Bacillus aryabhattai and application thereof in reduction of pentavalent vanadium

Abstract

The invention relates to bacillus aryabhattai and application of the bacillus aryabhattai to 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 bacillus aryabhattai ST-1, the bacterial strain is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20242964. The Pristia aryabhattai ST-1 has high tolerance to vanadium, iron and chromium in the environment, can adapt to survival conditions of high background pollution areas, can survive in soil and water environments, is simple in operation process for reducing pentavalent vanadium, has high reduction rate under the condition that electron donors do not need to be added and high-concentration vanadium can be achieved, and can be applied to the field of industrial production. On one hand, cost reduction is facilitated, and on the other hand, secondary pollution can be The bacillus aryabhattai ST-1 disclosed by the invention still has a relatively good removal effect on vanadium under different vanadium concentration conditions, is convenient for removing vanadium toxic substances in various extremely polluted water bodies, and has the advantages of simplicity, convenience and high efficiency.

Description

Technical Field

[0001] The present invention relates to Bacillus aryabhattai and its use for reducing pentavalent vanadium, and belongs to the field of microbial technology. Background Art

[0002] Mining, especially vanadium ore mining, has become an important industry in many regions of the world. However, the mining process is often accompanied by the release of a large number of heavy metal pollutants, posing a great threat to the ecological environment and human health. When vanadium ore (such as vanadium-titanium magnetite) is mined, wastewater, waste gas and solid waste containing pentavalent vanadium (V 5 +) are often produced. These pollutants not only affect the surrounding soil, water and air quality, but may also accumulate through the food chain and ultimately harm the health of organisms. Vanadium is a transition metal element with an atomic number of 23 and is 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 industrial production, such as metallurgy, atomic energy industry, chemistry and textile 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 natural waters in the oxidation states of +3, +4 and +5. The toxicity of vanadium increases with the increase of its valence state, and pentavalent vanadium has the highest toxicity.

[0003] There are usually two methods for treating and repairing heavy metal pollution: one is to use physical, chemical and biological methods to change the existing form of pollutants in soil and water, so that they are fixed or precipitated, reducing their migration ability and bioavailability in the environment and no longer expanding the pollution range; the other is to completely remove the pollutants from soil and water so that their residual concentration is close to or reaches the background value. Generally speaking, there are mainly three categories: physical, chemical and biological remediation. Biological remediation technology is mainly divided into microbial and plant remediation. Because of its simple operation and environmental friendliness, it has received extensive attention in the field of environmental governance. Compared with plants and animals, microorganisms can resist environmental stress through rapid mutation and evolution. Many microorganisms, especially some metal-tolerant microorganisms, can grow in an environment containing pentavalent vanadium and reduce it to tetravalent vanadium (VI). Tetravalent vanadium has low water solubility and usually exists in the form of insoluble oxides, which makes it not easy to enter water or soil and reduces its toxicity. Therefore, screening and applying microorganisms that can tolerate pentavalent vanadium and carry out reduction transformation can not only effectively reduce the environmental pollution of vanadium, but also provide a new idea for the ecological restoration of vanadium mining areas.

[0004] Patent CN114317369A discloses a Bacillus amyloliquefaciens SM01 that can reduce pentavalent vanadium, and patent CN 114292792A 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, and when these strains are applied, electron donors such as citric acid need to be added. Therefore, a new strain that can simply 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 Priestia aryabhattai ST-1.

[0006] Priestia aryabhattai ST-1 of the present invention is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20242964, and the deposit date is December 31, 2024.

[0007] The second technical problem solved by the present invention is to provide the application of Priestia aryabhattai ST-1 of the present invention in reducing pentavalent vanadium.

[0008] Priestia aryabhattai ST-1 of the present invention can reduce pentavalent vanadium to tetravalent vanadium, has a good function of reducing pentavalent vanadium, has a high tolerance to vanadium, chromium, and iron, and the reduction method is simple and does not require additional addition of electron donors. On the one hand, it is beneficial to reduce costs, and on the other hand, it can reduce secondary pollution.

[0009] The present invention also provides a vanadium toxicity remover.

[0010] The vanadium toxicity remover of the present invention has an active ingredient including Priestia aryabhattai ST-1 described in claim 1.

[0011] The present invention also provides a method for reducing pentavalent vanadium.

[0012] The method for reducing pentavalent vanadium of the present invention includes the following steps: introducing the above-mentioned Priestia aryabhattai ST-1 into the environment polluted by vanadium.

[0013] In one embodiment of the present invention, the introduction method is to directly introduce the bacterial liquid of Priestia aryabhattai ST-1 grown to the logarithmic phase or a preparation with Priestia aryabhattai ST-1 as the main active ingredient into the liquid environment for growth.

[0014] In one embodiment of the present invention, the pH value of the liquid environment is 5 - 11.

[0015] In one embodiment of the present invention, in the liquid environment, the concentration of pentavalent vanadium is less than 2000 mg·L -1 。

[0016] In some specific embodiments of the present invention, in the liquid environment, the concentration of pentavalent vanadium is 50 - 1000 mg·L -1 。

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. Priestia aryabhattai ST-1 of the present invention has high tolerance to vanadium, iron, and chromium in the environment. The tolerance concentration to vanadium is as high as 2000 mg·L -1 , the tolerance concentration to iron is as high as 400 mg·L -1 , and the tolerance concentration to chromium is 100 mg·L -1 . It can adapt to the survival conditions in high-background pollution areas and can survive in soil and water environments. This is the first report that Priestia aryabhattai ST-1 can reduce pentavalent vanadium to tetravalent vanadium, providing a bioremediation technology for vanadium pollution.

[0019] 2. The present invention only needs to simply culture Priestia aryabhattai ST-1 using NB liquid medium and then can carry out the reduction of pentavalent vanadium. The operation process is simple. Without adding an electron donor, a high reduction rate can also be achieved under high-concentration vanadium conditions. On the one hand, it is beneficial to reduce costs, and on the other hand, it can reduce secondary pollution.

[0020] 3. Priestia aryabhattai ST-1 of the present invention still has a good removal effect on vanadium under different vanadium concentration conditions, facilitating the removal of vanadium toxic substances in various extremely polluted waters, and having the advantages of simplicity, convenience, and high efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a colony photo of the strain Priestia aryabhattai ST-1 of the present invention.

[0022] Figure 2 This is the phylogenetic tree of the strain Priestia aryabhattai ST-1 of the present invention. Detailed implementation manners

[0023] In the present invention, soil samples were collected from the periphery of the Baoding mining area in the west district of Panzhihua City, Sichuan Province. A strain of bacteria was obtained through isolation and purification. Through morphological and 16S rDNA identification, this bacterium has 99% homology with Bacillus Priestia aryabhattai and is named Priestia aryabhattai ST-1. This bacterium was deposited with the China Center for Type Culture Collection on December 31, 2024, and the deposit number is CCTCC NO: M 20242964. The address of the China Center for Type Culture Collection is Wuhan University, China, Wuhan.

[0024] Research has found that this bacterium has high tolerance to vanadium and iron in the environment. The tolerance concentration to vanadium is as high as 2000 mg·L -1 , and the tolerance concentration to iron is as high as 400 mg·L -1 , and it can adapt to the survival conditions in high-background pollution areas and can survive in soil and water environments. Moreover, this bacterium can reduce pentavalent vanadium to tetravalent vanadium with a high reduction rate, providing a bioremediation technology for vanadium pollution.

[0025] The suitable culture conditions for this bacterium are: tryptone 10 g / L, yeast 5 g / L, sodium chloride 10 g / L, pH 7.0. Experiments have proved that a highly vanadium-tolerant microbial reducing agent can be prepared using this strain for the treatment of vanadium wastewater.

[0026] In an embodiment of the present invention, the nucleotide sequence of the 16S rDNA of the Bacillus aryabhattai Priestia aryabhattai ST-1 is shown as 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 shown as SEQ ID No.1, and this bacterium has 99% homology with Bacillus Priestiaaryabhattai. Therefore, this strain is named Priestia aryabhattai ST-1.

[0027] The Bacillus aryabhattai Priestia aryabhattai ST-1 of the present invention can reduce pentavalent vanadium to tetravalent vanadium, and the reduction effect of pentavalent vanadium is good.

[0028] The vanadium toxicity remover of the present invention has an active ingredient comprising the Bacillus aryabhattai Priestia aryabhattai ST-1 of the present invention.

[0029] The method for reducing vanadium in the present invention includes the following steps: introducing the above-mentioned Priestia aryabhattai ST-1 into the environment polluted by vanadium.

[0030] In one embodiment of the present invention, the introduction method is to directly introduce the bacterial solution of Priestia aryabhattai ST-1 grown to the logarithmic phase or a preparation with Priestia aryabhattai ST-1 as the main active ingredient into the liquid environment for growth.

[0031] In one embodiment of the present invention, the bacterial solution can be prepared by the following method: culturing the above-mentioned Priestia aryabhattai ST-1 in a basal medium with a formula of tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and pH 7.0. When it grows to the logarithmic phase, introduce the logarithmic-phase bacterial solution into the vanadium-polluted liquid environment to achieve the purpose of removing the toxicity of vanadium in the environment.

[0032] In one embodiment of the present invention, the pH value of the liquid environment is 5 - 11.

[0033] In one embodiment of the present invention, the treatment temperature is 10 - 40 °C, and the preferred treatment temperature is about 30 °C.

[0034] In one embodiment of the present invention, in the liquid environment, the concentration of pentavalent vanadium is less than 2000 mg·L -1 。

[0035] In some specific embodiments of the present invention, in the liquid environment, the concentration of pentavalent vanadium is 50 - 1000 mg·L -1 。

[0036] The following further describes the specific embodiments of the present invention in conjunction with the examples, and the present invention is not limited to the scope of the described examples.

[0037] Example 1 Isolation, purification and identification of Priestia aryabhattai ST-1

[0038] Collect soil samples around the Baoding mining area in the west district of Panzhihua City, Sichuan Province for isolation and purification. The specific steps are as follows:

[0039] 1. Isolation and purification of bacteria

[0040] (1) Weigh 10 g of soil sample and pour it into a conical flask. Then add 90 ml of sterile water and stir evenly to prepare a soil suspension. After mixing, place it in a constant temperature shaker at 30 °C and 180 r / min for shaking culture for 30 min, and let it stand for 10 min. Pipette 2 ml of the upper suspension and add it to a 250 ml stoppered conical flask containing 100 ml of pre-prepared liquid medium, and culture it at 150 r / min and 30 °C for 3 - 4 d for constant temperature expansion culture.

[0041] (2) Take 1 ml of the liquid medium cultured in (1) for 2 d and add it to 99 ml of liquid medium containing 200 mg / L of heavy metal vanadium, and culture it at 30 °C and 150 rpm for 2 d.

[0042] (3) Take 1 ml of the liquid medium cultured in (2) for 2 d and add it to 99 ml of liquid medium containing 400 mg / L of heavy metal vanadium, and culture it at 30 °C and 150 rpm for 2 d.

[0043] (4) Take 1 ml of the liquid medium cultured in (3) for 2 d and add it to 99 ml of liquid medium containing 800 mg / L of heavy metal vanadium, and culture it at 30 °C and 150 rpm for 2 d.

[0044] (5) Take 1 ml of the liquid medium cultured in (4) for 2 d and add it to 99 ml of liquid medium containing 1000 mg / L of heavy metal vanadium, and culture it at 30 °C and 150 rpm for 2 d.

[0045] (6) Take 1 ml of the liquid medium cultured in (5) for 2 d and add it to 99 ml of liquid medium containing 1200 mg / L of heavy metal vanadium, and culture it at 30 °C and 150 rpm for 2 d.

[0046] (7) Take 1 ml of the liquid medium cultured in (6) for 2 d and add it to 99 ml of liquid medium containing 1400 mg / L of heavy metal vanadium, and culture it at 30 °C and 150 rpm for 2 d.

[0047] (8) Take 1 ml of the liquid medium cultured in (7) for 2 d and add it to 99 ml of liquid medium containing 1800 mg / L of heavy metal vanadium, and culture it at 30 °C and 150 rpm for 2 d.

[0048] After appropriately diluting the bacterial liquid, coat it on a solid medium with a higher vanadium concentration and culture it in an inverted position for 3 d for re-screening. Use an inoculation loop to observe and pick different morphological single colonies and continue plate streaking. Repeat several times until pure colonies are obtained. Name it ST-1.

[0049] The formula of the above NB solid medium is: 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.

[0050] NB liquid medium formulation: 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.

[0051] 2. Bacterial identification

[0052] The colony morphology of ST-1 is round with neat edges, milky white, and smooth surface. The bacteria were Gram-stained and were purple, and had a short rod-shaped appearance, being Gram-positive bacteria. The colony photo is shown in Figure 1 .

[0053] 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 program 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 sample + 6 uL 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 spliced with ContigExpress, and the inaccurate parts at both ends were removed. The spliced sequence was compared in the NCBI database (blast.ncbi.nlm.nih.gov), and phylogenetic construction was performed using MEGA software. The results are shown in Figure 2 .

[0054] ST-1 has 99% homology with Bacillus Priestia aryabhattai, and this bacterium was named Priestia aryabhattai ST-1.

[0055] Example 2 Tolerance concentration test

[0056] The single bacterium Priestia aryabhattai ST-1 obtained in Example 1 was inoculated into NB liquid medium and cultured with shaking at 30 °C and 150 rpm for 24 h. The bacterial solution was collected, and the bacterial solution was diluted according to the ratios of 1, 10, 100, 1000, 10000 to prepare gradient bacterial solutions. The method of continuous streaking was used to inoculate them into the medium containing vanadium at concentrations of 1000 mg·L -1 , 1200 mg·L -1 , 1400 mg·L -1 , 1600 mg·L -1 , 1800 mg·L -1 , 2000 mg·L-1 with an iron concentration of 100 mg·L -1 ,200 mg·L -1 ,300 mg·L -1 ,400 mg·L -1 with a chromium concentration of 10 mg·L -1 ,50 mg·L -1 ,100 mg·L -1 ,cultured at a constant temperature of 30 °C for 48 h. Finally, at a vanadium concentration of 2000 mg·L -1 ,an iron concentration of 400 mg·L -1 ,a chromium concentration of 100 mg·L -1 the bacterial growth gradually weakened. It shows that the tolerance concentration of this strain of bacteria to vanadium reaches 2000 mg·L -1 ,the tolerance concentration of iron is 400 mg·L -1 ,the tolerance concentration of chromium is 100 mg·L -1 .

[0057] Example 3 Reduction effect test under different vanadium conditions

[0058] Inoculate Priestia aryabhattai ST-1 into NB liquid medium and expand the culture for 12 h at 30 °C and 150 rpm. Add the bacterial liquid to the NB medium containing 50 mg·L -1 ,100 mg·L -1 ,500 mg·L -1 ,1000 mg·L -1 of V(V) at an inoculation amount of 10% by volume, culture at a constant temperature of 30 °C and 150 rpm for 48 h, centrifuge at 4000 rpm, take the supernatant, filter it, and determine the pentavalent vanadium concentration in the solution by ultraviolet spectrophotometry, and determine the total vanadium content by ICP-MS. At the same time, set a group without adding V(V) as a control to determine whether the NB medium will affect the determination of pentavalent vanadium by ultraviolet spectrophotometry. The results are shown in Table 1.

[0059] Table 1

[0060]

[0061] Among them, Bacillus amyloliquefaciens SM01 is Bacillus amyloliquefaciens in Patent CN114317369A, and Bacillus pacificus SM02 is Bacillus pacificus in Patent CN 114292792A.

[0062] The results showed that Priestia aryabhattai ST-1 could effectively reduce vanadium(V), and could well reduce high-concentration vanadium(V) without adding electron donors such as citric acid. When the initial vanadium(V) was 100 mg·L -1 , the highest reduction rate was 40.98 ± 0.99%.

[0063] Among them, the calculation method of the reduction rate in the present invention is: R e =(c (0) -c) / c (0)

[0064] where c is the concentration of vanadium(V) in the solution after the reaction; c (0) is the initial concentration of vanadium(V) in the solution.

[0065] It can be seen that Priestia aryabhattai ST-1 in the present invention can adapt to high-concentration vanadium, and can well reduce vanadium(V) at different concentrations, with good reduction effect.

Claims

1. Priestia aryabhattai ST-1, characterized in that: It is deposited in the China Center for Type Culture Collection, with the accession number being CCTCC NO: M 20242964, and the deposit date being December 31, 2024.

2. Use of Priestia aryabhattai ST-1 according to claim 1 in reducing pentavalent vanadium.

3. A vanadium toxicity remover, characterized in that: The active ingredient comprises the Priestia aryabhattai ST-1 described in claim 1.

4. A method for reducing pentavalent vanadium, characterized in that: The method comprises the following steps: introducing the Priestia aryabhattai ST-1 described in claim 1 into an environment contaminated by vanadium.

5. The method for reducing pentavalent vanadium according to claim 4, characterized in that: The introduction method is to directly introduce the Priestia aryabhattai ST-1 bacterial solution grown to the logarithmic phase or the preparation with the Priestia aryabhattai ST-1 as the main active ingredient into the liquid environment for growth.

6. The method for reducing pentavalent vanadium according to claim 5, characterized in that: The pH value of the liquid environment is 5-11.

7. The method for reducing pentavalent vanadium according to claim 5, characterized in that: In liquid environment, the concentration of pentavalent vanadium is less than 2000 mg·L -1 .

8. The method for reducing pentavalent vanadium according to claim 7, characterized in that: In liquid environment, the concentration of pentavalent vanadium is 50-1000 mg·L -1 .

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