Arthrobacter arilaitensis strain CYL1-3, its bacterial agent and application in removing heavy metal ions

Abstract

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

Description

Technical Field

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

[0002] Heavy metals refer to metals with a specific gravity greater than 4 or 5, about 45 kinds in total. Usually, heavy metal pollution mainly refers to the environmental pollution of 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 treat. 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 (such as cadmium, copper, nickel, gold, etc.) wastewater is one of the industrial wastewaters that cause the most serious environmental pollution and the greatest harm to humans. If directly discharged 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 substances, causing great harm to organisms. Therefore, the treatment of heavy metal-containing wastewater has become the focus.

[0003] Existing heavy metal treatment methods can generally be divided into chemical methods, physical methods and biological methods. Chemical methods mainly include chemical precipitation method and electrolysis method, which are mainly applicable to the treatment of wastewater containing high-concentration heavy metal ions. Physical methods mainly include solvent extraction separation, ion exchange method, membrane separation technology and adsorption method. Biological method is a method of removing heavy metals in wastewater by means of flocculation, absorption, accumulation, enrichment, etc. of microorganisms or plants, including biological adsorption, biological flocculation, phytoremediation and other methods. Chemical methods and physical methods have problems such as technical limitations, high economic costs and extremely easy to cause secondary pollution. Biological method has become a research hotspot due to its safe, harmless and efficient characteristics.

[0004] Arthrobacter, a Gram-positive bacterium and an aerobic bacterium, lives in all corners of the earth. Investigations and studies show that it has many uses, such as degrading nicotine, desulfurizing, polyphosphorus, treating heavy metals in sewage, nitrogen fixation, etc. In existing research, Ge S et al. isolated Proteus sp. H24 from chromium-polluted seawater, which could successfully remove 1000 mg / L of Cr 6+ , and remove and reduce hexavalent chromium in industrial wastewater through ion exchange and extracellular polymers. Filali et al. isolated Proteus mirabilis from Moroccan industrial wastewater, which has strong tolerance to various heavy metals such as mercury, copper, zinc, cadmium and cobalt. Proteus has great development prospects in aspects such as immobilizing heavy metals, utilizing toxic pollutants and promoting plant growth due to its super metabolic characteristics and environmental adaptability.

[0005] In the prior art, no relevant applications of Arthrobacter sp. in degrading heavy metals cadmium and copper have been found. Summary of the Invention

[0006] Aiming at the problem that there is no relevant application of Arthrobacter sp. in the existing methods for treating heavy metals cadmium and copper, the present invention provides an Arthrobacter sp. CYL1-3, its bacterial agent, and its application in removing heavy metal ions to solve the above problems. The Arthrobacter sp. CYL1-3 of the present invention can bind free and unmetabolized heavy metal ions to the surface of the cell wall through positive and negative charges relying on the adsorption effect on the cell surface. Its cell wall is mainly composed of teichoic acid, peptidoglycan, and some neutral polysaccharides, etc. The polysaccharides 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 producing secondary pollution.

[0007] The technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides an Arthrobacter sp. CYL1-3, and the Arthrobacter sp. ( Arthrobacter arilaitensis ) CYL1-3 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 32526, the deposit date being November 06, 2024, and the address of the deposit institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] In the second aspect, the present invention provides a bacterial agent containing Arthrobacter sp. CYL1-3, and the bacterial agent is a liquid bacterial agent.

[0010] A method for preparing the above liquid bacterial agent includes the following steps:

[0011] Pick one loop of the activated Arthrobacter sp. CYL1-3 and inoculate it into the LB liquid medium, and culture it at a constant temperature of 30 °C with 200 rpm until the OD value = 1, then end the fermentation to obtain the liquid bacterial agent.

[0012] Furthermore, the composition of the LB liquid medium is as follows: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH = 7.0.

[0013] In the third aspect, the present invention provides an application of Arthrobacter sp. CYL1-3 in removing heavy metal ions.

[0014] Furthermore, the heavy metal ions are Cd 2+ or Cu 2+ .

[0015] Further, when removing heavy metal ions, a liquid bacterial agent containing Arthrobacter aurescens CYL1-3 is added, and the addition amount of the liquid bacterial agent is 10,000 - 12,700 CFU / L·mg based on the heavy metal ion content. The optimal addition amount is 10,000 CFU / L·mg when removing cadmium ions; the optimal addition amount is 12,700 CFU / L·mg when removing copper ions.

[0016] The beneficial effects of the present invention are as follows:

[0017] Arthrobacter aurescens CYL1-3 provided by the present invention can rapidly adsorb heavy metal ions in sewage, without secondary pollution, and is safe and effective. At the same time, the fermentation process of Arthrobacter aurescens CYL1-3 is simple, the preparation period is short, the action time is long, it is convenient to use, and it can play a role directly when added to sewage. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is the colony map of Arthrobacter aurescens CYL1-3 in Example 1 of the present invention.

[0020] Figure 2 It is the Gram staining map of Arthrobacter aurescens CYL1-3 in Example 1 of the present invention.

[0021] Figure 3 It is the trend graph of the influence of time on the removal rate when Arthrobacter aurescens CYL1-3 removes heavy metal ions in Example 2 of the present invention.

[0022] Figure 4 It is the trend graph of the influence of the inoculation amount on the removal rate when Arthrobacter aurescens CYL1-3 removes heavy metal ions in Example 2 of the present invention. Detailed Embodiments

[0023] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the 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. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] Example 1

[0025] 1. Sampling

[0026] On March 23, 2024, sewage aerobic tank sludge was taken from Sinopec Jinling Petrochemical Co., Ltd.

[0027] 2. Separation and screening

[0028] (1)Prepare Cd screening medium and Cu screening medium, and the preparation methods are as follows:

[0029] (1.1)Use analytical pure cadmium chloride (CdCl 2 ), copper chloride (CuCl 2 )reagents to prepare stock solutions containing heavy metal ions Cd 2+ 、Cu 2+ (concentration is 1000mg / L), sterilize and reserve for later use.

[0030] (1.2)Prepare LB medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH = 7.0. Sterilize and reserve for later use.

[0031] (1.3)Mix the sterilized stock solutions containing heavy metal ions Cd 2+ 、Cu 2+ with LB medium, and dilute them into Cd screening medium and Cu screening medium with concentrations of 200mg / L, 400mg / L, 600mg / L, 800mg / L and 1000mg / L, pH = 7.0.

[0032] (2)Weigh 10g of sludge, and put it into a 250ml conical flask containing sterilized glass beads together with 90ml of sterile water. After shaking in a shaker (30℃, 150r / min, 2h), let it stand and layer. Take the supernatant as the sludge physiological solution. In a laminar flow hood, use sterilized normal saline to gradient dilute this soil physiological solution to 10 -7 ~10 -9 times. Take 0.01ml of sludge dilution solution with concentrations of 10 -7 ~10 -9 times and spread it evenly on the LB solid medium by the plate coating method. There are 3 replicates for each gradient. Place it in a microbial incubator (30℃, 2 - 3d) for cultivation and observe regularly. After visible colonies grow, pick single colonies for plate streaking purification. Streak-culture the purified bacteria on the Cd screening medium and Cu screening medium with a concentration of 200mg / L respectively. After 2 days, eliminate the strains that cannot grow. Then streak-culture the strains with strong growth on the Cd screening medium and Cu screening medium with a concentration of 400mg / L respectively. After 2 days, eliminate the strains that cannot grow. And so on, select the strains with strong growth and gradually add them to the Cd screening medium and Cu screening medium with concentrations of 600mg / L, 800mg / L and 1000mg / L. Finally, strains that can degrade heavy metal ions Cd2+ and Cu 2+ Three strains of bacteria, numbered CYL1-1, CYL1-2, and CYL1-3 respectively. After the target bacteria were expanded in sterile LB liquid medium, they were stored in 50% glycerol tubes and preserved at -80°C.

[0033] (3)After activating the initially screened strains CYL1-1, CYL1-2, and CYL1-3 and expanding them in LB liquid medium, they were inoculated into Cd screening medium and Cu screening medium at a concentration of 1000 mg / L with an inoculation amount of 1% and cultured on a shaker (30°C, 200 r / min). The concentrations of metal ions were measured using an ICP spectrometer (manufacturer: Jiangsu Shipu Instrument Co., Ltd., model: ICP7000S) at 0 h, 12 h, 24 h, and 48 h respectively. By comparison, the effect of strain CYL1-3 was significantly stronger than that of strains CYL1-1 and CYL1-2. Therefore, strain CYL1-3 was retained for subsequent verification.

[0034] 3. Identification

[0035] Strain CYL1-3 formed regular round colonies on the LB plate, light yellow, turning golden yellow after long-term placement, slightly convex, opaque, with a smooth surface and easy to pick up, as Figure 1 shown. After Gram staining, it was positive, as Figure 2 shown. It was sent to Beijing Tsingke Biotechnology Co., Ltd. for identification, and the identification result was Arthrobacter arilaitensis. Arthrobacter arilaitensis ( Arthrobacter arilaitensis ) CYL1-3 was deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 32526, the deposit date being November 06, 2024, and the address of the deposit institution: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0036] Example 2

[0037] A bacterial agent containing Arthrobacter arilaitensis CYL1-3, the preparation method is as follows:

[0038] One loop of the activated Arthrobacter arilaitensis CYL1-3 was inoculated into LB liquid medium. The LB liquid medium included: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl, pH = 7.0. The fermentation was ended after culturing at 200 rpm and 30°C until the OD value = 1 to obtain a liquid bacterial agent. The viable bacteria concentration of Arthrobacter arilaitensis CYL1-3 in the liquid bacterial agent was 3×10 9 CFU / ml.

[0039] Example 3

[0040] Verification Experiment on the Removal Effect of Heavy Metal Ions by Arthrobacter sp. CYL1-3

[0041] 1. Preparation of Simulated Medium

[0042] (1)Cu Simulated 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.

[0043] (2)Cd Simulated 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.

[0044] 2. Calculation of Heavy Metal Ion Removal Rate

[0045] Removal Rate (%) = (c 0 -c q )÷c 0 × 100%;

[0046] In the formula:

[0047] c 0 is the initial concentration of heavy metal ions (mg / L), that is, the metal ion concentration at 0 h;

[0048] c q is the residual concentration of heavy metal ions measured at the time of sampling (mg / L).

[0049] 3. Simulation Experiment on the Influence of Time on Heavy Metal Ion Removal Rate

[0050] Take the bacterial agent containing Arthrobacter sp. CYL1-3 prepared in Example 2, inoculate it into the Cd simulated medium and the Cu simulated medium respectively according to a mass fraction of 1%, continue to shake culture (30 °C, 200 r / min), and sample at 0, 6, 12, 24, 36, 48 and 60 h under sterile conditions. Then centrifuge the samples at 12000 r / min for 10 min, take the supernatant, and measure the metal ion concentration with an ICP spectrometer (equipment manufacturer: Jiangsu Shipu Instrument Co., Ltd., instrument model: ICP7000S), and calculate the heavy metal ion removal rate. The experimental data are shown in Figure 3 .

[0051] From Figure 3It can be seen that Arthrobacter sp. CYL1-3 can continuously remove copper ions and cadmium ions within 60 h. As can be seen from the figure, the slope is larger before 30 h, and the removal rate is faster. After 30 h, the slope becomes smaller and the removal rate slows down. This is because the amount of binding between the negative charges on the bacterial surface and metal ions increases and approaches saturation, resulting in a slower adsorption rate. When applied in the actual field, the bacterial agent can be added every 48 h to maintain a high adsorption rate for removing heavy metal ions in sewage.

[0052] 4. Simulation experiment on the influence of inoculum size on the removal rate of heavy metal ions

[0053] Take the bacterial agent containing Arthrobacter sp. CYL1-3 prepared in Example 2 and inoculate it into the Cd simulated medium and Cu simulated medium at mass fractions of 0.01%, 0.1%, 1%, 2% and 3% respectively, and continue to culture on a shaker (30 °C, 200 r / min). Take aseptic samples after 48 h, then centrifuge the samples at 12000 r / min for 10 min, take the supernatant, and measure the concentration of metal ions with an ICP spectrometer (equipment manufacturer: Jiangsu Shipu Instrument Co., Ltd., instrument model: ICP7000S), and calculate the removal rate of metal ions. The experimental data are shown in Figure 4 .

[0054] It can be seen from Figure 4 that the removal rate of heavy metal ions does not always increase with the increase of inoculum size. When the inoculum size exceeds 2%, the removal rate of metal ions begins to decrease. This may be because a large amount of bacteria compete for nutrients continuously, resulting in the death of some bacteria and a decrease in the removal rate. The increase of the removal rate of heavy metal ions is slow when the inoculum size is between 0.01% and 1%. In actual application, an addition amount of 0.01% can be selected to reduce costs.

[0055] Although the present invention has been described in detail by referring 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, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention. / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. A strain of Arthrobacter arifrugiperda CYL1-3, characterized in that: The Arthrobacter afseri ( 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 arifuge CYL1-3 as claimed in claim 1 in removing heavy metal ions, characterized in that: The heavy metal ion is Cd 2+ Cu 2+ .

8. 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.

Images