A strain of Arthrobacter afzwei CYL1-3 and its bacterial agent and application in removing heavy metal ions

Abstract

The present invention relates to a strain of Arthrobacter ahreii CYL1‑3 and its bacterial agent and application in removing heavy metal ions, and belongs to the technical field of microorganisms. Arthrobacter ahreii CYL1‑3 is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with a deposit number of CGMCC No.32526, a deposit date of November 6, 2024, and a deposit address of the depository institution: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The present invention also provides an application of Arthrobacter ahreii CYL1‑3 in removing heavy metal ions. The Arthrobacter ahreii CYL1‑3 provided by the present invention can quickly adsorb heavy metal ions in sewage, without secondary pollution, and is safe and effective. At the same time, the fermentation process of Arthrobacter ahreii CYL1‑3 is simple, the preparation cycle is short, the action time is long, and it is easy to use, and it can play a role by directly adding it to sewage.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Arthrobacter arnoldii CYL1-3 and a bacterial agent thereof and an application thereof in removing heavy metal ions. Background Art

[0002] Heavy metals refer to metals with a specific gravity greater than 4 or 5, and there are about 45 kinds of them. Common heavy metal pollution mainly refers to environmental pollution caused by heavy metals with significant biological toxicity such as mercury, lead, cadmium, chromium and arsenic, and also includes heavy metals with certain toxicity such as zinc, copper, cobalt, nickel, tin, vanadium, etc. Heavy metal pollutants are difficult to control. When they accumulate to a certain limit in water bodies, they will cause serious harm to water bodies, aquatic plants, and aquatic animal systems, and may affect human health through the food chain. Heavy metal wastewater (such as cadmium, copper, nickel, gold, etc.) is one of the most serious industrial wastewaters that pollute the environment and are most harmful to humans. If it is discharged directly without treatment, heavy metals will not only cause serious damage to the adjacent environment, but also accumulate along the food chain, combine with other toxins to form more toxic organic matter, causing great harm to organisms. Therefore, the treatment of wastewater containing heavy metals has become a focus.

[0003] Existing heavy metal treatment methods can be roughly divided into chemical, physical and biological methods. Chemical methods mainly include chemical precipitation and electrolysis, which are mainly suitable for the treatment of wastewater containing high concentrations of heavy metal ions. Physical methods mainly include solvent extraction separation, ion exchange, membrane separation technology and adsorption. Biological methods are methods of removing heavy metals from wastewater by means of flocculation, absorption, accumulation, enrichment and other effects of microorganisms or plants, including biosorption, bioflocculation, phytoremediation and other methods. Chemical and physical methods have problems such as technical limitations, high economic costs and easy secondary pollution. Biological methods have become a hot topic of research due to their safety, harmlessness and high efficiency.

[0004] Arthrobacter, a Gram-positive aerobic bacterium, lives in every corner of the earth. Investigations and studies have shown that it has many uses, such as nicotine degradation, desulfurization, polyphosphate, treatment of heavy metals in sewage, nitrogen fixation, etc. In existing studies, Proteus sp. H24 isolated from chromium-contaminated seawater by Ge S et al. can successfully remove 1000 mg / L of Cr in LB culture medium. 6+ , remove and reduce high-valent chromium in industrial wastewater through ion exchange and extracellular polymers. Proteus mirabilis isolated from Moroccan industrial wastewater by Filali et al. has strong tolerance to various heavy metals such as mercury, copper, zinc, cadmium and cobalt. Proteus has great development prospects in the immobilization of heavy metals, utilization of toxic pollutants and promotion of plant growth due to its super strong metabolic characteristics and environmental adaptability.

[0005] In the prior art, no relevant application of Arthrobacter acherii in degrading heavy metals cadmium and copper has been found. Summary of the invention

[0006] In view of the problem that there is no application of Arthrobacter acherii in the existing methods for treating heavy metal cadmium and copper, the present invention provides an Arthrobacter acherii CYL1-3 and its bacterial agent and application for removing heavy metal ions to solve the above problem. The Arthrobacter acherii CYL1-3 of the present invention can bind free unmetabolized heavy metal ions to the surface of the cell wall through positive and negative charges by relying on the adsorption effect on the cell surface. The cell wall is mainly composed of teichoic acid, peptidoglycan and some neutral polysaccharides, and the polysaccharide and teichoic acid can form a network structure, which can adsorb and adhere to heavy metals, safely and efficiently reduce the heavy metal content in sewage without causing secondary pollution.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a strain of Arthrobacter ahreii CYL1-3, wherein the Arthrobacter ahreii ( Arthrobacter arilaitensis )CYL1-3 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32526, the deposit date is November 6, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] In a second aspect, the present invention provides a bacterial agent containing Arthrobacter achernobacter CYL1-3, wherein the bacterial agent is a liquid bacterial agent.

[0009] A method for preparing the above-mentioned liquid bacterial agent comprises the following steps: A loopful of activated Arthrobacter aeruginosa CYL1-3 was inoculated into LB liquid culture medium, and cultured at 200 rpm and 30°C until the OD value was 1, and then the fermentation was terminated to obtain a liquid bacterial agent.

[0010] Furthermore, the LB liquid culture medium has the following components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH=7.0.

[0011] In a third aspect, the present invention provides a use of Arthrobacter arnoi CYL1-3 in removing heavy metal ions.

[0012] Furthermore, the heavy metal ion is Cd 2+ or Cu 2+ .

[0013] Furthermore, when removing heavy metal ions, a liquid bacterial agent containing Arthrobacter agglomerans CYL1-3 is added, and the amount of the liquid bacterial agent added is 10,000-12,700 CFU / L·mg based on the heavy metal ion content. The optimal amount added when removing cadmium ions is 10,000 CFU / L·mg; the optimal amount added when removing copper ions is 12,700 CFU / L·mg.

[0014] The beneficial effects of the present invention are: The Arthrobacter arifuge CYL1-3 provided by the invention can quickly absorb heavy metal ions in sewage, has no secondary pollution, is safe and effective, and has a simple fermentation process, a short preparation cycle, a long action time, and is convenient to use, and can be directly added to sewage to play a role. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 It is a colony diagram of Arthrobacter acherii CYL1-3 in Example 1 of the present invention.

[0017] Figure 2 It is the Gram staining picture of Arthrobacter arnoi CYL1-3 in Example 1 of the present invention.

[0018] Figure 3 This is a trend diagram of the effect of time on the removal rate when Arthrobacter arnoi CYL1-3 removes heavy metal ions in Example 2 of the present invention.

[0019] Figure 4 This is a trend diagram showing the effect of inoculation amount on the removal rate when Arthrobacter arnoi CYL1-3 removes heavy metal ions in Example 2 of the present invention. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0021] Example 1 1. Sampling On March 23, 2024, sewage aerobic tank sludge was taken from Sinopec Jinling Petrochemical Co., Ltd.

[0022] 2. Separation and screening (1) Prepare Cd screening medium and Cu screening medium as follows: (1.1) Use analytically pure cadmium chloride (CdCl2) and copper chloride (CuCl2) reagents to prepare 2+ , Cu 2+ The mother solution (concentration is 1000 mg / L) is sterilized and set aside.

[0023] (1.2) Prepare LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH = 7.0. Sterilize and set aside.

[0024] (1.3) Sterilized heavy metal ion Cd 2+ , Cu 2+ The mother liquor was mixed with LB medium and diluted into 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L and 1000 mg / L Cd screening medium and Cu screening medium, pH=7.0.

[0025] (2) Weigh 10 g of sludge and 90 ml of sterile water into a 250 ml conical flask with sterilized glass beads. Shake on a shaker (30°C, 150 r / min, 2 h) and let stand to separate the layers. Take the supernatant as the sludge physiological solution and dilute the soil physiological solution to 10% with sterile saline in an ultra-clean workbench. -7 ~10 -9 times, take 10 each -7 ~10 -9 0.01 ml of the sludge dilution with a multiple concentration was evenly spread on the LB solid culture medium by the plate coating method, with 3 replicates for each gradient. It was placed in a microbial incubator (30°C, 2~3d) for culture and observed regularly. After the colony growth was visible to the naked eye, a single colony was picked for plate streaking purification. The purified bacteria were streaked on 200 mg / L Cd screening medium and Cu screening medium, respectively. After 2 days, the strains that could not grow were eliminated. Then the vigorously growing strains were inoculated and streaked on 400 mg / L Cd screening medium and Cu screening medium, respectively. After 2 days, the strains that could not grow were eliminated. Similarly, the vigorously growing strains were selected and added step by step to 600 mg / L, 800 mg / L and 1000 mg / L Cd screening medium and Cu screening medium. Finally, the degradable heavy metal ion Cd was obtained. 2+ and Cu 2+Three strains were obtained, numbered CYL1-1, CYL1-2, and CYL1-3. The target bacteria were cultured in sterile LB liquid medium and stored in 50% glycerol tubes at -80°C.

[0026] (3) After the initial screening strains CYL1-1, CYL1-2, and CYL1-3 were activated and inoculated into LB liquid culture medium for expansion, they were inoculated into 1000 mg / L Cd screening medium and Cu screening medium at a 1% inoculation rate for shaking culture (30°C, 200 r / min), and the concentration of metal ions was measured using an ICP spectrometer (equipment manufacturer: Jiangsu Shipu Instrument Co., Ltd., instrument model: ICP7000S) at 0h, 12h, 24h, and 48h. By comparison, the effect of strain CYL1-3 was significantly stronger than that of strain CYL1-1 and strain CYL1-2. Therefore, strain CYL1-3 was retained for subsequent verification.

[0027] 3. Identification The strain CYL1-3 appears as regular round colonies on the LB plate, light yellow, golden yellow after long-term storage, slightly raised, opaque, smooth and easy to pick up. Figure 1 As shown. Gram staining is positive, such as Figure 2 It was sent to Beijing Qingke Biotechnology Co., Ltd. for identification, and the result was Arthrobacter afrenii. Arthrobacter afrenii ( Arthrobacter arilaitensis )CYL1-3 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32526, the deposit date is November 6, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0028] Example 2 A bacterial agent containing Arthrobacter afrenii CYL1-3, the preparation method of which is as follows: A loop of activated Arthrobacter arifuge CYL1-3 was inoculated into LB liquid culture medium, which included: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH = 7.0. The culture was cultured at 200 rpm and 30 °C until the OD value was 1, and then the fermentation was terminated to obtain a liquid bacterial agent. The concentration of live Arthrobacter arifuge CYL1-3 in the liquid bacterial agent was 3 × 10 9 CFU / ml.

[0029] Example 3 Experimental verification of heavy metal ion removal effect of Arthrobacter agglomerans CYL1-3 1. Preparation of mock culture medium (1) Cu simulation culture medium: copper chloride 500 mg / L, anhydrous sodium acetate 2 g / L, ammonium sulfate 0.4 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 0.42 g / L, magnesium sulfate 0.2 g / L, pH = 7.0.

[0030] (2) Cd simulation culture medium: cadmium chloride 500 mg / L, anhydrous sodium acetate 2 g / L, ammonium sulfate 0.4 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 0.42 g / L, magnesium sulfate 0.2 g / L, pH = 7.0.

[0031] 2. Calculation of heavy metal ion removal rate Removal rate (%) = (c0-c q )÷c0×100%; Where: c0 is the initial concentration of heavy metal ions (mg / L), i.e., the metal ion concentration at 0 h; c q It is the residual concentration of heavy metal ions measured during sampling (mg / L).

[0032] 3. Simulation experiment on the effect of time on heavy metal ion removal rate Take the bacterial agent containing Arthrobacter acherii CYL1-3 prepared in Example 2, inoculate it into Cd simulated medium and Cu simulated medium at a mass fraction of 1%, continue to culture in a shaking table (30°C, 200r / min), and take samples at 0, 6, 12, 24, 36, 48 and 60h under sterile conditions, then centrifuge the samples at 12000r / min for 10min, take the supernatant and use ICP spectrometer (equipment manufacturer: Jiangsu Shipu Instrument Co., Ltd., instrument model: ICP7000S) to determine the concentration of metal ions, and calculate the metal ion removal rate. For experimental data, see Figure 3 .

[0033] Depend on Figure 3 It can be seen that Arthrobacter agglomerans CYL1-3 can continuously remove copper and cadmium ions within 60 hours. From the figure, it can be seen that the slope is larger and the removal rate is faster before 30 hours, and the slope becomes smaller and the removal rate slows down after 30 hours. This is because the amount of negative charge on the bacterial surface combined with metal ions increases, approaches saturation, and the adsorption rate slows down. In actual field applications, bacterial agents can be added every 48 hours to maintain the adsorption rate at a high level to remove heavy metal ions in sewage.

[0034] 4. Simulation experiment on the effect of inoculation volume on heavy metal ion removal efficiency The bacterial agent containing Arthrobacter acherii CYL1-3 prepared in Example 2 was inoculated into Cd simulated medium and Cu simulated medium at mass fractions of 0.01%, 0.1%, 1%, 2% and 3%, respectively, and continued to be cultured in a shaking table (30°C, 200r / min). After 48h, aseptic sampling was performed, and then the sample was centrifuged at 12000r / min for 10min. The supernatant was taken and the concentration of metal ions was determined by ICP spectrometer (equipment manufacturer: Jiangsu Shipu Instrument Co., Ltd., instrument model: ICP7000S), and the metal ion removal rate was calculated. For experimental data, see Figure 4 .

[0035] Depend on Figure 4 It can be seen that the removal rate of heavy metal ions does not always increase with the increase of inoculation amount. When the inoculation amount exceeds 2%, the metal ion removal rate begins to decline. This may be due to the large amount of bacteria, which constantly compete for nutrients, resulting in the death of some bacteria and the reduction of the removal rate. The removal rate of heavy metal ions increases slowly when the inoculation amount is between 0.01% and 1%. In practical applications, in order to reduce costs, an addition amount of 0.01% can be selected.

[0036] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A strain of Arthrobacter arifrugiperda CYL1-3, characterized in that: Arthrobacter afzini Arthrobacter arilaitensis )CYL1-3 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32526, the deposit date is November 6, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The Arthrobacter arifuge CYL1-3 according to claim 1, characterized in that The 16SrDNA sequence of Arthrobacter arnoi CYL1-3 is shown in SEQ ID NO.

1.

3. A bacterial agent containing Arthrobacter arifuge CYL1-3 as claimed in claim 1, characterized in that: The bacterial agent is a liquid bacterial agent.

4. A method for preparing the bacterial agent as claimed in claim 3, characterized in that: The following steps are involved: The activated Arthrobacter arnoi CYL1-3 was inoculated into LB liquid culture medium, and cultured at a constant temperature of 200 rpm and 30° C. until the OD value was 1, and then the fermentation was terminated to obtain a liquid bacterial agent.

5. The method according to claim 4, characterized in that The concentration of viable bacteria of Arthrobacter arifrugiperda CYL1-3 in the liquid bacterial agent was 3×10 9 CFU / ml.

6. The method according to claim 4, characterized in that The LB liquid culture medium had the following components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH=7.

0.

7. Use of the Arthrobacter ahreyi CYL1-3 as claimed in claim 1 in removing heavy metal ions.

8. The use according to claim 7, characterized in that The heavy metal ion is Cd 2+ Cu 2+ .

9. The use according to claim 7, characterized in that When removing heavy metal ions, a liquid bacterial agent containing Arthrobacter achernobacter CYL1-3 is added, and the amount of liquid bacterial agent added is 10000~12700 CFU / L·mg based on the heavy metal ion content.

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