Low-temperature resistant multifunctional strain and application thereof

Abstract

The present application belongs to the technical field of biological treatment of environmental pollutants, and particularly relates to a multifunctional strain and its application in remediation of soil and water pollution. The low-temperature-resistant multifunctional strain is a strain BSLT1 of Bacillus, which was preserved in the China General Microbiological Culture Collection Center on August 9, 2023, with a preservation number of GCMCC No: 28122. The microbial agent of the present application can be applied to stabilize heavy metal copper in water and soil environments, and is helpful to enrich the strain resource library of heavy metal and tetracycline antibiotic degrading bacteria. The purified strain is made into a bacterial suspension, which can effectively stabilize copper in different environmental media such as water and soil under low-temperature environment, and has the function of degrading oxytetracycline in different environmental inoculation such as water and soil. Compared with physical adsorption, chemical oxidation and other methods, the method has less by-products, high degradation efficiency, low cost and is easy to operate, and provides an effective biological-non-biological combined remediation technology for copper-oxytetracycline composite contaminated soil and water remediation.

Description

Technical Field

[0001] This invention belongs to the field of biological treatment technology for environmental pollutants, specifically relating to a multifunctional strain (which simultaneously adsorbs heavy metals and degrades antibiotics) and its application in the remediation of soil and water pollution. Background Technology

[0002] my country's livestock and poultry farming industry has developed rapidly, with its scale continuously expanding. However, the high-density farming model has also led to a series of livestock and poultry diseases, resulting in more frequent use of antibiotics for their prevention and treatment. However, antibiotics in these drugs are not completely absorbed by animals; over 70% are excreted in feces and urine. Especially in northern regions, where winter temperatures are low, livestock and poultry manure cannot be disposed of promptly, leading to large accumulations. With rising temperatures and increased rainfall in spring, heavy metals and antibiotics in the manure are released into the environment through rainwater runoff, polluting soil and water bodies, and even being absorbed by plants grown using manure as fertilizer. Heavy metals and antibiotics in manure can accumulate through the food chain, posing a threat to human health. Currently, copper and oxytetracycline are the main heavy metals and antibiotics found in manure. The dosage of copper used in pig farms can reach 1700 mg / kg, and oxytetracycline as high as 2.8 mg / kg. Therefore, it is necessary to conduct in-depth research on materials that simultaneously stabilize copper in soil and water and degrade oxytetracycline.

[0003] Biodegradation is one of the main pathways for the natural decay of antibiotics. Currently, among the methods for treating residual antibiotics in livestock and poultry manure, utilizing microorganisms to degrade antibiotics into smaller, less toxic or non-toxic molecules is one of the most economical and effective methods. However, microorganisms are specific; their growth rate is slow in low or unsuitable environments, often requiring a long time to reach the concentrations needed for soil and water remediation, thus affecting the efficiency of pollution treatment. Therefore, microorganisms that can adapt to different temperatures, possess good stability, and have the ability to stabilize heavy metals and degrade antibiotics can play an important role in the remediation of complex polluted soils and water bodies in northern regions. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the primary objective of this invention is to propose a low-temperature resistant, multifunctional strain.

[0005] Another object of the present invention is to provide the application of the strain in soil remediation.

[0006] Another object of the present invention is to provide the application of the strain in water remediation.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A low-temperature resistant, multifunctional strain, BSLT1, belonging to the genus Bacillus, was deposited on August 9, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No:28122.

[0009] The application of the strain, specifically the application of the Bacillus strain BSLT1 in the degradation environment as an antibiotic.

[0010] The application of the Bacillus strain BSLT1 in the low-temperature degradation of antibiotics in soil or water; wherein the antibiotic is oxytetracycline.

[0011] Application of the Bacillus strain BSLT1 in a stable environment for heavy metals.

[0012] The application of the Bacillus strain BSLT1 in a low-temperature stable environment with heavy metals; wherein the heavy metal is copper.

[0013] A low-temperature resistant, multifunctional bacterial agent containing the aforementioned bacterial strain.

[0014] The bacterial agent is a culture medium or culture suspension containing the bacterial strain.

[0015] The bacterial culture medium is obtained by culturing the strain BSLT1 in LB liquid medium at 10-15 degrees Celsius until the logarithmic growth phase; the culture medium is then centrifuged to collect the precipitate, which is then resuspended in sterile physiological saline to OD. 600 The bacterial suspension has a concentration of 0.7–1.4.

[0016] An application of the aforementioned bacterial agent, specifically the application of the Bacillus strain BSLT1 in the degradation of antibiotics in an environment;

[0017] Alternatively, the application of the Bacillus strain BSLT1 in a stable environment for heavy metals.

[0018] The bacterial agent is applied to the contaminated soil and water to be treated, and the OD of the bacterial agent... 600 When the concentration is 1.0, apply an inoculum of 3-5 wt% to the environment to be treated.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention isolates a strain with heavy metal tolerance and oxytetracycline antibiotic degradation activity from in-situ livestock and poultry farming soil contaminated with heavy metals and antibiotics. The strain was molecularly identified as *Bacillus subtilis*. This strain's ability to degrade oxytetracycline contributes to enriching the strain resource library of tetracycline-degrading bacteria, providing an effective biodegradation method for the remediation of oxytetracycline-contaminated soil. Simultaneously, this strain can adsorb high concentrations of copper ions, enriching the strain resource library for copper heavy metal stabilization, providing an effective treatment method for the remediation of copper-contaminated soil. This strain exhibits strong adaptability, low-temperature tolerance, safety, effectiveness, and environmental friendliness, enabling the degradation of oxytetracycline and the stabilization of copper in a low-temperature environment suitable for microbial adaptation. Attached Figure Description

[0021] Figure 1 This is a morphological diagram of the BSLT1 strain provided in an embodiment of the present invention.

[0022] Figure 2 The image shows the results of BSLT1 strain prepared with a 3% inoculum amount provided in this embodiment of the invention stabilizing copper in soil and removing oxytetracycline under low temperature (15℃) conditions.

[0023] Figure 3 The image shows the results of BSLT1 strain prepared with a 3% inoculum amount provided in this embodiment of the invention, stabilizing copper in soil at room temperature (25℃) and removing oxytetracycline.

[0024] Figure 4 The image shows the results of BSLT1 strain prepared with a 3% inoculum amount provided in this embodiment of the invention, stabilizing copper in soil and removing oxytetracycline under normal temperature (35℃).

[0025] Figure 5 The image shows the results of BSLT1 strain prepared with a 5% inoculum amount provided in this embodiment of the invention, which stabilizes copper in water at low temperature (10℃) and removes oxytetracycline.

[0026] Figure 6 The image shows the results of BSLT1 strain prepared with a 5% inoculum amount provided in this embodiment of the invention stabilizing copper in water and removing oxytetracycline under normal temperature (20℃).

[0027] Figure 7 The image shows the results of BSLT1 strain prepared with a 5% inoculum amount provided in this embodiment of the invention stabilizing copper in water and removing oxytetracycline under normal temperature (30℃).

[0028] Figure 8 The image shows the results of BSLT1 strain prepared with a 5% inoculum amount provided in this embodiment of the invention, stabilizing copper in soil at room temperature (30℃) and removing oxytetracycline. Detailed Implementation

[0029] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0030] This invention discloses a low-temperature resistant, multifunctional microorganism capable of simultaneously stabilizing copper and degrading oxytetracycline at varying temperatures. This discovery will provide a new solution for the remediation of water and soil contaminated with copper-oxytetracycline in northern China. Specifically, the strain can be used to stabilize heavy metal copper in water and soil environments, contributing to a richer strain resource library of bacteria that stabilize heavy metals and degrade tetracycline antibiotics. The purified strain is prepared into a bacterial suspension, which can effectively stabilize copper in different environmental media such as water and soil at low temperatures, while also degrading oxytetracycline inoculated in different environments. Compared with physical adsorption and chemical oxidation methods, this method produces fewer byproducts, has higher degradation efficiency, lower cost, and is easier to operate, providing an effective bio-abiotic combined remediation technology for the remediation of copper-oxytetracycline contaminated soil and water.

[0031] Example 1: Isolation and Identification of Strains:

[0032] In this invention, the Bacillus subtilis BSLT1 strain was obtained by screening and domestication from soil in livestock and poultry farms contaminated with heavy metals and antibiotics. The specific steps are as follows:

[0033] A. Screening for antibiotic-resistant bacteria in soil contaminated by livestock and poultry farming using oxytetracycline and copper:

[0034] The acclimatization process was as follows: Soil samples contaminated with livestock and poultry manure were collected (in this example, soil contaminated around a pig farm in Haicheng City, Anshan City, Liaoning Province was collected), and the initial concentration of oxytetracycline in the soil samples was measured. The soil samples were then diluted and separated. The soil was prepared into a suspension with water and added to an inorganic salt culture medium containing oxytetracycline for gradient acclimatization. The concentration of oxytetracycline in the culture medium was increased sequentially to 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, 1 mg / L, and 2 mg / L; simultaneously, the concentration of copper in the inorganic salt culture medium was increased sequentially to 20 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 300 mg / L. At each concentration, the samples were cultured at 15°C in a shaker at 150 r / min in the dark for 7 days. If the liquid in the culture medium changed from transparent to turbid, and the OD... 600 A value higher than 1 indicates that microorganisms tolerant to 2 mg / L oxytetracycline and 300 mg / L copper have been properly activated and can be further enriched.

[0035] A mixed bacterial culture obtained by screening at a concentration of 2 mg / L oxytetracycline and 300 mg / L copper was transferred to a solid inorganic salt medium containing 2 mg / L oxytetracycline and cultured at 15°C in the dark. This process was repeated 6 times to allow strains tolerant to copper and capable of degrading oxytetracycline to gain a dominant growth position.

[0036] The obtained mixed bacteria were inoculated into an inorganic salt medium containing 2 mg / L oxytetracycline and cultured at 15℃ and 150 rpm in the dark. Samples were taken at days 0 and 3, filtered through a 0.22 μm filter, and the degradation efficiency was determined by LC-MS. The degradation experiment showed that after 3 days, the removal efficiency of oxytetracycline reached 56.53%, and the stabilization rate of copper reached 41.67%. The obtained mixed bacteria were then inoculated into a liquid inorganic salt medium containing 2 mg / L and cultured until OD... 600 The concentration was 1.0. After resuspending in sterile physiological saline, a mixed microbial suspension that was tolerant to and degradable to oxytetracycline was finally obtained.

[0037] B. The mixed microbial suspension obtained after domestication was inoculated onto solid LB medium, solid inorganic salt medium and solid potato medium for streak culture. All three media contained 2 mg / L oxytetracycline and 300 mg / L copper. The microorganisms were cultured in the dark at 15°C under different media. The microorganisms were transferred every 7 days, and the process was repeated 6 times to isolate and purify the strains in each medium, and finally obtain a single strain.

[0038] C. Each isolated and purified single strain was inoculated into a culture medium containing 2 mg / L oxytetracycline antibiotic and 300 mg / L copper ions to conduct oxytetracycline degradation and copper ion adsorption experiments. The degradation ability of single strains of oxytetracycline in LB medium and the adsorption capacity of copper were investigated. Finally, the microorganism BSLT1 with the highest degradation effect of oxytetracycline among the single strains was selected.

[0039] The purified single strain obtained was *Bacillus subtilis*, named BSLT1, and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 9, 2023. The accession number is CGMCC No: 28122.

[0040] The obtained strain underwent DNA extraction, PCR amplification, sequence sequencing, and sequence alignment. The 16S rDNA sequence of strain BSLT1 showed over 99.9% homology with *Bacillus subtilis*, indicating a close phylogenetic relationship. Therefore, it was identified as *Bacillus subtilis*, abbreviated as BSLT1. Its morphology is shown in [image / description missing]. Figure 1 Components of each culture medium used for screening:

[0041] Inorganic salt culture medium (g / L): ((NH4)2SO4 1.2, KH2PO4 1.2, K2HPO4 1.2, NaCl 0.5, FeCl3 6H2O 0.1, CaCl2 0.05, anhydrous glucose 15, adjust pH=7.0), and bring the volume to 1L with water.

[0042] Basic culture medium (g / L): yeast extract 5, peptone 10, NaCl 10; sterilize the culture medium at 121℃ for 20 min before use.

[0043] Potato culture medium: Boil 200g of freshly cut potatoes on a stove for 20 minutes, filter out the potato pieces, add 20g of glucose to the supernatant, and bring the volume to 1L.

[0044] Inorganic salt solid culture medium (g / L): ((NH4)2SO4 1.2, KH2PO4 1.2, K2HPO4 1.2, NaCl 0.5, FeCl3 6H2O 0.1, CaCl2 0.05, anhydrous glucose 15, adjust pH=7.0), 20g agar powder heated to boiling.

[0045] LB medium (g / L): yeast extract 5g, peptone 10g, NaCl 10g; 15-20g agar powder heated to boiling.

[0046] Potato solid culture medium: Boil 200g of diced fresh potatoes on a stove for 20 minutes, filter out the potato pieces, add 20g of glucose to the supernatant, and bring the volume to 1L. Heat 20g of agar powder to a boil.

[0047] Before use, the culture medium should be sterilized at 115℃ (containing glucose) / 120℃ (without glucose) for 15-30 minutes.

[0048] The 16S rDNA sequence is as follows:

[0049] GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC

[0050] GGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGT

[0051] AACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACC

[0052] GGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAA

[0053] GGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAG

[0054] TTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGA

[0055] GAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTAC

[0056] GGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGG

[0057] AGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGT

[0058] TGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGG

[0059] TACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTA

[0060] ATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTC

[0061] GCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGG

[0062] GAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTG

[0063] GAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACC

[0064] AGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAA

[0065] AGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTA

[0066] AACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAG

[0067] CTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAA

[0068] ACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT

[0069] TTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTG

[0070] ACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTG

[0071] GTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC

[0072] GCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCAC

[0073] TCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACG

[0074] TCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATG

[0075] GACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACA

[0076] AATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGC

[0077] TGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCC

[0078] CGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACC

[0079] CGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGA

[0080] CAGATGATTGGGGTGAAGTCGT

[0081] Example 2

[0082] Preparation of bacterial agent: The obtained single strain BSLT1 was activated in sterile inorganic salt liquid medium. After activation, it was inoculated into liquid LB medium at an inoculation rate of 3 wt% and incubated in a constant temperature incubator at 15℃ until the logarithmic phase. Then, the bacterial strain was washed with sterile physiological saline to prepare OD. 600 A bacterial suspension with a concentration of 1.0 is a bacterial agent.

[0083] Experiments were conducted using the above-obtained degrading microbial agents to treat oxytetracycline and copper ions in a low-temperature soil environment (15℃):

[0084] The tested soil was taken from a contaminated livestock farm in Shenyang City, Liaoning Province. The soil was sandy loam with a pH of 7.30. The available forms of oxytetracycline and copper were 368 μg / kg and 246.7 mg / kg, respectively, and the soil passed through a 2 mm sieve. 100 g of soil was inoculated with 3 wt% microbial inoculant and incubated outdoors (8-10℃) for one month. Two treatments were set up for the oxytetracycline and copper contaminated soil remediation experiment:

[0085] Treatment 1: Control treatment: Control treatment 1 original soil (without bacteria) for 0 days; Control treatment 2 original soil (without bacteria) for 30 days.

[0086] Treatment 2: Microbial agent treatment (microbial agent inoculation amount accounts for 3% (w:w) of soil).

[0087] Each treatment was performed in triplicate, with soil samples collected at day 0 and day 30 for analysis of oxytetracycline and available copper residues. Soil moisture content was maintained at 16-22% throughout the experiment. The results showed that the BSLT1 strain was tolerant to oxytetracycline and copper, and exhibited a certain degradation effect on oxytetracycline, reducing the available copper content. The bacterial suspension showed a 57.7% decrease in oxytetracycline degradation and a 39.5% decrease in available copper content at day 30 compared to day 0. (See attached figures). Figure 2 .

[0088] Example 3

[0089] Experiments were conducted using the above-obtained degrading microbial agents to treat oxytetracycline and copper ions in a soil environment at room temperature (25℃):

[0090] The test soil was taken from contaminated soil at a livestock and poultry farm in Shenyang City, Liaoning Province. The soil was sandy loam with a pH of 7.35. The available contents of oxytetracycline and copper were 330 μg / kg and 259.9 mg / kg, respectively, and the soil was sieved through a 2 mm sieve. 100 g of soil was inoculated with 3 wt% of the microbial agent prepared in Example 2 above and cultured for one month in a normal temperature room (25℃). Two treatments were set up for the oxytetracycline and copper contaminated soil remediation experiment:

[0091] Treatment 1: Control treatment: Control treatment 1 original soil (without bacteria) for 0 days; Control treatment 2 original soil (without bacteria) for 30 days.

[0092] Treatment 2: Inoculum treatment (inoculum agent described in Example 2) (inoculum agent inoculation amount was 3% (w:w) of soil). Each treatment was set up in triplicate, and soil samples were taken at day 0 and day 30 for analysis of oxytetracycline and available copper residues. The soil moisture content was maintained at 16-22% throughout the experiment. The experimental results showed that the BSLT1 strain was tolerant to oxytetracycline and copper, and had a certain degradation effect on oxytetracycline, which could reduce the available copper. The degradation effect of the bacterial suspension on oxytetracycline and the effect on available copper decreased by 62.9% and 50.4% respectively at day 30 compared with day 0. The results are shown in […]. Figure 3 .

[0093] Example 4

[0094] Experiments were conducted using the above-obtained degrading microbial agents to treat oxytetracycline and copper ions in a soil environment at room temperature (35℃):

[0095] The tested soil was taken from a contaminated livestock farm in Shenyang City, Liaoning Province. The soil was sandy loam with a pH of 7.35. The available forms of oxytetracycline and copper were 345 μg / kg and 276.1 mg / kg, respectively, and the soil passed through a 2 mm sieve. 100 g of soil was inoculated with 3 wt% microbial inoculant and incubated at 35℃ for one month. Two treatments were set up for the oxytetracycline and copper contaminated soil remediation experiment:

[0096] Treatment 1: Control treatment: Control treatment 1 original soil (without bacteria) for 0 days; Control treatment 2 original soil (without bacteria) for 30 days.

[0097] Treatment 2: Inoculum treatment (inoculum agent described in Example 2) (inoculum agent inoculation amount was 3% (w:w) of soil). Each treatment was set up in triplicate, and soil samples were taken at day 0 and day 30 for analysis of oxytetracycline and available copper residues. The soil moisture content was maintained at 16-22% throughout the experiment. The experimental results showed that the BSLT1 strain was tolerant to oxytetracycline and copper, and had a certain degradation effect on oxytetracycline, which could reduce the available copper. The degradation effect of the bacterial suspension on oxytetracycline and the effect on available copper decreased by 76.2% and 49.8% respectively at day 30 compared with day 0. The results are shown in […]. Figure 4 .

[0098] Example 5:

[0099] Preparation of bacterial agent: The obtained single strain BSLT1 was activated in sterile inorganic salt liquid medium. After activation, it was inoculated into liquid LB medium at an inoculation rate of 5 wt% and incubated in a constant temperature incubator at 10℃ until the logarithmic phase. Then, the bacterial strain was washed with sterile physiological saline to prepare OD. 600 A bacterial suspension with a concentration of 1.0 is a bacterial agent.

[0100] Experiments were conducted using the above-obtained degrading bacterial agent to degrade oxytetracycline and stabilize copper in a low-temperature water environment (10℃):

[0101] The test water was a simulated polluted water body in the laboratory, with a pH of 6.8, an oxytetracycline content of 927.63 μg / kg, and a total copper content of 286 mg / L. 100 mL of the composite polluted water was inoculated with 5 wt% microbial agent and incubated at a constant temperature of 10℃ for three days. Two treatments were set up for the remediation of the polluted water:

[0102] Treatment 1: Control Treatment: Control Treatment 1: Original polluted water (without bacteria) for 0 days; Control Treatment 2: Original polluted water (without bacteria) for 3 days.

[0103] Treatment 2: Microbial agent treatment (microbial agent inoculation amount accounts for 5% of soil (w:w)).

[0104] Each treatment was performed in triplicate, with water samples collected on day 0 and day 3 to determine the residual oxytetracycline and total copper content. Results are shown below. Figure 5 Experimental results showed that the BSLT1 strain was tolerant to oxytetracycline and exhibited a certain ability to degrade it, as well as good adsorption capacity for copper. The bacterial suspension achieved a 51.0% degradation rate of oxytetracycline within 3 days, and maintained a stable copper adsorption rate of 67.6%.

[0105] Example 6:

[0106] Remediation experiment of oxytetracycline-copper co-contaminated water body using BSLT1 bacterial agent at room temperature (20℃):

[0107] The test water was a simulated polluted water body in the laboratory, with a pH of 6.8, an oxytetracycline content of 1113.95 μg / kg, and a total copper content of 255.70 mg / L. 100 mL of the composite polluted water was inoculated with 5 wt% microbial agent and incubated at a constant temperature of 20℃ for three days. Two treatments were set up for the remediation of the polluted water: Treatment 1: Control treatment; Control treatment 1: Original polluted water (without bacteria) for 0 days; Control treatment 2: Original polluted water (without bacteria) left to stand for 3 days.

[0108] Treatment 2: Microbial agent treatment (microbial agent inoculation amount accounts for 5% of soil (w:w)).

[0109] Each treatment was performed in triplicate, with water samples collected on day 0 and day 3 to determine the residual oxytetracycline and total copper content. Results are shown below. Figure 6 The experimental results showed that the BSLT1 strain was tolerant to oxytetracycline and had a certain ability to degrade oxytetracycline, as well as good adsorption capacity for copper. The bacterial suspension achieved a 70.6% degradation rate of oxytetracycline and a 65.6% stabilization capacity for copper within 3 days.

[0110] Example 7:

[0111] Remediation experiment of BSLT1 bacterial agent on oxytetracycline-copper co-contaminated water at room temperature (30℃):

[0112] The test water was a simulated polluted water body in the laboratory, with a pH of 6.8, an oxytetracycline content of 1622.86 μg / kg, and a total copper content of 279.5 mg / L. 100 mL of the composite polluted water was inoculated with 5 wt% microbial agent and incubated at a constant temperature of 20℃ for three days. Two treatments were set up for the remediation of the polluted water: Treatment 1: Control treatment; Control treatment 1: Original polluted water (without bacteria) for 0 days; Control treatment 2: Original polluted water (without bacteria) left to stand for 3 days.

[0113] Treatment 2: Microbial agent treatment (microbial agent inoculation amount accounts for 5% of soil (w:w)).

[0114] Each treatment was performed in triplicate, with water samples collected on day 0 and day 3 to determine the residual oxytetracycline and total copper content. Results are shown below. Figure 7 The experimental results showed that the BSLT1 strain was tolerant to oxytetracycline and had a certain ability to degrade it, as well as good adsorption capacity for copper. The bacterial suspension achieved an 80.0% degradation rate of oxytetracycline and a 66.6% stabilization capacity for copper within 3 days.

[0115] Example 8

[0116] Experiments were conducted using the above-obtained degrading microbial agents to treat oxytetracycline and copper ions in a soil environment at room temperature (30℃):

[0117] The tested soil was taken from a contaminated livestock farm in Shenyang City, Liaoning Province. The soil was sandy loam with a pH of 7.35. The available forms of oxytetracycline and copper were 342.8 μg / kg and 262.5 mg / kg, respectively, and passed through a 2 mm sieve. 100 g of soil was inoculated with 5 wt% microbial inoculant and incubated at room temperature (35℃) for one month. Two treatments were set up for the oxytetracycline and copper contaminated soil remediation experiment: Treatment 1: Control treatment; Control treatment 1: Original soil (without inoculant) for 0 days; Control treatment 2: Original soil (without inoculant) left to stand for 30 days.

[0118] Treatment 2: Inoculum treatment (inoculum agent described in Example 2) (inoculum agent inoculation amount was 5% (w:w) of soil). Each treatment was set up in triplicate, and soil samples were taken at day 0 and day 30 for analysis of oxytetracycline and available copper residues. The soil moisture content was maintained at 16-22% throughout the experiment. The experimental results showed that the BSLT1 strain was tolerant to oxytetracycline and copper, and had a certain degradation effect on oxytetracycline, which could reduce the available copper. The degradation effect of the bacterial suspension on oxytetracycline and the effect on available copper decreased by 81.2% and 71.4% respectively at day 30 compared to day 0. The results are shown in […]. Figure 8 .

[0119] In summary, the bacterial suspension provided by this invention can effectively degrade oxytetracycline in soil and aquatic environments and stabilize copper in the environment at different temperatures, exhibiting good antibiotic resistance and degradation potential. The strains screened by this invention grow rapidly and have strong adaptability, reaching the logarithmic growth phase within half a day. The biodegradation process is low-cost, fast-response, highly efficient, and environmentally friendly, making it worthy of widespread application.

Claims

1. A low-temperature resistant, multifunctional bacterial strain, characterized by: The low-temperature resistant multifunctional bacterium is Bacillus subtilis strain BSLT1, which was deposited at the China General Microbiological Culture Collection Center on August 9, 2023, with accession number CGMCC No: 28122.

2. The application of the strain according to claim 1, characterized in that: The application of the Bacillus strain BSLT1 in the degradation environment as an antibiotic; The antibiotic in question is oxytetracycline.

3. The application of the strain according to claim 2, characterized in that: The application of the Bacillus strain BSLT1 in the low-temperature degradation of antibiotics in soil or water; wherein the antibiotic is oxytetracycline.

4. The application of the strain according to claim 1, characterized in that: Application of the Bacillus strain BSLT1 in a stable environment with heavy metals; The heavy metal is copper.

5. The application of the strain according to claim 4, characterized in that: The application of the Bacillus strain BSLT1 in a low-temperature stable environment with heavy metals; wherein the heavy metal is copper.

6. A low-temperature resistant, multifunctional bacterial agent, characterized in that: The bacterial agent contains the strain described in claim 1.

7. The microbial agent according to claim 6, characterized in that: The bacterial agent is a culture medium or bacterial suspension containing the strain described in claim 1.

8. The microbial agent according to claim 7, characterized in that: The bacterial culture medium is obtained by culturing the strain BSLT1 described in claim 1 in LB liquid medium at 10-15 degrees Celsius until the logarithmic growth phase; the culture medium is centrifuged to collect the precipitate, and the precipitate is resuspended in sterile physiological saline to OD. 600 The bacterial suspension has a concentration of 0.7–1.

4.

9. The application of the microbial agent according to claim 6, characterized in that: The application of the Bacillus strain BSLT1 in the degradation environment as an antibiotic; Alternatively, the application of the Bacillus strain BSLT1 in a stable environment with heavy metals; The antibiotic in question is oxytetracycline; The heavy metal is copper.

10. The application of the microbial agent according to claim 9, characterized in that: The bacterial agent is applied to the contaminated soil and water to be treated, and the OD of the bacterial agent... 600 When the concentration is 1.0, apply an inoculum of 3-5 wt% to the environment to be treated.

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