Serratia marcescens with demercuration capability, demercuration agent and preparation method and application of demercuration agent

Abstract

The invention discloses serratia marcescens with demercuration capacity, a demercuration agent and a preparation method and application of the demercuration agent, and belongs to the technical field of microorganisms, the serratia marcescens WS-5 is preserved in China Center for Type Culture Collection on March 31, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2025622. The bacterium and a mercury removal agent for colonizing the bacterium on the surface of activated carbon through a carbon-loaded technology can reduce mercury (I ) in mercury-containing wastewater, so that the bacterium has the capability of removing mercury from the mercury-containing wastewater, realizes mercury recovery, has good economic benefits, and effectively solves the problems of high cost and poor effect of purifying the mercury-containing wastewater by the existing physical and chemical repair technology.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a Serratia marcescens with mercury removal ability, a mercury remover, a preparation method and application of the mercury remover. Background Art

[0002] With the development of modern industry, heavy metal pollution has become increasingly serious, causing great harm to the local ecological environment and the health of residents. Since heavy metals in the environment are difficult to remove, conventional physical and chemical remediation technologies not only have high costs and poor effects, but also are prone to secondary pollution. Therefore, developing efficient, safe and low-cost bioremediation technologies is of great significance.

[0003] Mercury (Hg) is a toxic heavy metal with high neurotoxicity and bioaccumulation effects. The cost of purifying mercury-containing wastewater by conventional physical and chemical methods is relatively high. Therefore, finding efficient microbial strains for mercury removal and recovery from mercury-containing wastewater has very important practical significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a Serratia marcescens with mercury removal ability, a mercury remover, a preparation method and application of the mercury remover, so as to solve the problems of high cost and poor effect of existing physical and chemical remediation technologies for purifying mercury-containing wastewater.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A Serratia marcescens WS-5 with mercury removal ability has been deposited at the China Center for Type Culture Collection on March 31, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025622.

[0007] Provide an application of Serratia marcescens WS-5 in mercury removal from mercury-containing wastewater.

[0008] A mercury remover, wherein the mercury remover comprises Serratia marcescens WS-5.

[0009] A preparation method of a mercury remover, comprising the following steps:

[0010] (1) Prepare a mercury-removing bacterial suspension with an OD 600 of 0.8, wherein the mercury-removing bacteria are Serratia marcescens WS-5;

[0011] (2) Weigh a certain amount of activated carbon and add it to the OD 600In the mercury-removing bacterial suspension with a concentration of 0.8, the mass-volume ratio of activated carbon to the mercury-removing bacterial suspension is 1:200 (w / v). The mixture is shaken and cultured at 30 °C and 150 rpm to enable the effective adsorption of mercury-removing bacteria on the surface of the activated carbon.

[0012] (3) Mix the activated carbon adsorbed with mercury-removing bacteria with a sterilized sodium alginate solution. After mixing, the concentration of sodium alginate is 3% (w / v).

[0013] (4) Drop the mixture in step (3) into a calcium chloride solution with a concentration of 2% (w / v) for crosslinking, and let it stand for hardening at 4 °C.

[0014] (5) After hardening is completed, an immobilized bacterial material is obtained. The immobilized bacterial material is repeatedly washed with 10 mM HEPES buffer to remove the excess calcium chloride on the surface.

[0015] (6) Place the prepared immobilized bacterial material in 10 mM HEPES buffer and store it at 4 °C for use as a mercury-removing agent.

[0016] Provide an application of a mercury-removing agent containing Serratia marcescens WS-5 in the mercury removal of mercury-containing wastewater.

[0017] To sum up, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0018] 1. In the present invention, the strain Serratia marcescens WS-5 can reduce mercury (II), thus having the ability to remove mercury from mercury-containing wastewater.

[0019] 2. In the present invention, the mercury-removing agent is based on a carbon-supported technology, enabling mercury-removing bacteria to colonize on the surface of activated carbon. The strain Serratia marcescens WS-5 is used to reduce mercury (II) to repair mercury-containing wastewater and recover mercury, which has good economic benefits and effectively solves the problems of high purification cost and poor effect of existing physical and chemical repair technologies for mercury-containing wastewater. Detailed implementation manners

[0020] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following combines examples to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are a part rather than all of the embodiments of the present invention. At the same time, the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0021] Isolation, screening and identification of Serratia marcescens WS-5:

[0022] Collect high-mercury soil samples from the Wanshan mining area in Tongren City, Guizhou Province, store them in sterile bags, and transport them with dry ice. Take 2.0 g of soil samples for biological cultivation. Dissolve mercuric chloride in ultrapure water to obtain a solution with a Hg concentration of 100 mg / L. Cool the sterilized R2A agar medium (121 °C, 30 minutes) to 55 °C, and then mix it with mercuric chloride to form a medium with a Hg concentration of 10 mg / L. Mix 1 g of soil sample with 10 mL of 0.9% sterile normal saline and sonicate for 10 minutes to form a -1 10-fold dilution. Perform serial dilution (10 -2 to 10 -7 ), and plate 200 μL of each dilution on mercury-rich agar and incubate at 37 °C for 72 hours. Perform streak pure culture with different colonies and then culture them in LB medium. Measure the optical density (OD 600 ) using a spectrophotometer to evaluate the growth.

[0023] Perform phylogenetic analysis using 16S rRNA gene sequencing to identify mercury-resistant strains, deposit the sequences into NCBI GenBank for BLASTN comparison. Use MEGA 5.0 and the neighbor-joining method to establish 1000 bootstrap replicates to determine the position of the isolates. The strain was identified as Serratia marcescens and named Serratia marcescens WS-5. Its sequence information:

[0024] GGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAA

[0025] GGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGAC

[0026] TCCAATCCGGACTACGACGTACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATACGCCATTGTAG

[0027] CACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTC

[0028] TCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCA

[0029] CAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCT

[0030] GGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTC

[0031] AATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGCCACGCCTC

[0032] AAGGGCACAACCTCCAAATCGACATCGTTTACAGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTT

[0033] CGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGC

[0034] TACACCTGGAATTCTACCCCCCTCTACGAGACTCTAGCTTGCCAGTTTCAAATGCAGTTCCCAGGTTGAGCCCGGGGA

[0035] TTTCACATCTGACTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATT

[0036] ACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCAATTGATGAACGTATTAAGTTCACCAC

[0037] CTTCCTCCTCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGCTTGCGCCC

[0038] ATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCT

[0039] CAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGC

[0040] AAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTC

[0041] CATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCAGGGAGCAAGCTCCCCTGTGCTACCGCTCGACTTGCATGTGTTAAGCCTGCCCGCCAGCGTTC。

[0042] Experiment on the mercury removal ability of the strain:

[0043] The reduction of mercury (II) by the strain was tested in an open system. 1.0 mL of the bacterial culture (OD 600 / 0.40 ± 0.05) was added to a 50 mL test tube, and 7.5 mL of LB medium and 7.5 mL of Hg(II) solution (354.1 μg / L) were added. After dark incubation for 6.5 hours, 1.0 mL of the supernatant was taken to measure the remaining mercury concentration.

[0044] The experimental data are shown in Table 1. Serratia marcescens WS-5 was able to reduce Hg(II).

[0045] Strain HgS dissolution ability Hg(II) reduction ability Control group - 1.19 0.5％ WS-5 Serratia marcescens 278.62 50.5％

[0046] Table 1

[0047] A preparation method of a mercury removal agent, comprising the following steps:

[0048] (1) Prepare OD 600In the mercury-removing bacterial suspension with an OD of 0.8, the mercury-removing bacterium is Serratia marcescens WS-5;

[0049] (2) Weigh a certain amount of activated carbon and add it to the mercury-removing bacterial suspension with an OD 600 of 0.8. The mass-volume ratio of the activated carbon to the mercury-removing bacterial suspension is 1:200 (w / v). Under the conditions of 30 °C and 150 rpm, shake and culture for 2 hours to effectively adsorb the mercury-removing bacteria on the surface of the activated carbon;

[0050] (3) Mix the activated carbon adsorbed with mercury-removing bacteria with the sterilized sodium alginate solution. After mixing, the concentration of sodium alginate is 3% (w / v);

[0051] (4) Drop the mixture in step (3) into a calcium chloride solution with a concentration of 2% (w / v) through a syringe for cross-linking, and let it stand and harden at 4 °C for 24 hours;

[0052] (5) After hardening is completed, obtain the immobilized bacterial material, and repeatedly wash the immobilized bacterial material with 10 mM HEPES buffer solution to remove the excess calcium chloride on the surface;

[0053] (6) Place the prepared immobilized bacterial material in 10 mM HEPES buffer solution and store it at 4 °C for use as a mercury-removing agent.

[0054] Collect the mercury-containing wastewater from a certain place. Take a sample and measure the total mercury concentration of the mercury-containing wastewater to be 54.8 mg / L. Put 6.0 g of the mercury-removing agent of the present invention into 20 mL of the mercury-containing wastewater, and measure the total mercury concentration of the mercury-containing wastewater at intervals of 24 hours, 48 hours, 72 hours, and 96 hours respectively. The results are shown in Table 2. As time goes by, the total mercury concentration of the mercury-containing wastewater continues to decrease. The mercury removal rate of the mercury-containing wastewater at 96 hours is 55.8%.

[0055]

[0056] Table 2

[0057] The above is the embodiment of the present invention. The foregoing are the preferred embodiments of the present invention. If the preferred implementation manners in each preferred embodiment are not obviously self-contradictory or based on a certain preferred implementation manner, each preferred implementation manner can be arbitrarily superimposed and combined. The embodiments and the specific parameters in the embodiments are only for clearly expressing the verification process of the invention, and are not used to limit the patent protection scope of the present invention. The patent protection scope of the present invention still takes its claims as the criterion. Any equivalent structural changes made by using the content of the specification of the present invention should similarly be included in the protection scope of the present invention.

Claims

1. A Serratia marcescens WS-5 with mercury removal ability, characterized in that, Serratia marcescens WS-5 was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2025622.

2. Use of Serratia marcescens WS-5 according to claim 1 in mercury removal from mercury-containing wastewater.

3. A mercury removal agent, characterized in that, The mercury removal agent comprises Serratia marcescens WS-5 according to claim 1.

4. A preparation method of a mercury removal agent as described in claim 3, characterized in that, Comprising the following steps: (1) Preparation of OD 600 in the mercury-removing bacterial suspension with an OD of 0.8, the mercury-removing bacterium is Serratia marcescens WS-5; (2) Weigh a certain amount of activated carbon and add it to the mercury-removing bacterial suspension with an OD 600 of 0.

8. The mass-volume ratio of the activated carbon to the mercury-removing bacterial suspension is 1:200 (w / v). Incubate it with shaking at 30 °C and 150 rpm to enable the effective adsorption of the mercury-removing bacteria on the surface of the activated carbon; (3) Mix the activated carbon adsorbed with mercury-removing bacteria with a sterilized sodium alginate solution, and the concentration of sodium alginate after mixing is 3% (w / v); (4) Drop the mixture in step (3) into a calcium chloride solution with a concentration of 2% (w / v) for crosslinking, and let it stand for hardening at 4°C; (5) After the hardening is completed, an immobilized bacterial material is obtained, and the immobilized bacterial material is repeatedly washed with 10 mM HEPES buffer to remove the excess calcium chloride on the surface; (6) Place the prepared immobilized bacterial material in 10 mM HEPES buffer and store it at 4°C for use as a mercury removal agent.

5. Use of the mercury removal agent according to claim 3 in mercury removal from mercury-containing wastewater.

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