigerator before the experiment.

[0040] S2, adding microplastic particles and triclosan solution to the sediment sample and incubating.

[0041] The beaker experiment was incubated for 60 days, specifically: 300g of sediment was added to a 500ml beaker, and the original polyvinyl chloride microplastic (PVC) content was 1% (W / W), and triclosan (TCS) was 10mg / kg. In order to simulate the submerged state of river sediments in shallow freshwater, we added deionized water to make the water surface about 2mm higher than the sediment surface, and replenished water regularly to maintain its state. Put into 25℃ incubator, dark condition, incubation experiment.

[0042] In order to explore the combined pollution of microplastics and triclosan on the structure and ecological function of sediment microbial community, different treatment conditions were set, Figure 1 Among them, CK (original sediment), P (CK+1% PVC), T (CK+10mg / kg TCS), PT (CK+1% PVC+10mg / kg TCS). All treatments are uniform, and one is three times, according to the above method.

[0043] S3, extract microbial DNA for 16S rRNA. After extracting the quality and concentration of DNA, polymerase chain reaction (PCR) is carried out for DNA amplification. The primer 338F (the gene sequence of 338F is specifically shown in SEQ ID NO. 1, specifically: 5'-ACTCCTACGGGGGGGGAGGCAGCA-3') and 806R (the gene sequence of 806R is specifically shown in SEQ ID NO. 2, specifically: 5'-GGACTACHVGGGTWTCTAAT-3') are used to amplify the V3-V4 region of bacterial 16S rRNA gene. Then the product obtained is prepared for high-throughput sequencing by Illumina MiSeq platform (Illumina, San Diego, CA, USA). Sequences with 97% similarity are clustered into ASV.

[0044] S4, based on the sequencing results, the microbial community diversity, community composition and ecological function evaluation.

[0045] After detection and evaluation, it is confirmed that the existence of polyvinyl chloride microplastics and triclosan significantly changes the composition and function of sediment microbial community.

[0046] Experiment one: investigate the PCOA analysis chart of each group of sediment at ASV level, door level column chart and genus level heat map.

[0047] Based on the results of 16S rRNA sequencing, the microbial community structure and composition of each group of samples were analyzed.

[0048] Figure 2Figure 1 is a PCoA analysis plot, a door level column chart and an genus level heat map of each group at the ASV level. It can be seen from the figure that: the PCoA analysis based on the Bray-Curtis distance matrix shows that the phylogenetic structure of the microbial community in the sediment under different pollutant treatments is significantly different. The isolated cluster indicates that the exposure of pollutants changes the microbial community structure. The changes in T group and PT group are more obvious than those in P group. In addition, the microbial community structure in PT group and T group is significantly separated from the control. Specifically, at the door and genus levels, some microorganisms are significantly enriched in different pollution groups (Fig. b-c), we found that Acidobacteriota was significantly enriched in T group, Gemmatimonadota and Verrucomicrotiota were significantly enriched in P group, and Myxococcota was significantly enriched in PT group.

[0049] Experiment two: investigate the microbial co-occurrence network diagram of the sediment in each group.

[0050] Figure 3 Figure 2 is the microbial co-occurrence network diagram of the sediment in the embodiment of the application. It can be seen from the figure that TCS increases the network index of the bacterial co-occurrence network, including the edge number, the positive nature, the average degree and the graph density, which increases by 36.49%, 0.61%, 36.49% and 36.47% respectively, which indicates that a bacterial co-occurrence network with higher interconnection density and more complex connectivity is developed adaptively, and then the modular degree is reduced by 51.38%, which shows that under the action of triclosan alone, the stability of the co-occurrence network structure is reduced, resulting in the decrease of the community stability, reflecting the decrease of the adaptability and recovery ability of the bacterial community to pollution stress. Compared with the control group, P group and PT group reduce the edge number, positive nature, average degree and graph density of the bacterial co-occurrence network, and increase the modular degree at the same time. The increase of the modular degree helps to form a more stable co-occurrence network structure, indicating that the community stability is improved, reflecting the adaptability and recovery ability of the bacterial community to microplastic pollution.

[0051] Experiment three: investigate the change of the relative abundance of genes in the FAPROTAX database of each group of sediment.

[0052] Figure 4is a plot of the relative abundance of genes in the FAPROTAX database of the embodiment of the present application. As can be seen from the figure: different microbial communities play a unique ecological role in the sediment, and the structure and external environmental changes affect the conversion of matter and energy. The control group CK maintains a relatively stable carbon cycle function spectrum at all time points, indicating that the microbial community maintains functional homeostasis under the absence of pollution pressure. Under the addition of triclosan or microplastics, the function of carbon cycle oxidative cooperation is reduced, and methane action and ureolysis in nitrogen cycle are enhanced, nitrogen respiration and nitrogen fixation. Specifically, microplastics enhance methanol oxidation, and reduce aromatic compound degradation function. Triclosan enhances the function of methanotrophy, and under co-contamination, it is shown that triclosan can offset the effect of microplastics and further affect its carbon cycle function. In the nitrogen cycle function, triclosan reduces nitrification, triclosan enhances the function of nitrogen fixation, and microplastics reduces its function, but when microplastics and triclosan coexist, its function changes little, indicating that microplastics can weaken the effect of triclosan, indicating that the interaction offsets the respective influences.

[0053] The above results show that the combined effect of pollutants changes the influence of individual pollutants on the composition of microbial communities. Through PCoA and community composition analysis, it is found that the diversity and structure of microbial communities have changed significantly, specifically, at the level of phylum and genus, some bacteria are significantly enriched in different pollution groups. The advantage in co-contamination not only reduces the complexity of the co-contamination network. FAPROTAX analysis shows that co-contamination significantly changes the abundance of carbon and nitrogen cycle related functions.

[0054] The above description is only a preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification of the above embodiments made according to the technical essence of the present application, without departing from the technical solutions of the present application, all still belong to the protection scope of the technical solutions of the present application.

Claims

1. A method of detecting the effects of microplastics and triclosan on the structure and function of sediment microbial communities, characterized in that, The method comprises the following steps: S1, obtaining a sediment sample at a sampling point; S2, adding microplastic particles and triclosan solution to the sediment sample and incubating; S3, extracting microbial DNA for 16S rRNA sequencing; S4, based on the sequencing results, evaluating microbial community diversity and ecological function.

2. The method of claim 1, wherein, The S1 comprises: Sediment sampling is performed at a depth of 25 m in a selected study area; after sampling, the sediment sample is thoroughly mixed and uniformly mixed, then subjected to air drying treatment, impurities are removed and sieved, and then stored in a 4℃ refrigerator for standby.

3. The method of claim 1, wherein, The S2 comprises: S2-1, adding 1% by mass of microplastic particles and 10 mg / kg of triclosan solution to the pretreated sediment sample; adding deionized water to make the water surface higher than the sediment surface by about 2 mm, and replenishing every 3 days to maintain its state; S2-2, incubating for 60 days under 25℃ dark conditions to simulate the long-term action process of microplastics and triclosan in the sediment.

4. The method according to any one of claims 1 to 3, characterized in that, The S3 comprises: Extracting the DNA of microorganisms in the sediment, and performing PCR amplification and high-throughput sequencing with 338F and 806R primers to specifically target the 16S rRNA V3-V4 region, the gene sequence of the 338F being as shown in SEQ ID NO. 1, and the gene sequence of the 806R being as shown in SEQ ID NO.

2.

5. The method according to any one of claims 1 to 3, characterized in that, In the S4, the Bray-Curtis distance combined with principal coordinate analysis method is used to analyze the community diversity of the sequencing results, and to evaluate the differences in microbial community composition among different treatments.

6. The method according to any one of claims 1 to 3, characterized in that, In the S4, the community composition changes are characterized by door level column chart and genus level heat map.

7. The method according to any one of claims 1 to 3, characterized in that, In the S4, the FAPROTAX ecological function prediction method is used to predict the ecological function of microorganisms in the sediment according to the microbial community structure information.

8. The method according to any one of claims 1 to 3, characterized in that, In the S4, a co-occurrence network diagram is constructed to analyze the correlation between microbial species and the interaction relationship of key species.

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