Method for screening soil oxytetracycline high-efficiency degrading bacteria by utilizing metagenome
By screening for highly efficient oxytetracycline-degrading strains in soil using metagenomic technology, the problems of low screening efficiency and long cycle in existing technologies have been solved, achieving efficient degradation and rapid remediation of oxytetracycline in soil.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-12
- Publication Date
- 2026-03-13
AI Technical Summary
Existing technologies suffer from low screening efficiency, long screening cycles, and inability to identify microorganisms carrying degradation functional genes when screening oxytetracycline-degrading strains in soil. This results in the omission of a large number of potentially highly efficient degrading bacteria, making it difficult to meet the needs of soil oxytetracycline pollution remediation.
Metagenomics was used to determine the structure of soil microbial communities and the relative abundance of resistance genes. Combined with typical soils from the north and south, highly efficient degradation strains carrying specific functional genes were screened.
It has achieved efficient degradation of oxytetracycline in soil, screened out highly efficient degradation strains, shortened the screening cycle, improved screening efficiency, and met the actual needs of soil pollution remediation.
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Figure CN121653271A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial screening technology, and more specifically to a method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics. Background Technology
[0002] Tetracycline antibiotics, as broad-spectrum antibiotics, are widely used in livestock and poultry farming, agricultural disease control, and human medical fields. However, a large amount of unabsorbed antibiotics enter the soil environment through feces, sewage, and other routes, leading to excessive antibiotic residues in the soil. This not only damages the soil microbial community structure and inhibits soil ecological functions, but may also pose a potential threat to human health through bioaccumulation in the food chain.
[0003] Currently, the main methods for eliminating oxytetracycline residues in soil include physical adsorption, chemical oxidation, and biodegradation. Among these, biodegradation has become a research hotspot due to its advantages such as environmental friendliness, low cost, and lack of secondary pollution. The core of biodegradation lies in obtaining highly efficient microbial strains that can degrade oxytetracycline.
[0004] Existing methods for screening oxytetracycline-degrading bacteria in soil mostly employ a combination of traditional enrichment culture and isolation / purification. This involves preparing a selective medium containing oxytetracycline, enriching soil samples with the medium, and then isolating and purifying single strains to verify their degradation capabilities. However, this method has significant limitations: firstly, most microorganisms in soil are difficult to culture on traditional media, leading to the omission of a large number of potentially highly efficient degrading bacteria; secondly, traditional methods cannot specifically target bacterial communities carrying degradation functional genes, resulting in low screening efficiency and long cycles, which is insufficient to meet the actual needs of oxytetracycline-contaminated soil remediation.
[0005] Metagenomics technology can directly analyze the genomes of all microorganisms in environmental samples without relying on microbial culture. It can comprehensively resolve the genetic information and community structure of soil microorganisms, providing the possibility for targeted screening of microorganisms carrying specific functional genes. Therefore, developing a screening method for highly efficient soil oxytetracycline-degrading bacteria based on metagenomics technology is of great significance for overcoming the bottlenecks of traditional screening techniques and rapidly obtaining highly efficient degrading strains. Summary of the Invention
[0006] A method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics can be used to screen out soil oxytetracycline-efficient degrading bacteria through metagenomic technology.
[0007] The soils used for the soil degradation experiment were Qingzi clay from Zhejiang and black soil from Heilongjiang.
[0008] To achieve the objectives of this invention, the experimental scheme adopted is as follows:
[0009] (1) The initial pollution concentration was set at 20 mg / kg. After mixing and equilibration, the soil was treated for 2 months, with 3 replicates. The dry soil samples were ground with a soil mill and passed through a 100-mesh sieve for the determination of oxytetracycline.
[0010] (2) Oxytetracycline in soil was determined by enzyme-linked immunosorbent assay (ELISA) kit. The detection limit for oxytetracycline in soil was 2 ppb. The specific assay method was performed according to the instructions of the kit.
[0011] (3) Metagenomics was used to determine the structure of soil microbial communities and the relative abundance of resistance genes.
[0012] (4) The present invention also achieves the screening of oxytetracycline-efficient degrading bacteria.
[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0014] This invention provides a method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics. It can utilize typical soils from both northern and southern China, and combined with metagenomics technology, it can comprehensively analyze the genetic information and community structure of soil microorganisms, providing the possibility for targeted screening of microorganisms carrying specific functional genes and providing technical support for screening microorganisms that efficiently degrade oxytetracycline. Attached Figure Description
[0015] Figure 1 This refers to the degradation of oxytetracycline in bluish-purple mud and black soil in Example 1;
[0016] Figure 2 The relative abundance of soil microbial communities at the phylum level in oxytetracycline contaminated in Example 2;
[0017] Figure 3 The relative abundance of soil microbial communities at the family level in oxytetracycline contamination in Example 2;
[0018] Figure 4 The relative abundance of soil microbial communities at the genus level in the oxytetracycline-contaminated soil in Example 2;
[0019] Figure 5 The relative abundance of the functional pathway of soil bacteria contaminated with oxytetracycline in Example 2;
[0020] Figure 6 The relative abundance of antibiotic resistance genes in soil microorganisms contaminated with oxytetracycline in Example 2 is shown. Detailed Implementation
[0021] The present invention will be further explained below with reference to specific embodiments.
[0022] The detection methods used in the experiment are as follows:
[0023] Detection method for oxytetracycline: enzyme-linked immunosorbent assay (ELISA) kit;
[0024] Methods for detecting microbial community structure and relative abundance of antibiotic resistance genes: high-throughput sequencing.
[0025] Example 1
[0026] This example is an experiment on the degradation of oxytetracycline in soil.
[0027] 1. Soil sample collection and pretreatment
[0028] Typical farmland soils from both northern and southern China were selected, with two types chosen: Zhejiang Qingzi clay and Heilongjiang black soil. Soil samples were collected from the 0-40cm soil layer at each sampling point. After mixing, roots and stones were removed, and the samples were passed through a 10-mesh sterile sieve. Then, 1000g of the air-dried, sieved soil was accurately weighed, and a prepared oxytetracycline solution (20mg / kg) was added. The mixture was thoroughly mixed and left to stand in the dark for 24 hours. After two months of natural environmental treatment, the soil was aliquoted into sterile self-sealing bags and stored at -20℃ for later use.
[0029] 2. Determination of oxytetracycline content in soil
[0030] Soil samples treated with oxytetracycline for two months were selected, milled, and sieved through a 100-mesh sieve. The oxytetracycline content in the soil was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The detection limit for oxytetracycline in the soil was 2 ppb. Specific assay procedures were performed according to the kit instructions.
[0031] 3. Test Results
[0032] Using black soil from Heilongjiang and bluish-purple clay from Zhejiang as research subjects, and soil without antibiotics as a control, the remaining soils were treated with 20 mg / kg of oxytetracycline. Samples were taken after two months of treatment, and the oxytetracycline content in the different soils was analyzed. Figure 1 It can be seen that the content of oxytetracycline in the soil gradually decreases over time, and the degradation rate of oxytetracycline varies significantly in different soil types. After adding oxytetracycline to different soils for two months, the degradation rate in blue-purple clay can reach over 95%; the degradation rate in black soil can reach over 98%. The degradation rate in black soil is greater than that in blue-purple clay.
[0033] Example 2
[0034] This example is a preliminary screening of microorganisms that degrade oxytetracycline in soil.
[0035] 1. Total DNA extraction and metagenomic sequencing of soil microorganisms
[0036] Total DNA from soil microorganisms was extracted using the EZNA® Soil DNA Kit, followed by DNA testing: (1) DNA purity was detected using NanoDrop2000, and DNA concentration was detected using TBS-380; (2) DNA integrity was tested using 1% agarose gel electrophoresis at 5V / cm for 20min. The DNA was broken into fragments of approximately 350bph and 400bp using a Covaris M220 ultrasonic disruptor for constructing a PE library.
[0037] The specific process for library construction using NEXTFLEX Rapid DNA-Seq is as follows:
[0038] (1) Connector link;
[0039] (2) Use magnetic beads to screen and remove self-connected segments at the joints;
[0040] (3) Enrichment of library templates using PCR amplification;
[0041] (4) PCR products were recovered by magnetic beads to obtain the final library.
[0042] Metagenomic sequencing was performed using the Illumina NovaSeq sequencing platform. The specific workflow is as follows:
[0043] (1) One end of the library molecule is complementary to the primer bases. After one round of amplification, the template information is fixed on the chip.
[0044] (2) The other end of the molecule fixed on the chip is randomly complementary to another nearby primer and is also fixed, forming a "bridge";
[0045] (3) PCR amplification produces DNA clusters;
[0046] (4) DNA amplicon linearizes into a single strand.
[0047] (5) Add modified DNA polymerase and dNTPs with 4 fluorescent labels, and synthesize only one base per cycle;
[0048] (6) Use a laser to scan the surface of the reaction plate and read the types of nucleotides that were polymerized in the first round of reaction for each template sequence;
[0049] (7) Chemically cleave the "fluorescent group" and "terminator group" to restore the 3' end stickiness and continue to polymerize the second nucleotide;
[0050] (8) Statistically analyze the fluorescence signal results collected in each round to obtain the sequence of the template DNA fragment.
[0051] 2. Metagenomic data analysis to identify candidate degradative bacterial communities
[0052] The amino acid sequences of the non-redundant gene set were compared with the NR, KEGG and ARDB databases using Diamond (http: / / www.diamondsearch.org / index.php, version 0.8.35). Species annotations were obtained from the taxonomic information database corresponding to the NR database. The abundance of a species was then calculated by summing the gene abundances corresponding to that species.
[0053] 3. Test Results
[0054] exist Figures 2 to 6 In the figures, BCK represents black soil control; BOTC represents black soil with added oxytetracycline (20 mg·kg⁻¹); PCK represents bluish-purple mud control; and POTC represents bluish-purple mud with added oxytetracycline (20 mg·kg⁻¹).
[0055] Door level: by Figure 2 It was found that the dominant bacterial groups in the black soil control group were Actinobacteria, Proteobacteria, Chlorophyta, Planctomyces, Acidobacteria, and Archaea. After the addition of oxytetracycline, the relative abundance of Actinobacteria, Proteobacteria, and Firmicutes all increased significantly, with Proteobacteria showing the largest increase, while the abundance of Chlorophyta, Planctomyces, Acidobacteria, and Archaea decreased significantly. In contrast, the dominant bacterial groups in the purple clay control group were Actinobacteria, Firmicutes, Proteobacteria, Chlorophyta, and Planctomyces. After the addition of oxytetracycline, the relative abundance of Firmicutes increased significantly, while other bacterial groups showed a certain degree of decrease. Therefore, the degradation effect of oxytetracycline may be related to the presence of Proteobacteria, Firmicutes, and Actinobacteria in the soil.
[0056] Science level: by Figure 3It can be seen that, at the taxonomic level, the dominant bacteria in black soil and bluish-purple mud containing added oxytetracycline were mainly Gemmatimonadaceae, Geminicoccaceae, and Anaerolinraceae. In black soil, oxytetracycline contamination reduced the proportion of Nitrososphaeraceae, JG30-KF-CM45, and Rubrobactericeae, while increasing the proportion of 67-14, norank_o_norank_c_Subgroup_6, and Geminicoccaceae. In bluish-purple mud, oxytetracycline contamination reduced the proportion of ammonia-oxidizing archaea, Anaerolinaceae, and Burkholderiaceae, while increasing the proportion of Baxillaceae and unclassified_o_Bacillales.
[0057] Attribute level: by Figure 4 It can be seen that the addition of oxytetracycline increased the relative abundance of gene sequences of *Gelleria*, *Bacillus*, and 67-14 in black soil, while the relative abundance of gene sequences of unclassified genera such as ammonia-oxidizing archaea and JG30-KF-CM45 decreased significantly. In the purplish clay, the relative abundance of gene sequences of unclassified *Bacillus*, *Bacillus*, *Bacillus fictitidis*, and *Syngonium* in the experimental group with added oxytetracycline increased significantly, while the relative abundance of gene sequences of unclassified genera such as *Isoproteroides*, KD4-96, *Anaerobic*, and *Gaia* decreased significantly.
[0058] Depend on Figure 5 It can be seen that, based on the two different soil types, black soil and bluish-purple mud, the six biological metabolic pathways of soil bacteria in the two types of soil before and after oxytetracycline treatment were: Metabolism, Genetic information Processing, Environmental information Processing, Cellular Processes, Human Diseases, and Organismal Systems. The overall difference was not significant, which suggests that oxytetracycline pollution does not have a significant impact on the metabolic pathways of bacteria in the soil.
[0059] Relative abundance of resistance genes: by Figure 6It can be seen that, based on the two different soil types, black soil and bluish-purple mud, the resistance gene in the soil is dominated by baca. Oxytetracycline pollution did not increase the relative abundance of tetracycline antibiotic resistance genes in black soil; however, for bluish-purple mud, after oxytetracycline treatment, the relative abundance of fosb and tet in bluish-purple mud increased.
[0060] In summary, we can identify Bacillus subtilis as the dominant genus for oxytetracycline degradation in purple mud, and Gaiellales (actinomycetes) as the dominant degrading genus in black soil.
Claims
1. A method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics, characterized in that, The method includes the following steps: conducting soil oxytetracycline degradation experiments, using metagenomics to screen for highly efficient soil oxytetracycline degrading bacteria, and achieving preliminary screening of soil oxytetracycline degrading bacteria.
2. The method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics as described in claim 1, characterized in that, The soil samples were taken from Heilongjiang black soil and Zhejiang blue-purple clay. The soil was topsoil of 0-40 cm. It was tested and found to be free of antibiotics. After removing impurities from the soil, it was naturally air-dried in a well-ventilated, dark, and cool place. The air-dried soil was then milled and passed through a 10-mesh sieve for storage.
3. The method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics as described in claim 2, characterized in that, The antibiotics mentioned include oxytetracycline.
4. The method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics as described in claim 1, characterized in that, The steps of the soil oxytetracycline degradation experiment include: accurately weighing 1000 g of air-dried and sieved soil, adding oxytetracycline solution with a concentration of 20 mg / kg, mixing evenly, letting it stand in the dark for 24 h for later use, subjecting it to two months of natural environmental treatment, collecting soil samples, grinding the air-dried soil and passing it through a 100-mesh sieve for later use, and using a portion for oxytetracycline content determination, and extracting bacterial DNA from the soil for soil microbial metagenomic determination.
5. The method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics as described in claim 4, characterized in that, The determination of the oxytetracycline content in the soil includes the following steps: using an enzyme-linked immunosorbent assay (ELISA) kit to determine the oxytetracycline content in the soil, with a detection limit of 2 ppb.
6. The method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics as described in claim 4, characterized in that, Bacterial DNA extraction from the soil was performed using the EZNA® Soil DNA Kit. After extraction, DNA concentration and purity were measured, DNA integrity was assessed using 1% agarose gel electrophoresis, and DNA fragmentation was performed using a Covaris M220 to select 400bp fragments for constructing a PE library.
7. The method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics as described in claim 4, characterized in that, The determination of the soil microbial metagenomics includes the following steps: constructing a PE library, bridging PCR, sequencing, and data analysis.
8. The method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics as described in claim 7, characterized in that, The steps for constructing the PE library include: library construction using NEXTFLEX Rapid DNA-Seq, the specific process of which is as follows: (1) Connector link; (2) Use magnetic beads to screen and remove self-connected segments at the joints; (3) Enrichment of library templates using PCR amplification; (4) PCR products were recovered by magnetic beads to obtain the final library.
9. The method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics as described in claim 7, characterized in that, The bridge PCR and sequencing process includes the following steps: metagenomic sequencing using the Illumina NovaSeq sequencing platform, with the specific procedure as follows: (1) One end of the library molecule is complementary to the primer bases. After one round of amplification, the template information is fixed on the chip; (2) The other end of the molecule fixed on the chip is randomly complementary to another nearby primer and is also fixed, forming a bridge; (3) PCR amplification produces DNA clusters; (4) DNA amplicon linearizes into a single strand; (5) Add modified DNA polymerase and dNTPs with 4 fluorescent labels, and synthesize only one base per cycle; (6) Use a laser to scan the surface of the reaction plate and read the types of nucleotides that were polymerized in the first round of reaction for each template sequence; (7) Chemically cleave the fluorescent group and the terminator group to restore the 3' end stickiness and continue to polymerize the second nucleotide; (8) Statistically analyze the fluorescence signal results collected in each round to obtain the sequence of the template DNA fragment.
10. The method for screening soil oxytetracycline-efficient degrading bacteria using metagenomics as described in claim 7, characterized in that, The data analysis includes the following steps: using Diamond to align the amino acid sequences of the non-redundant gene set with the NR, KEGG, and ARDB databases, obtaining species annotations through the taxonomic information database corresponding to the NR database, and then calculating the abundance of the species using the sum of the gene abundances corresponding to the species.