Klebsiella oxytoca and application thereof in reduction of pentavalent vanadium

Abstract

The invention relates to klebsiella oxytoca and application thereof in reduction of pentavalent vanadium, and belongs to the technical field of microorganisms. The first technical problem to be solved by the invention is to provide a bacterial strain for reducing a high-concentration vanadium solution, namely Klebsiella oxytoca G-17. The Klebsiella oxytoca G-17 disclosed by the invention is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the Klebsiella oxytoca G-17 is CCTCC M 20242962. The Klebsiella oxytoca G-17 has high tolerance to vanadium in the environment, the tolerance concentration to vanadium reaches up to 2000 mg.L <-1 >, the Klebsiella oxytoca G-17 can adapt to the survival condition of a high background pollution area, the Klebsiella oxytoca G-17 is reported for the first time to reduce pentavalent vanadium into tetravalent vanadium, and a bioremediation technology is provided for vanadium pollution. The vanadium reduction operation process is simple, the high reduction rate can be achieved under the condition that an electron donor does not need to be added, the reduction rate of 84.72 + / -0.17% of pentavalent vanadium can be achieved under the concentration of 500 mg / L, and the method has the advantages of being convenient to use and efficient.

Description

Technical Field

[0001] The present invention relates to Klebsiella oxytoca and its application in reducing pentavalent vanadium, belonging to the field of microbial technology. Background Art

[0002] Vanadium is a transition metal element widely present in the earth's crust. Because of its excellent properties such as high melting point, hard texture, and good toughness, it is widely used in the atomic energy industry, metallurgy, textile, and chemical industries. With the development of science and technology, the demand for vanadium products is increasing continuously. The combustion of vanadium-containing fuels and the extraction and smelting of vanadium-containing minerals produce a large amount of vanadium, which migrates into the environment in the form of the atmosphere and solution. Vanadium exists in various valence states of +3, +4, and +5 in the natural system. Vanadate species, pentavalent vanadium, is thermodynamically stable under oxidative conditions, while tetravalent vanadium is stable under suboxic conditions, and trivalent vanadium stably exists in anoxic environments. The toxicity of vanadium increases with the increase of valence state, and pentavalent vanadium has the highest toxicity. It can be seen that the metal toxicity of high-valence vanadium is much greater than that of low-valence vanadium. Therefore, the reduction method is usually used to treat vanadium-polluted groundwater to reduce its harm to the environment. However, the current physical and chemical remediation technologies are costly and can cause secondary pollution to the environment. Most chemical materials have good reduction effects under laboratory operating conditions, but the actual environment is complex, and whether they can achieve ideal remediation effects in the actual environment requires further research.

[0003] Microbial remediation technology is a kind of bioremediation technology. Because of its simple operation and environmental friendliness, it has received extensive attention in the field of environmental governance. Microbial remediation mainly goes through the following processes: Microorganisms can absorb through their own functional metabolism or reduce high-valence metals, thereby achieving the remediation of heavy metals in soil and water bodies. Some specific microorganisms can reduce the content of heavy metals in the environment through cell surface adsorption, reduction, and intracellular bioaccumulation. The extracellular polysaccharides present on the cell surface have specific functional groups, such as amino, carboxyl, and hydroxyl groups. These functional groups can absorb metal cations or oxygen anions through electrostatic forces; under the action of an external electron donor, microorganisms conduct electrons through their own enzymes, and heavy metals, as the final electron acceptors, are reduced to low-valence metal cations; microorganisms absorb heavy metal ions through active transport, and these ions combine with lipids in the cells and thus remain in the cells. Through the above processes, microorganisms can effectively achieve the remediation of heavy metals in the environment. In addition, these microorganisms all come from polluted areas. Microorganisms screened in situ can better adapt to the local environment, and the addition of these microorganisms can coordinate the in-situ microbial community structure and better achieve the in-situ remediation of heavy metals.

[0004] Currently, the microorganisms that can degrade vanadium are mainly Bacillus species. For example, Patent CN114317369A discloses a Bacillus amyloliquefaciens SM01 that can reduce pentavalent vanadium, and Patent CN114292792A discloses a Bacillus pacificus SM02 that can reduce pentavalent vanadium. These strains have a certain tolerance to vanadium, with a maximum tolerance of up to 1200 mg·L -1 , however, when these strains are used for the reduction of high-concentration vanadium, the effect is not good. Therefore, a new strain that can reduce high-concentration vanadium solution is needed. Summary of the Invention

[0005] Aiming at the above defects, the first technical problem solved by the present invention is to provide a strain for reducing high-concentration vanadium solution, namely Klebsiella oxytoca G-17.

[0006] The Klebsiella oxytoca G-17 of the present invention is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC M 20242962 and the preservation date of December 31, 2024.

[0007] In one embodiment of the present invention, the nucleotide sequence of the 16S rDNA of the Klebsiella oxytoca G-17 is shown as SEQ ID No.1.

[0008] The second technical problem solved by the present invention is to provide the application of the Klebsiella oxytoca G-17 of the present invention in reducing pentavalent vanadium.

[0009] The Klebsiella oxytoca G-17 of the present invention can reduce pentavalent vanadium to tetravalent vanadium and has a good function of reducing pentavalent vanadium. Among them, at a high concentration of 500 mg / L, the reduction rate of pentavalent vanadium can reach 84.72 ± 0.17%.

[0010] The present invention also provides a vanadium toxicity remover.

[0011] The active ingredient of the vanadium toxicity remover of the present invention contains the Klebsiella oxytoca G-17 of the present invention.

[0012] The present invention also provides a method for reducing pentavalent vanadium.

[0013] The method for reducing pentavalent vanadium in the present invention includes the following steps: adding the above-mentioned Klebsiella oxytoca G-17 to water bodies or soil containing pentavalent vanadium.

[0014] In one embodiment of the present invention, in the water body containing pentavalent vanadium, the concentration of pentavalent vanadium is less than 2000 mg·L -1 .

[0015] In a specific embodiment of the present invention, in the water body containing pentavalent vanadium, the concentration of pentavalent vanadium is 500 mg·L -1 .

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. Klebsiella oxytoca G-17 in the present invention has a high tolerance to vanadium in the environment, and the tolerance concentration to vanadium is as high as 2000 mg·L-1, and it can adapt to the survival conditions in high-background pollution areas. And this is the first report that Klebsiella oxytoca G-17 can reduce pentavalent vanadium to tetravalent vanadium, providing a bioremediation technology for vanadium pollution.

[0018] 2. The present invention only needs to simply culture Klebsiella oxytoca G-17 in NB liquid medium for 48 - 72 h until it grows to the logarithmic phase, and rely on the medium to provide nutrients for the reduction of pentavalent vanadium. The operation process is simple, and without the need to add an electron donor, a high reduction rate can also be achieved under high-concentration vanadium conditions.

[0019] 3. The vanadium toxicity remover described in the present invention can achieve a reduction rate of 84.72 ± 0.17% for pentavalent vanadium at a concentration of 500 mg / L. The reduction rate is much higher than the reduction rate of bacteria for pentavalent vanadium under high-concentration conditions reported previously, and it has the advantages of convenience and high efficiency. Specific Embodiments

[0020] In the present invention, soil groundwater in a heavily vanadium-polluted area near a vanadium-titanium magnetite smelter in Panzhihua was collected, and a strain of bacteria was obtained through separation and purification. Through morphological and 16S rDNA identification, this bacteria has 99% homology with Klebsiella oxytoca and is named Klebsiella oxytoca G-17. This bacteria was submitted for biological preservation to the China Center for Type Culture Collection on January 2, 2025, and the preservation number is CCTCC M 20242962. The address of the China Center for Type Culture Collection is Wuhan University, China, Wuhan.

[0021] The research found that this bacterium has a high tolerance to vanadium in the environment, with a tolerance concentration to vanadium up to 2000 mg·L-1, and it can adapt to the survival conditions in high-background pollution areas. Moreover, this bacterium can reduce pentavalent vanadium to tetravalent vanadium, with a high reduction rate, providing a bioremediation technology for vanadium pollution.

[0022] The suitable culture conditions for this bacterium are: tryptone 10 g / L, yeast 5 g / L, sodium chloride 10 g / L, pH 7.2, anaerobic culture.

[0023] In one embodiment of the present invention, the nucleotide sequence of the 16srDNA of Klebsiella oxytoca G-17 is as shown in SEQ ID No.1. After extracting the 16S rDNA of this bacterium and performing PCR amplification and sequencing, it is found that the nucleotide sequence of the 16s rDNA of this bacterium is as shown in SEQ ID No.1, and this bacterium has 99% homology with Klebsiella oxytoca. Therefore, this strain is named Klebsiella oxytoca G-17.

[0024] Klebsiella oxytoca G-17 of the present invention can reduce pentavalent vanadium to tetravalent vanadium and has a good function of reducing pentavalent vanadium. Among them, the reduction rate of pentavalent vanadium can reach 84.37±0.17%, with a high reduction rate.

[0025] The vanadium toxicity remover of the present invention has an active ingredient comprising Klebsiella oxytoca G-17 described in the present invention.

[0026] The method for reducing valueless vanadium of the present invention includes the following steps: adding Klebsiella oxytoca G-17 described in the present invention into a water body or soil containing pentavalent vanadium.

[0027] In one embodiment of the present invention, Klebsiella oxytoca G-17 grown to the logarithmic phase is added, and the inoculation amount of Klebsiella oxytoca G-17 is 1%.

[0028] In one embodiment of the present invention, in the water body containing pentavalent vanadium, the concentration of pentavalent vanadium is less than 2000 mg·L -1 .

[0029] In a specific embodiment of the present invention, in the water body containing pentavalent vanadium, the concentration of pentavalent vanadium is 500 mg·L -1 .

[0030] The following further describes the specific implementation manners of the present invention in conjunction with embodiments, and the present invention is not limited to the scope of the described embodiments accordingly.

[0031] Example 1

[0032] Soil groundwater in a heavily vanadium-polluted area near a Panzhihua vanadium-titanium magnetite smelter was selected. According to the test results, the highest content of vanadium in the local soil can reach 4156.47 mg·kg -1 .

[0033] 1. Isolation and purification of bacteria

[0034] (1) 1 ml of soil groundwater in the polluted area was added to 9 ml of sterile water and mixed evenly. The above steps were repeated to obtain concentrations of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 times the original solution concentration. 10 -3 , 10 -4 , 10 -5 times the original solution concentration were spread on the NB solid medium and anaerobically cultured at 30 °C for 48 h.

[0035] (2) Single bacteria strains in the solid medium in step (1) were separately selected and inoculated again into the solid medium containing 100 mg·L -1 of vanadium, and anaerobically cultured at 30 °C for 48 h.

[0036] (3) The bacteria with good growth in step (2) were inoculated into the solid medium with vanadium concentrations of 300 mg·L -11 respectively, and anaerobically cultured at 30 °C for 48 h.

[0037] (4) The bacteria with good growth in step (3) were inoculated into the solid medium with vanadium concentrations of 500 mg·L -1 respectively, and anaerobically cultured at 30 °C for 48 h.

[0038] (5) The bacteria with good growth in step (4) were inoculated into the solid medium with vanadium concentrations of 700 mg·L -11 respectively, and anaerobically cultured at 30 °C for 48 h.

[0039] (6) The bacteria with good growth in step (5) were inoculated into the solid medium with vanadium concentrations of 900 mg·L -1 respectively, and anaerobically cultured at 30 °C for 48 h.

[0040] (7) As the inoculation concentration gradually increased, a tolerant bacterium was finally obtained and named G-17. This bacterium was streaked and purified on a heavy metal-free plate, and a single colony was picked and inoculated into NB liquid medium, followed by anaerobic culture at 30 °C for 48 h.

[0041] The formula of the above NB solid medium is as follows: add 10 g of tryptone, 5 g of yeast, 10 g of sodium chloride, and 15 g of agar to 1 L of water, and adjust the pH to 7.0.

[0042] The formula of NB liquid medium: add 10 g of tryptone, 5 g of yeast, and 10 g of sodium chloride to 1 L of water, and adjust the pH to 7.0.

[0043] 2. Bacterial identification

[0044] The colony morphology of G-17 is round, milky white, with a smooth and moist surface and a viscous shape. The bacterium was subjected to Gram staining, showing red color and a short rod-shaped appearance, indicating that it is a Gram-negative bacterium.

[0045] After extracting the bacterial 16S rDNA, PCR amplification and sequencing were performed. The universal primers were 27F (SEQ ID No.2, AGTTTGATCMTGGCTCAG) and 1492R (SEQ ID No.3, GGTTACCTTGTTACGACTT), and the amplification length was about 1500 bp. The PCR reaction procedure was as follows: ① Pre-denaturation at 94 °C for 5 min; ② Denaturation at 94 °C for 30 s; ③ Annealing at 54 °C for 30 s; ④ Extension at 72 °C for 1 min 30 s; Repeat steps ②, ③, and ④ 39 times; ⑤ Extension at 72 °C for 10 min; Store at 4 °C. The amplified PCR product was subjected to agarose gel electrophoresis (2 uL of sample + 6 uL of bromophenol blue) at 300 V for 12 min. The PCR product was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing results were assembled using ContigExpress, and the inaccurate parts at both ends were removed. The assembled sequence was compared in the NCBI database (blast.ncbi.nlm.nih.gov), and phylogenetic construction was performed using MEGA software.

[0046] G-17 has 99% homology with Klebsiella oxytoca, and this bacterium was named Klebsiella oxytoca G-17.

[0047] Example 2 Tolerance concentration test

[0048] Klebsiella oxytoca G-17 was inoculated onto a plate containing vanadium at a concentration of 100 mg·L -1 , 1000 mg·L -1 , 2000 mg·L-1 , 3000 mg·L -1 in a solid medium, and anaerobically cultured at 30 °C for 48 h. Finally, when the vanadium concentration was 2000 mg·L -1 , the growth of bacteria gradually weakened. It indicates that the tolerance concentration of this strain of bacteria to vanadium reached 2000 mg·L -1 .

[0049] Example 3 Reduction Effect Test

[0050] Inoculate Klebsiella oxytoca G-17 into NB liquid medium and expand the culture for 48 h. Anaerobically culture it statically at 30 °C. Then wash the bacteria twice with sterile water and adjust the OD 600 to 1. Add it to the V(V) solution containing 500 mg·L -1 at an inoculation amount of 1%, and anaerobically culture it at 30 °C for 48 h. Centrifuge at 5000 rpm, filter the supernatant, and measure the pentavalent vanadium concentration in the solution by ultraviolet spectrophotometry, and determine the total vanadium content by ICP-OES. At the same time, set up an inactivated bacteria group as a control to determine whether the bacteria have an adsorption effect. The results are shown in Table 1:

[0051] Table 1

[0052]

[0053] The results show that Klebsiella oxytoca G-17 can reduce pentavalent vanadium, and the reduction rate of pentavalent vanadium is 84.72 ± 0.17%. Among them, the calculation method of the reduction rate in the present invention is: R e =(c (0) -c) / c (0)

[0054] c is the concentration of pentavalent vanadium in the solution after the reaction; c (0) is the initial pentavalent vanadium concentration in the solution.

[0055] It can be seen that Klebsiella oxytoca G-17 in the present invention can adapt to high concentrations of vanadium, and can well reduce pentavalent vanadium without adding an electron donor, and has a relatively high reduction rate.

Claims

1. Klebsiella oxytoca G-17, characterized in that: Deposited with the China Center for Type Culture Collection, deposit number CCTCC M 20242962.

2. Use of Klebsiella oxytoca G-17 according to claim 1 in reducing pentavalent vanadium.

3. Vanadium toxicity remover, characterized in that: Its active ingredient includes Klebsiella oxytoca G-17 according to claim 1.

4. A method for reducing pentavalent vanadium, characterized in that, Comprising the following steps: adding Klebsiella oxytoca G-17 according to claim 1 to water or soil containing pentavalent vanadium.

5. The method for reducing pentavalent vanadium according to claim 4, wherein: In water containing vanadium(V), the concentration of vanadium(V) is less than 2000 mg·L -1 .

6. The method for reducing pentavalent vanadium according to claim 5, characterized in that: In the water body containing vanadium pentoxide, the concentration of vanadium pentoxide is 500 mg·L -1 .

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