Heavy metal contaminated soil bioremediation method and system based on soil metagenome sequencing

Abstract

The invention belongs to the technical field of soil heavy metal pollution remediation, and discloses a heavy metal pollution soil bioremediation method and system based on soil metagenome sequencing, and the method comprises the following steps: carrying out gene sequencing on experimental microorganism class groups in heavy metal pollution soil to determine functional microorganisms containing heavy metal resistance genes; heavy metal resistant microorganism species in the soil can be accurately identified by judging whether functional genes are contained or not, and ecological unbalance possibly caused by blind introduction of alien species is avoided; a remediation strategy taking a microbial resistance mechanism as a core can effectively reduce the bioavailability of heavy metals in the soil, and also can improve the physicochemical properties of the soil through metabolic activity to form a sustainable remediation effect; the method has the advantages of being environmentally friendly, controllable in cost, lasting in effect and the like, and an innovative path is provided for soil ecological function restoration.

Description

Technical Field

[0001] The present invention belongs to the technical field of soil heavy metal pollution remediation, and relates to a biological remediation method and system for heavy metal contaminated soil based on soil metagenomic sequencing. Background Art

[0002] With the mining, industrial sewage discharge, and application of pesticides and fertilizers, copper pollution in the environment is becoming increasingly serious. Abandoned mines, electronic factories, landfills, etc. are all heavy disaster areas of copper pollution and have become pollution sources affecting surrounding farmland or forest land. Long-term copper pollution not only leads to the loss of soil functions and the decline of soil environmental quality, but also magnifies its toxicity through the food chain, posing a potential threat to human health. As an important part of the ecosystem, the health status of soil is directly related to ecological balance and human sustainable development. Therefore, the importance of soil copper pollution control has become increasingly prominent. Generally speaking, the treatment of metal pollution has become the focus of global attention. On the one hand, through strict environmental monitoring and technological innovation, efforts are made to cut off the emission sources of copper-containing pollutants and reduce the ways for them to enter the soil; on the other hand, for the already contaminated soil, active remediation work is carried out to restore the ecological functions of the soil.

[0003] Microbial remediation technology has become one of the important means for metal pollution treatment due to its low cost, environmental friendliness, and strong sustainability. The metabolic action of microorganisms is used to transform copper in the soil, thereby reducing its toxicity. The key to microbial remediation lies in screening and domesticating highly efficient microbial strains. For example, certain bacteria and fungi can fix or transform heavy metals into low-toxic forms through biosorption, precipitation, or redox reactions. In addition, by adding specific functional microorganisms to the contaminated soil, promoting plant growth or increasing the microbial biomass further improves the absorption of copper by organisms to achieve the purpose of reducing the metal content in the soil.

[0004] The strains or microbial agents obtained by the above traditional screening and domestication methods fail to consider the following situations: First, the growth environment requirements of different types of microbial communities are different; second, microorganisms may not grow well at the set screening concentrations, but in the actual natural environment, they can effectively reduce the activity of metals or help plant roots absorb metals, thereby achieving the effect of controlling heavy metal pollution. Therefore, it is impossible to efficiently and accurately screen strains suitable for large-scale field application, resulting in poor effects or inability to adapt to the field environment when laboratory-screened or domesticated strains are implemented in the wild. Traditional laboratory screening or domestication of remediation strains usually takes a long time, which makes the cycle of biological remediation long and the cost high, thus restricting its popularization and application to a certain extent. Summary of the Invention

[0005] The purpose of the present invention is to solve the problem that the traditional laboratory screening or domestication of repair strains for bioremediation in the prior art has a long cycle and high cost, and it is impossible to efficiently and accurately screen strains suitable for large-scale, field application for heavy metal contaminated soil remediation, and to provide a method and system for bioremediation of heavy metal contaminated soil based on soil metagenome sequencing.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Bioremediation methods for heavy metal contaminated soil based on soil metagenomic sequencing include:

[0008] Collect soil samples contaminated by heavy metals and perform genetic sequencing on the soil samples;

[0009] Annotate microbial classification information and heavy metal resistance genes in contaminated soil based on gene sequencing sequences;

[0010] Based on the annotation results, the microorganisms and their species information with heavy metal resistance genes in the contaminated soil were determined;

[0011] Microbial species containing heavy metal resistance genes are released into heavy metal contaminated soil for remediation to obtain remediated soil.

[0012] A further improvement of the present invention is:

[0013] Furthermore, soil samples contaminated by heavy metals were collected, specifically: several sampling points were randomly arranged in the soil contaminated by heavy metals, 0-30 cm of soil was collected avoiding plant roots, the soil was placed in a sterile polyethylene ziplock bag, and after being processed with a 2 mm sieve, 15 g of soil was taken and stored at 4°C for later use.

[0014] Furthermore, genetic sequencing was performed on soil samples, specifically: a unit weight of soil was taken and mixed thoroughly, and a DNA extraction kit was used to extract the total soil DNA; and the gene sequence was determined using a sequencing instrument to obtain the metagenome of all soil samples in the target restoration area, with a sequencing depth of at least 20G.

[0015] Furthermore, a gene sequencer is used to determine the gene sequences to obtain the metagenomes of all soil samples in the target repair area. Specifically: 2 μL of total soil DNA is taken and detected by 1% agarose gel electrophoresis; the DNA quality is detected by Nanodrop; the total soil DNA sample is randomly fragmented into small fragments by an ultrasonic crusher; the ends of the DNA small fragments are repaired to blunt ends; a polymerase is used to ligate an adenosine acid after the DNA small fragments; a DNA ligase is used to ligate the sequencing adapter to the end of the small fragment DNA; the DNA small fragments are fixed on the flow cell; fluorescently labeled nucleotides are gradually added to form complementary strands with the sample; the sequencer is turned on, and the base types at each position are determined by detecting the intensity and position feedback of these fluorescent labels to obtain the original sequencing data.

[0016] Furthermore, annotate the microbial taxonomic information in the contaminated soil. Specifically: use the software MetaBAT2 to perform homologous "binning" on the metagenomic sequences, and each "binned" genome is regarded as the complete genome of a microbial species; use CheckM to evaluate the integrity and contamination degree of all bins to obtain all high-quality "binned" genomes in the soil; among them, the high-quality judgment criteria are that the completeness is greater than 90% and the contamination degree is less than 10%; it is obtained by aligning with the Genome Taxonomy Database and sequencing data, and all sequenced genomes are annotated on GTDB and standardized taxonomic labels are provided; then the species taxonomic information is statistically analyzed at each taxonomic level of kingdom, phylum, class, order, family, genus, and species.

[0017] Furthermore, annotate the heavy metal resistance genes. Specifically: the high-quality "binned" genomes are aligned in the BactMet library, and a high-quality potential heavy metal remediation microbial genome is determined according to the matching of one or more genes of cinA, copR, copS, copD, copC, fpvA, cueA, copA, copB, golT, copG, czcC, copF, mmco, copP, ctpG, ctpV; the potential heavy metal remediation microbial genome is a "binned" genome containing heavy metal resistance genes.

[0018] Furthermore, heavy metal contaminated soil refers to soil with heavy metal content exceeding 50 mg / kg.

[0019] Furthermore, the dosage of high-quality potential heavy metal remediation microorganisms is 10 8 cells / gram, and the repair treatment time is 15 days.

[0020] A biological remediation system for heavy metal contaminated soil based on soil metagenomic sequencing, comprising:

[0021] A collection module, which collects soil samples contaminated by heavy metals and performs gene sequencing on the soil samples;

[0022] An annotation module, which annotates the microbial classification information and heavy metal resistance genes in contaminated soil based on gene sequencing sequences;

[0023] A determination module, which determines the microorganisms containing heavy metal resistance genes and their species information in contaminated soil based on the annotation results;

[0024] A remediation module, which puts the microbial species containing heavy metal resistance genes into heavy metal contaminated soil for remediation to obtain the remediated soil.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention uses gene sequencing of experimental microbial groups in heavy metal contaminated soil to determine functional microorganisms containing heavy metal resistance genes, and can accurately identify heavy metal resistant microbial species in soil by whether they contain functional genes, avoiding potential ecological imbalance caused by blindly introducing foreign species; The remediation strategy centered on the self-resistance mechanism of microorganisms can not only effectively reduce the bioavailability of heavy metals in soil, but also improve the physical and chemical properties of soil through metabolic activities, forming a sustainable remediation effect; The present invention has the advantages of environmental friendliness, controllable cost, and long-lasting effect, providing an innovative path for the restoration of soil ecological functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is a schematic flow chart of the biological remediation method for heavy metal contaminated soil based on soil metagenomic sequencing of the present invention;

[0029] Figure 2 It is a schematic structural diagram of the biological remediation system for heavy metal contaminated soil based on soil metagenomic sequencing of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] In order to make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0031] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0032] It should be noted that like reference numerals and letters denote like items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0033] In the description of the embodiments of the present invention, it should be noted that if terms such as "upper", "lower", "horizontal", "inner", etc. are used to indicate the orientation or positional relationship, it is based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship in which the invention product is usually placed during use. This is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation of the present invention. In addition, terms such as "first", "second", etc. are only used for distinguishing descriptions and cannot be construed as indicating or implying relative importance.

[0034] In addition, if the term "horizontal" appears, it does not mean that the component is required to be absolutely horizontal, but it can be slightly inclined. For example, "horizontal" only means that its direction is more horizontal relative to "vertical", and does not mean that the structure must be completely horizontal, but it can be slightly inclined.

[0035] In the description of the embodiments of the present invention, it should also be noted that unless otherwise clearly specified and limited, if terms such as "set", "installed", "connected", "connected" are used, they should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0036] The present invention will be further described in detail below with reference to the accompanying drawings:

[0037] See Figure 1 , the present invention discloses a method for bioremediation of heavy metal contaminated soil based on soil metagenomic sequencing, including:

[0038] S101, collecting soil samples contaminated by heavy metals and performing gene sequencing on the soil samples;

[0039] Randomly arrange several sampling points in the soil contaminated by heavy metals, avoid plant roots and collect the soil at 0 - 30 cm, put it in a sterile polyethylene self-sealing bag, after processing with a 2 mm sieve, take 15 g of soil and store it at 4 °C for later use.

[0040] Perform gene sequencing on the soil samples. Specifically: take a unit weight of soil and mix it thoroughly, use a DNA extraction kit for extraction to obtain the total soil DNA; and use a sequencing instrument to determine the gene sequence to obtain the metagenome of all soil samples in the target repair area, with a sequencing depth of at least 20 G.

[0041] The determination of the gene sequence using a sequencing instrument to obtain the metagenome of all soil samples in the target repair area is specifically as follows: take 2 μL of the total soil DNA and detect it on a 1% agarose gel electrophoresis; use Nanodrop to detect the DNA quality; randomly break the total soil DNA sample into small fragments with an ultrasonic disruptor; repair the ends of the DNA small fragments to blunt ends; use polymerase to ligate an adenosine acid after the DNA small fragments; use DNA ligase to ligate the sequencing adapter to the end of the small fragment DNA; fix the DNA small fragments on the flow cell; gradually add fluorescently labeled nucleotides to form complementary strands with the sample; turn on the sequencing instrument, and determine the base type at each position by detecting the intensity and position feedback of these fluorescent labels to obtain the raw sequencing data.

[0042] S102, annotate the microbial classification information and heavy metal resistance genes in the contaminated soil based on the gene sequencing sequence;

[0043] The annotation of the microbial classification information in the contaminated soil is specifically as follows: use the software MetaBAT2 to perform homologous "binning" on the metagenomic sequence, and regard each "binned" genome as the complete genome of a microbial species; use CheckM to evaluate the integrity and contamination degree of all bins to obtain all high-quality "binned" genomes in the soil; among them, the high-quality judgment criteria are that the completeness is greater than 90% and the contamination degree is less than 10%; it is obtained by comparing with the Genome Taxonomy Database and the sequencing data, annotate all sequenced genomes on GTDB, and provide standardized classification labels; then count the species classification information at each taxonomic level of kingdom, phylum, class, order, family, genus, and species.

[0044] Heavy metal resistance gene annotation, specifically: The high-quality "binned" genomes are aligned against the BactMet database, and high-quality potential heavy metal-remediating microbial genomes are determined based on the matching of one or more genes including cinA, copR, copS, copD, copC, fpvA, cueA, copA, copB, golT, copG, czcC, copF, mmco, copP, ctpG, ctpV; the potential heavy metal-remediating microbial genomes are "binned" genomes containing heavy metal resistance genes.

[0045] S103, determining the microorganisms and their species information of heavy metal resistance genes in contaminated soil based on the annotation results;

[0046] Microorganisms in contaminated soil have high-quality genomes; the microbial genomes contain metal resistance genes. When there are multiple high-quality microorganisms containing heavy metal resistance genes in contaminated soil, microorganisms with a larger number of resistance gene categories in the genome are preferably selected as the microbial species for addition in pollution treatment. The species classification of heavy metal resistance gene microorganisms is thus determined.

[0047] S104, putting the microbial species containing heavy metal resistance genes into heavy metal contaminated soil for remediation to obtain the remediated soil.

[0048] Heavy metal contaminated soil refers to soil with heavy metal content exceeding 50 mg / kg. The dosage of high-quality potential heavy metal-remediating microorganisms is 10 8 cells / gram, and the remediation treatment time is 15 days.

[0049] See Figure 2 In the present invention, a biological remediation system for heavy metal contaminated soil based on soil metagenomic sequencing is disclosed, including:

[0050] A collection module, which collects soil samples contaminated by heavy metals and performs gene sequencing on the soil samples;

[0051] An annotation module, which annotates the microbial classification information and heavy metal resistance genes in contaminated soil based on the gene sequencing sequences;

[0052] A determination module, which determines the microorganisms and their species information of heavy metal resistance genes in contaminated soil based on the annotation results;

[0053] A remediation module, which puts the microbial species containing heavy metal resistance genes into heavy metal contaminated soil for remediation to obtain the remediated soil.

[0054] Example 1:

[0055] Field investigations were carried out at the No. 1 abandoned land in a severely Cu-polluted metal mining area. 30 soil samples were randomly collected with a soil layer depth of 0 - 30 cm, placed in sterile polyethylene self-sealing bags, stored in a 4°C in-vehicle refrigerator, and then taken back to the laboratory and stored in a 4°C refrigerator. 1 g of each bag of the collected soil samples was taken to extract the total DNA of the soil samples. The metagenomic sequences were determined using an Illumina 3rd-generation sequencing instrument with a sequencing depth of 20 G and a library was constructed. The software "MetaBAT2" was used to perform homologous "binning" on the metagenomic sequences to obtain all high-quality (completeness > 90%, contamination < 10%) "binned" genomes in the soil. The high-quality "binned" genomes were compared in the BactMet library, and the "binned" genomes containing copper (Cu) resistance genes were the potential Cu-remediating microorganisms. For the microbial species containing Cu resistance genes identified by the comparison, the "GTDB-Tk" tool was then used to annotate the target genomes in the genome classification database to obtain the species classification information of the Cu-resistant microorganisms (see Table 1). Pseudomonadaceae was the potential Cu-remediating microorganism in this Cu-polluted soil. Pseudomonadaceae was added to the mine soil at a dosage of 10 8 cells / gram for metal pollutant removal treatment. The treatment was carried out in an aerated environment at 30°C for more than 15 days. After centrifuging to remove the culture medium, the remediation effect was calculated (see Table 2).

[0056] Example 2

[0057] For the remediation of contaminated forest soil around a severely Cu-polluted metal mining area, 30 soil samples were randomly collected with a soil layer depth of 0 - 30 cm, placed in sterile polyethylene self-sealing bags, stored in a 4°C in-vehicle refrigerator, and then taken back to the laboratory and stored in a 4°C refrigerator. 1 g of each bag of the collected soil samples was taken to extract the total DNA of the soil samples. The metagenomic sequences were determined using an Illumina 3rd-generation sequencing instrument with a sequencing depth of 20 G and a library was constructed. The software "MetaBAT2" was used to perform homologous "binning" on the metagenomic sequences to obtain all high-quality (completeness > 90%, contamination < 10%) "binned" genomes in the soil. The high-quality "binned" genomes were compared in the BactMet library, and the "binned" genomes containing copper (Cu) resistance genes were the potential Cu-remediating microorganisms. For the microbial species containing Cu resistance genes identified by the comparison, the "GTDB-Tk" tool was then used to annotate the target genomes in the genome classification database to obtain the species classification information of the Cu-resistant microorganisms as shown in Table 1. Burkholderiacea was the potential Cu-remediating microorganism with Cu-resistant functional genes. Burkholderiacea was added to the mine soil at a dosage of 10 8 cells / gram for metal pollutant removal treatment. The treatment was carried out in an aerated environment at 30°C for more than 15 days, and the remediation effect was calculated as shown in Table 2.

[0058] Comparative Example 1:

[0059] Collect the contaminated forest soil around the metal mining area seriously polluted by Cu, add the microorganism Bacillus licheniformis detected in Example 1 that does not contain the Cu-resistant gene to the soil, and perform pollutant removal treatment. Calculate the remediation effect after 15 days of treatment, and the results are shown in Table 2.

[0060] Table 1 Information of Cu-resistant microorganisms screened by the method of the present invention

[0061]

[0062] Table 2 Treatment effects of metal pollutants in soil

[0063]

[0064] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for bioremediation of heavy metal contaminated soil based on soil metagenomic sequencing, characterized in that, include: Collect soil samples contaminated by heavy metals and perform genetic sequencing on the soil samples; Annotate microbial classification information and heavy metal resistance genes in contaminated soil based on gene sequencing sequences; Based on the annotation results, the microorganisms and their species information with heavy metal resistance genes in the contaminated soil were determined; Microbial species containing heavy metal resistance genes are released into heavy metal contaminated soil for remediation to obtain remediated soil.

2. The method for bioremediation of heavy metal contaminated soil based on soil metagenomic sequencing according to claim 1, wherein The method of collecting soil samples contaminated by heavy metals comprises the following steps: randomly arranging a number of sampling points in the soil contaminated by heavy metals, collecting 0-30 cm soil away from plant roots, placing the soil in a sterile polyethylene ziplock bag, treating the bag with a 2 mm sieve, and taking 15 g of the soil and storing it at 4° C. for later use.

3. The bioremediation method of heavy metal contaminated soil based on soil metagenomic sequencing according to claim 2, characterized in that, The gene sequencing of soil samples is specifically performed as follows: a unit weight of soil is mixed thoroughly, and a DNA extraction kit is used to extract the soil total DNA; and a sequencing instrument is used to determine the gene sequence to obtain the metagenome of all soil samples in the target restoration area, with a sequencing depth of at least 20G.

4. The bioremediation method for heavy metal contaminated soil based on soil metagenomic sequencing according to claim 3, characterized in that, The method uses a sequencing instrument to determine the gene sequence and obtain the metagenome of all soil samples in the target restoration area, specifically: take 2 microliters of total soil DNA and place it in 1% agarose gel electrophoresis for detection; use Nanodrop to detect DNA quality; use an ultrasonic crusher to randomly break the total soil DNA sample into small fragments; repair the ends of the small DNA fragments to be blunt ends; use polymerase to insert an adenylate after the small DNA fragment; use DNA ligase to connect the sequencing adapter to the end of the small fragment DNA; fix the small DNA fragment in a flow cell; gradually add fluorescently labeled nucleotides to form a complementary chain with the sample; turn on the sequencer, determine the base type at each position by detecting the intensity and position feedback of these fluorescent labels, and obtain original sequencing data.

5. The bioremediation method for heavy metal contaminated soil based on soil metagenomic sequencing according to claim 4, characterized in that, The annotation of microbial classification information in contaminated soil is specifically as follows: using software MetaBAT2 to perform homologous "binning" of metagenomic sequences, and each "binned" genome is regarded as the whole genome of a microbial species; using CheckM to evaluate the integrity and contamination degree of all bins to obtain all high-quality "binned" genomes in the soil; wherein, the high-quality evaluation criteria are that the integrity is greater than 90% and the contamination is less than 10%; using a genome classification database and sequencing data comparison, all sequenced genomes are annotated on GTDB, and standardized classification labels are provided; and then species classification information is counted at each taxonomic level of kingdom, phylum, class, order, family, genus, and species.

6. The method for bioremediation of heavy metal contaminated soil based on soil metagenomic sequencing according to claim 5, characterized in that, Heavy metal resistance gene annotation, specifically: high-quality "binned" genomes are compared in the BactMet library, and high-quality potential heavy metal remediation microbial genomes are determined based on matching one or more genes of cinA, copR, copS, copD, copC, fpvA, cueA, copA, copB, golT, copG, czcC, copF, mmco, copP, ctpG, and ctpV; the potential heavy metal remediation microbial genome is a "binned" genome containing heavy metal resistance genes.

7. The bioremediation method for heavy metal contaminated soil based on soil metagenomic sequencing according to claim 6, characterized in that, Heavy metal contaminated soil refers to soil with heavy metal content exceeding 50 mg / kg.

8. The bioremediation method for heavy metal contaminated soil based on soil metagenomic sequencing according to claim 7, wherein The dosage of high-quality potential heavy metal-removing microorganisms is 10 8 individuals / gram, and the remediation treatment time is 15 days.

9. Heavy metal contaminated soil bioremediation system based on soil metagenomic sequencing, including: A collection module, wherein the collection module collects soil samples contaminated by heavy metals and performs gene sequencing on the soil samples; An annotation module, which annotates microbial classification information and heavy metal resistance genes in contaminated soil based on gene sequencing sequences; A determination module, which determines the microorganisms and species information of heavy metal resistance genes in the contaminated soil based on the annotation results; The repair module releases microbial species containing heavy metal resistance genes into heavy metal contaminated soil for repair to obtain repaired soil.

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