Lead-resistant bacterium, repairing agent prepared from lead-resistant bacterium, preparation method and application of repairing agent

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a lead-resistant bacterium, a repairing agent prepared from the lead-resistant bacterium, a preparation method and application. According to the specific technical scheme, the Paracoccus marcusii HMP-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on December 06, 2023, and the preservation number is CGMCC NO.29251. According to the invention, the Paracoccus marcusii HMP-1 can be used for preparing the paracoccus marcusii HMP-1, and the preservation number is CGMCC NO.29251. The invention further provides a lead repairing agent based on the HMP-1, and the repairing agent comprises the paracoccus martensii and humic acid at the same time. The repairing agent provided by the invention is high in removal rate of Pb < 2 + > in wastewater and stable in removal effect, and the Pb < 2 + > cannot be released into the environment again along with the prolonging of time.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a lead-resistant bacterium, a repair agent prepared using the same, and a preparation method and application thereof. Background Art

[0002] With the development of industries such as mining, metal smelting and processing, lead pollution has gradually become a problem that cannot be ignored in endangering the ecological environment and human health.

[0003] Bioremediation technology is an economical and effective method for lead remediation. Microorganisms have a strong affinity for heavy metal ions and can effectively remove heavy metals through extracellular complexation, extracellular precipitation and intracellular accumulation. However, on the one hand, lead has strong biological toxicity, and most microorganisms find it difficult to tolerate and treat high concentrations of lead; on the other hand, liquid bacterial agents are prone to breed bacteria during long-term storage and transportation, reducing the treatment effect, and the storage and transportation costs are high.

[0004] Therefore, if we can screen a new microorganism with high tolerance to lead and select a suitable carrier material to fix the microorganism, it is expected to solve the above problems and provide a new agent for the repair of lead in the environment. Summary of the invention

[0005] The purpose of the invention is to provide a lead-resistant bacteria, a repair agent prepared using the same, and a preparation method and application thereof.

[0006] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a strain of Paracoccus marcusii HMP-1 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 6, 2023, with a deposit number of CGMCC NO.29251.

[0007] Correspondingly, a strain of Paracoccus marcusii HMP-1, the 16SrDNA sequence of the strain HMP-1 is shown in SEQ ID NO:1.

[0008] Accordingly, the Paracoccus martensii is used in non-disease diagnosis and treatment in lead treatment.

[0009] Correspondingly, the bacterial suspension and freeze-dried powder prepared by using the Paracoccus martensii are used in non-disease diagnosis and treatment in lead treatment.

[0010] Correspondingly, a repair agent is provided, wherein the repair agent includes the Paracoccus martensii.

[0011] Preferably, the repair agent also includes humic acid.

[0012] Preferably, in the repair agent, Paracoccus martensii HMP-1 is prepared as OD 600 For bacterial suspensions with a mass ratio of ≥1.0, the mass ratio of humic acid to bacterial suspension is 1:2.5 to 1:20.

[0013] Correspondingly, the repair agent is used in removing lead ions.

[0014] The present invention has the following beneficial effects:

[0015] The present invention provides a new strain of Paracoccus martensii HMP-1, which has strong lead tolerance and processing ability and can efficiently process lead ions in the environment. Based on the Paracoccus martensii HMP-1, the present invention also provides a lead-resistant bacteria-humic acid composite repair bacterial agent, which uses humic acid as a carrier material to fix microorganisms, effectively solving the problems of liquid bacterial agents being prone to breeding of miscellaneous bacteria, difficult to ensure quality, and high storage costs during long-term storage and transportation.

[0016] Humic acid is a natural high molecular organic matter, rich in porous structure, with good biocompatibility and stability, which can provide more sufficient living space for Paracoccus martensii, and can also serve as the only carbon source for good growth of microorganisms. At the same time, humic acid is rich in carboxyl, alcohol hydroxyl, phenolic hydroxyl, quinone carbonyl and keto carbonyl and other active functional groups, with high reactivity, and can adsorb, complex and redox with metal ions in the environment, further removing heavy metals.

[0017] The composite repair bacterial agent provided by the present invention is effective for the treatment of Pb in wastewater. 2+ The removal rate is high and the removal effect is stable, Pb 2+ Will not be released back into the environment over time. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the growth of Paracoccus martensii HMP-1 on plates with different lead concentrations;

[0019] Figure 2 This is the colony morphology of Paracoccus martensii HMP-1. DETAILED DESCRIPTION

[0020] The present invention provides a new strain of Paracoccus marcusii HMP-1, which was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on December 6, 2023, with a deposit number of CGMCCNO.29251. The 16S rDNA sequence of the strain HMP-1 is shown in SEQ ID NO:1.

[0021] The Paracoccus martensii HMP-1 can tolerate high concentrations of Pb 2+, and has good lead enrichment and removal capabilities. The Paracoccus martensii HMP-1 (including the bacterial suspension or seed liquid or freeze-dried powder of the Paracoccus martensii HMP-1 or other forms of bacterial preparations containing live Paracoccus martensii HMP-1) can be directly used to repair, enrich, and remove lead ions in the environment, or can be prepared as a repair agent for further application. The application method includes: directly applying the Paracoccus martensii HMP-1 or the bacterial preparation or repair agent prepared therefrom to the environment to be repaired.

[0022] Based on the Paracoccus martensii HMP-1, the present invention also provides a repair agent for removing lead ions in the environment. The repair agent comprises Paracoccus martensii HMP-1 and humic acid. A preferred solution is: in the repair agent, Paracoccus martensii HMP-1 is prepared as OD 600 For bacterial suspensions with a mass ratio of ≥1.0, the mass ratio of humic acid to bacterial suspension is 1:2.5 to 1:20.

[0023] The present invention also provides a method for preparing the repair agent, which comprises mixing the bacterial suspension of Paracoccus martensii HMP-1 with humic acid. 600 ≥1.0 bacterial suspension, humic acid and the bacterial suspension are mixed at a mass ratio of 1:2.5 to 1:20 to obtain a liquid repair agent. In practice, the liquid repair agent can also be dried at 30°C for 24 hours to obtain a solid repair agent as needed. A more preferred scheme is: humic acid and bacterial suspension are evenly mixed for 24 to 48 hours, 28 to 30°C, 150 to 160r / min, and dried under shaking conditions. The liquid repair agent can also be prepared into freeze-dried powder and other preparations before use.

[0024] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0025] Example 1: Screening and identification of microorganisms

[0026] 1. Screening.

[0027] Take samples from heavy metal lead contaminated soil and collect them in sterile bags. Prepare soil sample dilution: weigh 10g of the soil sample to be tested in a 250mL conical flask, add 90mL of sterile water, and shake on a rotary shaker at 150r / min and 30℃ for 6h to fully suspend the bacteria. -1Soil dilution. After standing, take 1 mL of the supernatant and transfer it to a test tube. Then dilute it with sterile water in 10-fold ratios to obtain 10 -2 ~10 -8 Soil dilution, take 0.1mL 10 -2 ~10 -8 The diluent is spread on the solid culture medium, inverted and placed in an incubator for culture at 28-30°C. The solid culture medium is: 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L NaCl, 18 g / L agar, dissolved in deionized water, pH = 7.4-7.6, sterilized at 121°C for 30 min. The solid culture medium without agar is the liquid culture medium used in the present invention.

[0028] Observe the growth of the colonies, select strains with different morphologies and good growth, and purify them repeatedly by streaking them onto new solid culture media. The isolated and purified strains are gradually transferred to 200-1000 mg / L Pb 2+ In the transfer, strains suitable for growth were continuously selected for multiple screening, separation and purification, and finally the strain HMP-1 that could tolerate 500 mg / L lead ion concentration was obtained and stored in a new solid culture medium.

[0029] strain HMP-1 in Pb 2+ It grew well on the plates with concentrations of 300 and 500 mg / L. The growth photos are as follows: Figure 1 As shown, Figure 1 Pb on the left 2+ Concentration 300mg / L plate, right side is Pb 2+ Concentration 500mg / L plate. Among the strains screened, only strain HMP-1 could 2+ Growth on the plate with a concentration of 500 mg / L (although the growth was not as good as on the plate with Pb 2+ The other microorganisms could not grow on the Pb 2+ It survives and grows on plates with a concentration of 500 mg / L.

[0030] 2. Identification.

[0031] The colony morphology of the strain HMP-1 is as follows Figure 2 As shown, it is a Gram-negative bacterium. The microbial morphology is: the colony is orange-yellow, the surface is smooth and moist, the edge is regular, slightly convex, opaque, the growth temperature is: 5-30℃, the optimal growth temperature is: 15-30℃, the growth pH is: 6.8-8.5, and the optimal growth pH is: 7.0.

[0032] The 16S rDNA sequence of the strain HMP-1 is shown in SEQ ID NO: 1. Sequencing identified it as Paracoccus marcusii, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (CGMCC) on December 6, 2023, with a deposit number of CGMCC NO.29251.

[0033] Example 2: Preparation of repair agent and effect demonstration

[0034] 1. Inoculate the Paracoccus martensii HMP-1 into the liquid culture medium of Example 1, and culture at 30°C and 150 r / min for 24 h. Divide the culture into 50 mL sterile centrifuge tubes, centrifuge at 6000 r / min for 15 min, remove the supernatant, and retain the bacteria. Wash the bacteria three times with sterile water, then pick an appropriate amount of bacteria and use the liquid culture medium to prepare the OD 600 =1.0 bacterial suspension.

[0035] Humic acid and bacterial suspension were mixed at a mass ratio of 1:2.5, 1:5, 1:10, and 1:20, respectively, and mixed at 30°C and 150r / min for 24h, then centrifuged at 10000r for 10min, the supernatant was removed, washed with 20mL sterile water, centrifuged again at 10000r for 10min, the supernatant was removed, and the washing was repeated 3 times, and dried at 30°C for 24h to obtain lead-resistant bacteria-humic acid composite repair agents 1 to 4. At the same time, a group using only humic acid was set as a blank control group, and only OD 600 =1.0 was treated in the same way and dried as the positive control group. The humic acid used in this example was from Hefei BASF Biotechnology Co., Ltd.

[0036] 2. Add 0.2 g of each of the repair agents 1 to 4 prepared in step 1, humic acid (blank control), and bacterial suspension (positive control) to the initial theoretical Pb 2+ In aqueous solutions with concentrations of 500 mg / L and 800 mg / L respectively. 2+ From 2g / L Pb(NO3)2 solution.

[0037] Each group was treated in a constant temperature shaker at 30°C and 150 r / min for 24 h, and the supernatant of each group was taken at 6, 12, 18, and 24 h for detection of Pb 2+ Concentration. Each treatment was repeated 3 times and the results were averaged. The results are shown in the following table. 2+ The concentration unit is mg / L.

[0038] Table 1 Comparison of the ability of each repair agent to treat lead ions with an initial theoretical concentration of 500 mg / L

[0039]

[0040] Table 2 Comparison of the ability of each repair agent to treat lead ions with an initial theoretical concentration of 800 mg / L

[0041]

[0042]

[0043] The results showed that although humic acid alone can also play a good lead removal effect in a short period of time, after 12 hours, the lead ion concentration in the solution gradually recovered, and the bound lead was gradually released into the solution again. Therefore, when humic acid is used alone, the lead ion removal ability is unstable and not long-lasting. After combining with Paracoccus martensii HMP-1, although the lead ion removal ability did not increase significantly in a short period of time, after 12 hours, the lead ion concentration in the solution still basically maintained a downward or stable trend, and this stabilization effect was more obvious in the group with more Paracoccus martensii HMP-1 added.

[0044] At the same time, according to the positive control group, the effect of using the suspension of Paracoccus martensii HMP-1 alone to treat the lead ions in the solution was not very ideal, and after 18 hours of treatment, the lead ions in the solution also rebounded. However, after the combination of Paracoccus martensii HMP-1 and humic acid, the groups with more Paracoccus martensii HMP-1 (repair agents 3 and 4) had an ideal overall lead ion treatment effect, which was not weakened by the reduction in the amount of humic acid, and a new effect of stable lead ion removal effect appeared. This shows that Paracoccus martensii HMP-1 and humic acid form an organic whole after combining, and it is the bacteria-humic acid as a whole that treats lead, rather than a single component.

[0045] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Paracoccus marcusii HMP-1, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 6, 2023, with the deposit number CGMCC NO.29251.

2. A strain of Paracoccus marcusii HMP-1, characterized in that: The 16SrDNA sequence of the strain HMP-1 is shown in SEQ ID NO:

1.

3. Use of Paracoccus martensii as claimed in claim 1 or 2 in non-disease diagnosis and treatment in lead treatment.

4. A bacterial suspension or freeze-dried powder prepared by using the Paracoccus martensii according to claim 1 or 2, characterized in that: The bacterial suspension and freeze-dried powder are used in non-disease diagnosis and treatment in lead treatment.

5. A repairing agent, characterized in that: The repair agent includes the Paracoccus martensii described in claim 1 or 2.

6. The repair agent according to claim 4, characterized in that: The repairing agent also includes humic acid.

7. The repair agent according to claim 5, characterized in that: In the repair agent, Paracoccus martensii HMP-1 was prepared as OD 600 For bacterial suspensions with a mass ratio of ≥1.0, the mass ratio of humic acid to bacterial suspension is 1:2.5 to 1:

20.

8. Use of the repair agent according to any one of claims 5 to 7 in removing lead ions.

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