Copper-resistant providencia sp. And application thereof

Abstract

The invention discloses copper-resistant providencia sp. And application thereof in the technical field of copper adsorption. The copper-resistant providencia sp. Provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.32624. The copper-resistant providencia sp. The copper-resistant providencia sp. Is separated from fresh chicken manure, can tolerate high-concentration copper, has a relatively high adsorption rate on copper, and can remove a copper metal element in a matrix, and the matrix can be manure, a water body, a culture medium and other substances needing copper removal, so that the problems of copper poisoning and copper pollution in animal husbandry are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of copper adsorption, and particularly relates to a copper-tolerant Providencia bacterium and its application. Background Art

[0002] Regarding the environmental pollution problem caused by high copper, after pigs consume high-copper feed, only no more than 10% of the copper is absorbed by the pig intestine and deposited in the body, and 90% is excreted out of the body with feces. As a heavy metal element, this discharged copper is non-degradable and exists for a long time. Therefore, the use of high-copper diets not only causes waste of copper resources, but also damages the original ecological chain on the earth due to the large amount of discharged copper. Pig manure containing high copper causes an increase in the copper content in plants, which can not only lead to slow growth and reduced yield of plants, but also affect the content and absorption and utilization of other nutrient elements in plants. For example, when high-copper organic manure is applied to pastures in large amounts for a long time, when the copper content in the forage reaches 15 - 20 mg / kg, it may cause poisoning symptoms in copper-sensitive sheep. The source change of the food chain has an ecological magnification effect of biological chain transmission. Excessive copper can also lead to directional selection of regional plant species, that is, plants adapted to high copper survive advantageously, and plant species that cannot adapt are eliminated, thus causing the extinction of some species due to lack of food. For example: when the copper in water is excessive, some algae will be severely affected or die in large numbers, which in turn causes the extinction of many lower organisms and leads to the death of many fish that use these as food sources. This vicious cycle of the ecological chain must attract our high attention.

[0003] The treatment of copper pollution mainly includes precipitation method, adsorption method, ion exchange method, and biological method. The biological method removes copper ions from wastewater through the metabolism of microorganisms, and has the advantages of simple operation, low energy consumption, and no secondary pollution. There are many types of microorganisms that can adsorb copper biologically, including bacteria, fungi, and algae, etc. Regarding bacteria, among the existing technologies, Providencia bacteria, the genus Providencia is a group of Gram-negative bacteria that produce urease in the Enterobacteriaceae family. The patent applications with publication numbers CN111088197A and CN118497079 disclose Providencia strain TC1 and SCAU-2 strains, and the patent application with publication number CN117247864 discloses Providencia A2, all of which do not have the ability to adsorb copper.

[0004] Therefore, it is of great ecological significance to find copper-tolerant Providencia bacteria for the treatment of copper pollution. Summary of the Invention

[0005] The first object of the present invention is to provide a copper-tolerant Providencia sp. The Cu001 strain was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 14, 2024. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The taxonomic name is Providencia sp., and the deposit number is CGMCC NO. 32624. This strain can survive in a high-concentration copper environment and has a strong adsorption effect on copper, and can be used to remove copper metal elements from the matrix.

[0006] The second object of the present invention is to provide the application of the above-mentioned copper-tolerant Providencia sp. in adsorbing copper-contaminated matrix.

[0007] The third object of the present invention is to provide the application of the above-mentioned copper-tolerant Providencia sp. in preparing a copper adsorbent;

[0008] The fourth object of the present invention is to provide a copper adsorbent, which includes the above-mentioned copper-tolerant Providencia sp.

[0009] The fifth object of the present invention is to provide the application of the above-mentioned copper-tolerant Providencia sp. in inhibiting Staphylococcus aureus.

[0010] In order to achieve the above-mentioned invention objects, the following technical solutions are adopted for the copper-tolerant Providencia sp. and its application of the present invention:

[0011] A copper-tolerant Providencia sp., with the taxonomic name Providencia sp., was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 14, 2024, and the deposit number is CGMCC NO. 32624.

[0012] Preferably, the bile salt concentration tolerated by the copper-tolerant Providencia sp. is 0.2%.

[0013] Preferably, the maximum copper concentration tolerated by the copper-tolerant Providencia sp. is 200 mg / L.

[0014] The application of the above-mentioned copper-tolerant Providencia sp. in adsorbing copper-contaminated matrix.

[0015] Preferably, the copper-tolerant Providencia sp. is added to the copper-contaminated matrix, and the pH of the matrix is controlled to be 4 - 7.

[0016] The application of the above-mentioned copper-tolerant Providencia sp. in preparing a copper adsorbent.

[0017] A copper adsorbent, which includes the copper-tolerant Providencia sp.

[0018] The application of the above-mentioned copper-tolerant Providencia sp. in inhibiting Staphylococcus aureus.

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

[0020] 1. A new copper-tolerant Providencia bacterium is isolated from fresh chicken manure in the present invention. It can tolerate high concentrations of copper and has a high adsorption rate for copper, and can remove copper metal elements in the matrix. This matrix can be manure, water body, culture medium and other substances that need to remove copper, thus solving the problems of copper poisoning and copper pollution in the livestock industry.

[0021] 2. The copper-tolerant Providencia bacterium obtained in the present invention can tolerate 0.2% bile salt and has an inhibitory effect on Staphylococcus aureus, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the colony morphology diagram of the copper-tolerant Providencia bacterium of the present invention;

[0023] Figure 2 It is the Gram staining result diagram of the copper-tolerant Providencia bacterium of the present invention;

[0024] Figure 3 It is the phylogenetic tree constructed by the copper-tolerant Providencia bacterium of the present invention according to the 16s rRNA sequence;

[0025] Figure 4 It is the test result diagram of the maximum copper tolerance of the copper-tolerant Providencia bacterium of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be further clarified below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent forms of modification of the present invention fall within the scope defined by the appended claims of the present application. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through the market or can be prepared by existing methods.

[0027] A copper-tolerant Providencia bacterium, classified and named as Providencia sp., was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 14, 2024, with the deposit number CGMCC NO. 32624.

[0028] Furthermore, the bile salt concentration tolerated by the copper-tolerant Providencia bacterium is 0.2%.

[0029] Furthermore, the maximum copper concentration that the copper-tolerant Providencia bacterium can tolerate is 200 mg / L.

[0030] The application of the above-mentioned copper-tolerant Providencia bacterium in adsorbing copper-polluted matrix.

[0031] Further, Providencia sp. resistant to copper was added to the copper-polluted substrate, and the pH of the substrate was controlled to be 4 - 7.

[0032] Use of the above-mentioned Providencia sp. resistant to copper in the preparation of a copper adsorbent.

[0033] A copper adsorbent comprising Providencia sp. resistant to copper.

[0034] Use of the above-mentioned Providencia sp. resistant to copper in inhibiting Staphylococcus aureus.

[0035] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0036] Example 1: Isolation of Providencia sp. resistant to copper

[0037] Sample source: Fresh feces of healthy laying hens.

[0038] Isolation steps: Take 1 g of fresh feces of healthy laying hens and serially dilute it with a sterile physiological saline solution to 10 -5 , respectively take 100 μL of each dilution and evenly coat it on an LB solid medium containing 50 mg / L of Cu. After culturing at 37°C for 24 h, pick single colonies and continue to inoculate them on an LB solid medium containing 100 mg / L of Cu. After continuing to culture for 24 h, pick single colonies and repeat the inoculation on an LB solid medium containing 100 mg / L of Cu. The single colonies obtained after culturing for 24 h are strains resistant to 100 mg / L of copper, named Cu001. This strain is Providencia sp. resistant to copper. As Figure 1 shown, it is the colony morphology diagram of the strain of the present invention.

[0039] The obtained Providencia sp. resistant to copper was subjected to microbial preservation. The preservation information is: classified and named as Providencia sp., preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 14, 2024, at the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC NO. 32624.

[0040] The obtained Providencia sp. resistant to copper was applied to remove copper metal elements from the substrate. Such a substrate can be manure, water body, culture medium and other substances that need to remove copper, or can be used to make a copper adsorbent, or can be applied to inhibit Staphylococcus aureus.

[0041] Example 2: Identification of Providencia sp.

[0042] (1) Gram staining

[0043] The single Providencia sp. Cu001 isolated in Example 1 of the present invention was placed in 10 μL of sterile physiological saline solution, mixed well, spread on a sterile glass slide, quickly fixed over a flame, and after cooling, the operation was carried out according to the Gram staining steps. Observation was made under an oil immersion lens of a microscope, and it was determined to be a Gram-negative bacillus. The bacterial cells were short rod-shaped, with blunt ends at both ends and scattered, as Figure 2 shown.

[0044] (2) 16S rRNA biological identification

[0045] The single Providencia sp. Cu001 isolated in Example 1 of the present invention was cultured with shaking at 37 °C in LB liquid medium for 24 h. Then, 1 mL of the culture solution was taken, and the bacterial DNA was extracted according to the requirements of the TIANGEN Bacterial DNA Extraction Kit (No: DP302). The 16S rRNA gene of the strain was amplified by PCR using the universal primers for bacterial 16S rRNA gene. The PCR amplification program was: 95 °C: 1 min, 95 °C: 30 s, 52 °C: 30 s, 72 °C: 2 min, 35 cycles, 16 °C. 5 μL of the PCR amplification product was subjected to 1% agarose gel electrophoresis, and the remaining PCR product was sent to a biotechnology company for sequencing. The sequencing result was compared with the NCBI database to determine the copper-resistant bacterial species.

[0046] Among them, the above PCR amplification primers are shown in Table 1 below.

[0047] Table 1: Universal primer sequences for 16S rRNA gene

[0048]

[0049] The sequencing result is as follows (SEQ ID NO.1):

[0050] GCAATGGCGGCAGGCTACACATGCAGTCGAGCGGTAACAGGGGAAGCTTGCTTCTCGCTGACGAGCGGCGGACGGGTGAGTAATGTATGGGGATCTGCCCGATAGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAATCTCTTAGGAGCAAAGCAGGGGAACTTCGGTCCTTGCGCTATCGGATGAACCCATATGGGATTAGCTAGTTGGTGAAGGTAATGGCTCATCCAAGGCGAGCCGATTCTCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCCTAGGGTTGTAAAGTACTTTCAGTCGGGAGGAAGGCGTTGATGCTAATATCATCAACGATTGACGTTACCGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTGATTAAGTTAGATGTGAAATCCCCGGGCTTAACCTGGGAATGGCATCTAAGACTGGTCAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGAAGGTTGTTCCCTAGAGGAGTGGCTTTCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTAGGTGCCTTCGGGAACTCTGAGACAGGTGCCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGATTCGGTCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGAAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAAAGAGAAGCGGACCTCCGCGAGGAGCAAGCGGAAAACATAAACAACGACGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTACCACTTGATTAGGC

[0051] The phylogenetic tree constructed based on the 16S rRNA sequence is as shown in Figure 3 It can be seen that the copper-resistant Providencia sp. Cu001 isolated in Example 1 of the present invention is a new strain of Providencia sp. different from the existing ones.

[0052] Example 3: Maximum copper tolerance of copper-resistant Providencia sp.

[0053] The Providencia sp. Cu001 isolated in Example 1 of the present invention was inoculated into 5 mL of LB liquid medium at an inoculation amount of 1% and cultured for 24 h. The absorbance was adjusted to 1 (OD600), and it was serially diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 . 100 μL was taken respectively and spread on LB solid media containing 100 mg / L, 120 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, and 300 mg / L of copper. The results were observed after culturing at 37 °C for 48 h to determine the maximum copper concentration tolerated by the Providencia sp. The Providencia sp. strain with the maximum copper tolerance concentration was inoculated into LB liquid media containing 0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 300 mg / L of copper and cultured with shaking at 37 °C for 24 h. The results showed that except for the medium containing 300 mg / L of copper which remained clear and transparent, the others all had turbid flocculents, indicating that it could tolerate a maximum copper concentration of 200 mg / L, as Figure 4 shown.

[0054] Example 4: Adsorption of copper ions in solution by copper-tolerant Providencia sp. at different adsorption times

[0055] The Providencia sp. Cu001 isolated in Example 1 of the present invention was inoculated into 5 mL of LB liquid medium and cultured for 18 h. Then it was centrifuged at 10000 r / min for 10 minutes. The supernatant was removed, and the precipitate was washed with ultrapure water. After that, the absorbance was adjusted to 1 (OD600). 0.5 mL of the solution was still centrifuged at 10000 r / min for 10 minutes. The supernatant was removed and 0.5 mL of 5 mg / L copper solution with a pH of 5.0 was added to suspend the precipitate. It was shaken at medium speed at 37 °C for 1 h, 2 h, 6 h, and 12 h. Then it was centrifuged at 10000 r / min for 10 minutes. The supernatant was taken, and after appropriate dilution, the copper content in the solution was determined by atomic absorption spectrometry. The copper adsorption rate of the Providencia sp. was calculated using formula (1).

[0056] Adsorption rate (%) = 100% - (C 1 / C 0 ) × 100% (1)

[0057] where C 0 is the initial copper solution concentration, and C 1 is the copper solution concentration in the final supernatant.

[0058] The results shown in Table 2 below indicate that the copper adsorption rate of this Providencia sp. for the 5 mg / L copper solution (pH = 5) was greater than 85% at different times. The adsorption rate reached 96.4% at 12 h, and the average adsorption rate was 90.5%.

[0059] Table 2 Determination of copper adsorption capacity of Cu001

[0060]

[0061] Example 5: Adsorption of copper ions in solution by copper-resistant Providencia bacteria at different pH values

[0062] The Providencia bacteria Cu001 isolated in Example 1 of the present invention were inoculated into 5 mL of LB liquid medium at an inoculation amount of 1% and cultured for 24 h. Then, they were centrifuged at 10,000 r / min for 10 minutes. After removing the supernatant, the precipitate was washed with ultrapure water. The absorbance was adjusted to 1 (OD600). 0.5 mL of the bacterial cell dilution was taken and still centrifuged at 10,000 r / min for 10 minutes. After removing the supernatant, 0.5 mL of 5 mg / L copper solution with pH values of 2.5, 4.0, 5.0, and 7.0 was added to suspend the precipitate. After shaking at medium speed at 37°C for 2 h, it was centrifuged at 10,000 r / min for 10 minutes. The supernatant was taken, diluted by an appropriate multiple, and the copper content in the solution was measured by flame atomic absorption spectrometry. The copper adsorption rate of Providencia bacteria was calculated using formula (1).

[0063] Adsorption rate (%) = 100% - (C 1 / C 0 ) × 100% (1)

[0064] Among them, C 0 is the initial copper solution concentration, and C 1 is the copper solution concentration in the final supernatant.

[0065] The results are shown in Table 3 below. It shows that the pH value has a great influence on the copper adsorption of the strain. When the pH value is 2.5, the copper adsorption rate of Providencia bacteria on the 5 mg / L copper solution is less than 40%. As the pH value increases, the copper adsorption rate increases. When the pH is 7.0, the copper adsorption rate is the highest, up to 92.8%. When the pH is 4.0 and 5.0, the copper adsorption rates can both reach 85%.

[0066] Table 3 Adsorption rate of Cu001 on copper solution

[0067]

[0068] Example 6: Bacteriostatic properties of copper-resistant Providencia bacteria

[0069] The Providencia sp. Cu001 isolated in Example 1 of the present invention was inoculated into 5 mL of LB liquid medium at an inoculation amount of 1% and cultured for 18 h. The absorbance was adjusted to 1 (OD600). 1 mL of the LB culture solution was taken and placed in a sterile centrifuge tube. Sterile filter paper with a diameter of 8 mm was immersed in the LB culture solution respectively. The absorbances of Staphylococcus aureus, Escherichia coli, and Salmonella were adjusted to 0.08 - 0.1 (OD600). 50 μL of Staphylococcus aureus, Escherichia coli, and Salmonella were taken respectively and evenly spread on the LB medium. The filter papers were placed on the bacterial culture plates respectively. Staphylococcus aureus, Escherichia coli, Salmonella pullorum, Salmonella typhimurium, and Salmonella enteritidis were cultured at 37 °C for 24 h to determine the antibacterial property of the Providencia sp. The results showed that the Providencia sp. had no antibacterial effect on Escherichia coli and Salmonella, but had a good antibacterial effect on Staphylococcus aureus. The results are shown in Table 4.

[0070] Table 4 Determination of the antibacterial ability of Cu001

[0071]

[0072] Example 7: Bile salt tolerance of copper-tolerant Providencia sp.

[0073] The Providencia sp. Cu001 isolated in Example 1 of the present invention was inoculated into LB broth medium at an inoculation amount of 2% and cultured for 18 h. After centrifugation at 8000 rpm for 10 min, the supernatant was removed. The cells were resuspended with LB broth medium containing 0.2%, 0.4%, and 0.8% bile salts respectively. After culturing for 2 h and 4 h respectively, 1 mL of the bacterial solution was serially diluted with sterile normal saline to 10 -5 , 100 μL of the diluted solution was evenly spread on the LB agar medium and cultured at 37 °C for 36 h. The total number of colonies was counted and the survival rate was calculated to determine the bile salt concentration tolerated by the Providencia sp.

[0074] The results shown in Table 5 below indicate that the survival rates after treatment with 0.4% and 0.8% bile salts were 0, and this Providencia sp. could tolerate 0.2% bile salts.

[0075] Table 5 Bile salt tolerance of copper-tolerant Providencia sp. to 0.2% bile salts

[0076]

[0077] In summary, the maximum copper concentration tolerated by this Providencia sp. is 200 mg / L, the average copper adsorption rate to 5 mg / L copper solution (pH = 5) is 90.5%, it can tolerate 0.2% bile salts, and can inhibit the growth of Staphylococcus aureus.

Claims

1. A copper-resistant Providencia bacterium, characterized in that It was classified and named Providencia sp. and deposited in the General Microbiology Center of China Culture Collection Administration on November 14, 2024 with the deposit number CGMCC NO.32624.

2. The copper-resistant Providencia according to claim 1, characterized in that: The copper-resistant Providencia strain tolerates bile salt concentration of 0.2%.

3. The copper-resistant Providencia according to claim 1, characterized in that: The copper-resistant Providencia bacteria can tolerate a maximum copper concentration of 200 mg / L.

4. Use of the copper-resistant Providencia according to claim 1 in adsorbing copper-contaminated substrates.

5. The use of the copper-resistant Providencia bacteria in adsorbing copper-contaminated substrates according to claim 4, characterized in that: Copper-resistant Providencia were added to the copper-contaminated substrate, and the pH of the substrate was controlled at 4-7.

6. Use of the copper-resistant Providencia according to claim 1 in the preparation of a copper adsorbent.

7. A copper adsorbent, characterized in that it comprises the copper-resistant Providencia according to claim 1.

8. Use of the copper-resistant Providencia according to claim 1 in inhibiting Staphylococcus aureus.

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