CN114317369B - Bacillus and application thereof in reduction of pentavalent vanadium and hexavalent chromium - Google Patents
Bacillus and application thereof in reduction of pentavalent vanadium and hexavalent chromium Download PDFInfo
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Abstract
The invention relates to bacillus and application thereof in reduction of pentavalent vanadium and hexavalent chromium, belonging to the technical field of microorganisms. The first technical problem solved by the invention is to provide a bacterium capable of reducing vanadium and chromium simultaneously, namely bacillus Bacillus amyloliquefaciens SM, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: m20211038. The bacillus Bacullus amyloliquefaciens SM01 has higher tolerance to vanadium and chromium in the environment, and the tolerance concentration to the vanadium and the chromium is up to 1200 mg.L ‑1 And 1000 mg.L ‑1 The bacteria can completely reduce hexavalent chromium, realize 98.42+/-0.11% reduction rate on pentavalent vanadium, have high reduction rate and simple operation flow, are convenient and efficient, and lay a foundation for the vanadium and chromium pollution bioremediation technology.
Description
Technical Field
The invention relates to bacillus and application thereof in reduction of pentavalent vanadium and hexavalent chromium, belonging to the technical field of microorganisms.
Background
Chinese mineral resources are rich, and although the mineral resources have important promotion effect on the social and economic development, heavy metals and other harmful substances are released in the development process of the resources, so that the ecological environment is damaged. Excessive heavy metal content in soil and water can reduce productivity and quality of soil, and on one hand, influence plant growth, and on the other hand, influence functional circulation of microbial community diversity, metabolic potential and related ecological systems. Heavy metals exist in the environment for a long time, and the heavy metals can be enriched into the human body through a food chain to finally threaten the human body health. Acute inhalation of vanadium, for example, may cause bronchopneumonia, chronic inhalation may cause damage to the nervous system and respiratory system, and severe may lead to toxic kidney disease and abnormal protein metabolism. Exposure to chromium can lead to kidney disease and inhibition of expression of related genes may induce cancer.
Vanadium is a transition metal element with an atomic number of 23 and is widely present in the crust. Has excellent characteristics of high melting point, hard texture, good toughness and the like, and is widely used in industrial production, such as metallurgy, atomic energy industry, chemistry, textile and other fields. With the development of science and technology, there is an increasing demand for vanadium products, the combustion of vanadium-containing fuels and the extraction and smelting of vanadium-containing minerals producing a large amount of vanadium, which migrates into the environment through the atmosphere and in the form of solutions. Vanadium is present in the natural water domain in the +3, +4 and +5 oxidation states. The vanadate species pentavalent vanadium is thermodynamically stable under oxidizing conditions, while tetravalent vanadium is stable under sub-oxygen conditions, trivalent vanadium being found in anoxic environments. The toxicity of vanadium increases with the increase of valence state, and the toxicity of pentavalent vanadium is highest.
Chromium, as a common contaminant in soil, has several oxidation states, from-2 to +6. Wherein the element chromium Cr (0) is inert in biological materials and does not naturally exist in soil. Chromium pollution is mainly caused by artificial activities, and besides mineral exploitation and smelting, serious chromium pollution is caused by the discharge of industrial wastewater of tanneries, paper mills and the like. In nature, chromium generally exists in two forms, trivalent chromium Cr (III) and hexavalent chromium Cr (VI), and can be converted to each other under specific conditions. Trivalent chromium is relatively stable, is one of trace elements necessary in organisms, is easy to form precipitation in water, and achieves the purpose of removing by solid-liquid separation; hexavalent chromium has high toxicity, strong migration capability and strong oxidizing property, is one of recognized carcinogens, and has toxicity about 100 times that of trivalent chromium. Therefore, reduction of Cr (VI) with strong toxicity to Cr (III) is a major approach to reduce chromium pollution in the environment.
Since vanadium and chromium both belong to the valence-changing element and are located in the same main group of the periodic table of elements. High-valence metals are far more toxic than low-valence metals, so groundwater treated with vanadium and chromium pollution is usually reduced to reduce the harm of both metals to the environment. However, the physical and chemical restoration technology adopted at present has high cost and can cause secondary pollution to the environment. Most chemical materials have good reduction effect under laboratory operation conditions, but the conditions in the actual environment are complex, and further research is needed to achieve ideal restoration effect in the actual environment.
Bioremediation techniques are mainly classified into microbial and phytoremediation, and are of great interest in the field of environmental remediation due to their simplicity of operation and environmental friendliness. Microorganisms and plants absorb or reduce high-valence metals through autogenous function metabolism, so that the repair of heavy metals in soil and water is realized. Certain specific microorganisms are capable of reducing the content of heavy metals in the environment by adsorption, reduction and intracellular bioaccumulation on the cell surface. Extracellular polysaccharides present on the cell surface have specific functional groups such as amino groups, carboxyl groups, hydroxyl groups, etc., which are capable of absorbing metal cations or oxyanions by electrostatic force; under the action of an external electron donor, the microorganism conducts electrons through enzyme of the microorganism, heavy metals are taken as final electron acceptors, and the heavy metals are reduced into low-valence metal cations; microorganisms absorb heavy metal ions through active transport, and these ions are bound to lipids in cells to be retained in the cells. Through the three processes, the microorganisms can effectively repair heavy metals in the environment. In addition, the microorganisms come from the polluted area, the microorganisms screened in situ can be better suitable for the local environment, and the addition of the microorganisms can coordinate the in-situ microbial community structure, so that the in-situ repair of heavy metals can be better realized.
The Chinese patent application No. 201910681909.0 discloses a white bacillus strain and application thereof in hexavalent chromium reduction, wherein the tolerance of the strain to hexavalent chromium reaches 30g/L, and the culture of the strain LYC-2 and the hexavalent chromium reduction process can be performed under aerobic, normal pressure, a wider temperature range (25-35 ℃) and a wider pH range (pH 7-11) conditions, so that high-toxicity hexavalent chromium can be effectively reduced to low-toxicity trivalent chromium.
The Chinese patent application No. 201610723745.X discloses bacillus with tolerance to vanadium and its use, the bacillus has high tolerance to both heavy metals such as Cd, cr, pb, ni, co and Zn, and the tolerance to vanadium is up to 2000mg/L. Meanwhile, the strain has the capability of reducing pentavalent vanadium into tetravalent vanadium in a basic culture medium, the conversion rate is 46.7%, the characteristic of reducing the toxicity of the pentavalent vanadium in the solution can be achieved, and the strain can be especially used for developing a microbial inoculum for removing the toxicity of vanadium in a high-vanadium-toxicity liquid environment containing other heavy metal pollutants and has good application prospect in bioremediation of heavy metal polluted environments.
It can be seen that at present, no good practical solution exists for the research on the biological reduction of two valence-changing elements vanadium and chromium. The prior disclosed bacteria can rarely reduce vanadium and chromium at the same time, and the reduction rate of vanadium is not high, so that the bacteria need to be further improved.
Disclosure of Invention
In view of the above drawbacks, a first technical problem to be solved by the present invention is to provide a bacterium capable of reducing vanadium and chromium simultaneously, namely bacillus Bacillus amyloliquefaciens SM01.
The bacillus Bacillus amyloliquefaciens SM disclosed by the invention is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: m20211038, the preservation date is 2021, 8 and 16.
In one embodiment of the present invention, the nucleotide sequence of 16s rDNA of the Bacillus Bacillus amyloliquefaciens SM01 is shown in SEQ ID No. 1.
The second technical problem solved by the invention is to provide the application of the bacillus Bacillus amyloliquefaciens SM01 in reducing pentavalent vanadium or hexavalent chromium.
The bacillus Bacillus amyloliquefaciens SM disclosed by the invention can reduce pentavalent vanadium into tetravalent vanadium and hexavalent chromium into trivalent chromium, and has a good function of reducing pentavalent vanadium and hexavalent chromium, wherein the reduction rate of the pentavalent vanadium can reach 98.42+/-0.11%, the reduction rate of the hexavalent chromium can reach 100%, and the reduction rate is high.
The invention also provides a vanadium-chromium toxicity remover.
The active ingredient of the vanadium-chromium toxicity remover comprises bacillus Bacillus amyloliquefaciens SM01.
The invention also provides a method for simultaneously reducing vanadium and chromium.
The method for simultaneously reducing vanadium and chromium comprises the following steps: the bacillus Bacillus amyloliquefaciens SM01 disclosed by the invention is added into water or soil containing pentavalent vanadium and hexavalent chromium.
In one embodiment of the invention, citric acid is added as an electron donor.
Compared with the prior art, the invention has the beneficial effects that:
1. the bacillus sp SM01 has higher tolerance to vanadium and chromium in the environment, and the tolerance concentration to the vanadium and the chromium is up to 1200 mg.L -1 And 1000 mg.L -1 Can adapt to the living condition of the high background pollution area. And it is the first report Bacillus amyloliquefaciens SM01 that can reduce pentavalent vanadium to tetravalent vanadium at the same timeAnd hexavalent chromium to trivalent chromium, thereby providing a bioremediation technique for vanadium and chromium pollution.
2. According to the invention, bacillus amyloliquefaciens SM01 is simply cultured by adopting LB liquid medium for 16-20 hours to grow to a logarithmic phase, and citric acid is added as a nutrient substance and an electron donor to reduce pentavalent vanadium and hexavalent chromium by bacteria after washing.
3. The vanadium-chromium toxicity remover disclosed by the invention can completely reduce hexavalent chromium, realizes 98.42+/-0.11% reduction rate on pentavalent vanadium, has the reduction rate far higher than the reduction rate of bacteria on pentavalent vanadium and hexavalent chromium in the previous report, and has the advantages of convenience and high efficiency.
Detailed Description
The invention collects the soil in the area with serious vanadium and chromium pollution near the Panzhihua vanadium titano-magnetite smelter, and obtains a strain of bacteria by separation and purification, and the bacteria has 100 percent homology with bacillus Bacillus amyloliquefaciens and is named Bacillus amyloliquefaciens SM01 after morphological and 16S rDNA identification. The bacteria have submitted to China Center for Type Culture Collection (CCTCC) for biological preservation at 2021, 8 months and 16 days, with the preservation number of CCTCC NO: m20211038. The Chinese typical culture collection is addressed to China, the university of Wuhan, and Wuhan.
The research shows that the bacteria has higher tolerance to vanadium and chromium in the environment, and the tolerance concentration to vanadium and chromium is as high as 1200 mg.L -1 And 1000 mg.L -1 Can adapt to the living condition of the high background pollution area. The bacteria can reduce pentavalent vanadium into tetravalent vanadium, reduce hexavalent chromium into trivalent chromium, and provide a bioremediation technology for vanadium and chromium pollution.
Suitable culture conditions for the bacteria are: 10g/L tryptone, 5g/L yeast, 10g/L sodium chloride and pH 7.2.
In one embodiment of the present invention, the nucleotide sequence of 16s rDNA of the Bacillus Bacillus amyloliquefaciens SM01 is shown in SEQ ID No. 1. After the 16S rDNA of the bacterium was extracted and subjected to PCR amplification sequencing, the nucleotide sequence of the 16S rDNA of the bacterium was found to be shown as SEQ ID No.1, and the bacterium had 100% homology with Bacillus Bacillus amyloliquefaciens, and therefore, the strain was named Bacillus amyloliquefaciens SM.
The bacillus Bacillus amyloliquefaciens SM disclosed by the invention can reduce pentavalent vanadium into tetravalent vanadium and hexavalent chromium into trivalent chromium, and has a good function of reducing pentavalent vanadium and hexavalent chromium, wherein the reduction rate of the pentavalent vanadium can reach 98.42+/-0.11%, the reduction rate of the hexavalent chromium can reach 100%, and the reduction rate is high.
The active ingredient of the vanadium-chromium toxicity remover comprises bacillus Bacillus amyloliquefaciens SM01.
The method for simultaneously reducing vanadium and chromium comprises the following steps: the bacillus Bacillus amyloliquefaciens SM01 disclosed by the invention is added into water or soil containing pentavalent vanadium and hexavalent chromium.
In one embodiment of the invention, citric acid is added as an electron donor.
In one embodiment of the invention Bacillus amyloliquefaciens SM01, which grows to log phase, is added, the inoculum size of Bacillus amyloliquefaciens SM01 is 1%.
The following describes the invention in more detail with reference to examples, which are not intended to limit the invention thereto.
Example 1Bacillus amyloliquefaciens SM01 isolation and purification and identification
Selecting a region with serious vanadium and chromium pollution near a Panzhihua vanadium titano-magnetite smelting plant, wherein the content of vanadium and chromium in local soil can reach 4156.47 mg.kg according to the test result -1 And 1377.15 mg.kg -1 。
1. Isolation and purification of bacteria
(1) 1g of soil in a polluted area is selected and added into a conical flask containing 99mL of sterilized deionized water, after shaking for two hours at 38 ℃ and 150rpm, 1mL of supernatant is taken and added into 9mL of sterile water, the steps are uniformly mixed, and the steps are repeated to obtain 10 percent of stock solution respectively -1 ，10 -2 ，10 -3 ，10 -4 ，10 -5 Take 10 -3 ，10 -4 ，10 -5 The concentration of the stock solution is coated in LB solid medium, and the stock solution is cultured for 24 hours at the constant temperature of 30 ℃.
(2) Selecting single bacteria in the solid culture medium in the step (1), and inoculating again to 100 mg.L of vanadium and chromium -1 Culturing in solid culture medium at 30deg.C for 24 hr.
(3) Inoculating the bacteria with good growth condition in the step (2) to the concentration of vanadium and chromium respectively being 300 mg.L -1 And 100 mg.L -1 Is cultured at a constant temperature of 30 ℃ for 24 hours.
(4) Inoculating the bacteria with good growth condition in the step (3) to the concentration of vanadium and chromium of 500 mg.L respectively -1 And 300 mg.L -1 Is cultured at a constant temperature of 30 ℃ for 24 hours.
(5) Inoculating the bacteria with good growth condition in the step (4) to the concentration of vanadium and chromium of 700 mg.L respectively -1 And 500 mg.L -1 Is cultured at a constant temperature of 30 ℃ for 24 hours.
(6) Inoculating the bacteria with good growth condition in the step (5) to the concentration of vanadium and chromium of 900 mg.L respectively -1 And 700 mg.L -1 Is cultured at a constant temperature of 30 ℃ for 24 hours.
(7) With increasing inoculation concentration, a resistant bacterium, designated SM01, was finally obtained. The bacteria are streaked and purified on a plate without heavy metals, single colony is selected and inoculated into LB liquid medium, and the bacteria are cultured for 20 hours at 30 ℃ and 150 rpm.
The formula of the LB solid medium is as follows: to 1L of water was added 10g of tryptone, 5g of yeast, 10g of sodium chloride and 15g of agar, and the pH was adjusted to 7.2.
LB liquid medium formula: to 1L of water was added 10g of tryptone, 5g of yeast and 10g of sodium chloride, and the pH was adjusted to 7.2.
2. Identification of bacteria
SM01 colony is round, milky white, smooth and moist on the surface and sticky. Bacteria were gram-stained purple and in the shape of a short bar, gram-positive bacteria.
After bacterial 16S rDNA is extracted, PCR amplification and sequencing are carried out, the general primers are 27F (SEQ ID No.2, AGTTTGATCMTGGCTCAG) and 1492R (SEQ ID No.3, GGTTACCTTGTTACGACTT), and the amplification length is about 1500 bp. The PCR reaction procedure was: (1) pre-denaturation at 94℃for 5min; (2) denaturation at 94℃for 30s; (3) annealing at 54 ℃ for 30s; (4) extending at 72℃for 1min 30s; repeating the steps (2), (3) and (4) 39 times (5) extending for 10min at 72 ℃; preserving at 4 ℃. The amplified PCR products were subjected to agarose gel electrophoresis (2 uL sample+6 uL bromophenol blue) at 300V for 12min. And (3) sending the PCR product to Beijing engine biotechnology limited company for sequencing, splicing the sequencing result by using Contigexpress, removing inaccurate parts at two ends, comparing the spliced sequences in an NCBI database (blast.ncbi.lm.nih.gov), and carrying out systematic development construction by adopting MEGA software.
SM01 has 100% homology with bacillus Bacillus amyloliquefaciens, and this bacterium is designated Bacillus amyloliquefaciens SM01.
EXAMPLE 2 tolerance concentration test
Bacillus amyloliquefaciens SM01 was inoculated to a concentration of 1000 mg.L containing vanadium by continuous streaking -1 ，1100mg·L -1 ，1200mg·L -1 ，1300mg·L -1 The concentration of chromium is 900 mg.L -1 ，1000mg·L -1 ，1100mg·L -1 ，1200mg·L -1 Is cultured at a constant temperature of 30 ℃ for 24 hours. Finally, the concentration of vanadium and chromium is 1200 mg.L -1 And 1000 mg.L -1 The bacterial growth gradually weakens. Shows that the tolerance concentration of the strain of bacteria to vanadium and chromium reaches 1200 mg.L respectively -1 And 1000 mg.L -1 。
Example 3 test of reduction Effect
Inoculating Bacillus amyloliquefaciens SM01 into LB liquid medium, culturing for 20 hr at 30deg.C and 150rpm, washing with sterile water twice, and regulating OD 600 1. Added to a feed containing 50 mg.L according to an inoculum size of 1% -1 V (V) and 50mg.L -1 Adding citric acid as electron donor into Cr (VI) solution, culturing at 38deg.C and 150rpm for 24 hr, centrifuging at 4000rpm, filtering supernatant, and measuring pentavalent vanadium and hexavalent chromium concentration in the solution by ultraviolet spectrophotometry, ICP-OESAnd measuring the content of all vanadium and all chromium. And meanwhile, an inactivated bacteria group control is arranged to determine whether bacteria have adsorption effect. The results are shown in Table 1:
TABLE 1
Where "-" indicates no data, the data were also very different after dilution because the concentration was too high and the spectrophotometric concentration was inaccurate.
The result shows that Bacillus amyloliquefaciens SM01 can completely reduce hexavalent chromium, and the reduction rate of pentavalent vanadium is 98.42+/-0.11%. The method for calculating the reduction rate comprises the following steps: r is R e ＝(c (0) -c)/c (0)
c is the concentration of pentavalent vanadium and hexavalent chromium in the reacted solution; c (0) Is the initial pentavalent vanadium and hexavalent chromium concentration in the solution.
Therefore, the method Bacillus amyloliquefaciens SM01 can adapt to high-concentration vanadium and chromium, can well reduce hexavalent chromium and pentavalent vanadium, and has high reduction rate.
Claims (5)
1. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SM01, characterized in that: the culture medium is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: m20211038.
2. Use of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SM01 according to claim 1 for reducing pentavalent or hexavalent chromium.
3. The vanadium-chromium toxicity remover is characterized in that: an active ingredient comprising bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SM01 according to claim 1.
4. A method for simultaneously reducing vanadium and chromium, comprising the steps of: the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SM01 of claim 1 is added to a body of water or soil containing pentavalent vanadium and hexavalent chromium.
5. The method for simultaneously reducing vanadium and chromium according to claim 4 wherein: citric acid is added into water or soil containing pentavalent vanadium and hexavalent chromium as an electron donor.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009207404A (en) * | 2008-03-04 | 2009-09-17 | Central Res Inst Of Electric Power Ind | Microorganism reducing hexa-valent chromium, and method for environmental clean-up utilizing the same microorganism |
| CN106399153A (en) * | 2016-08-25 | 2017-02-15 | 成都理工大学 | Bacillus with tolerance to vanadium and use thereof |
| CN106676045A (en) * | 2017-02-16 | 2017-05-17 | 武汉科技大学 | Antibiotic-resistant hexavalent chromium reducing bacterium and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009207404A (en) * | 2008-03-04 | 2009-09-17 | Central Res Inst Of Electric Power Ind | Microorganism reducing hexa-valent chromium, and method for environmental clean-up utilizing the same microorganism |
| CN106399153A (en) * | 2016-08-25 | 2017-02-15 | 成都理工大学 | Bacillus with tolerance to vanadium and use thereof |
| CN106676045A (en) * | 2017-02-16 | 2017-05-17 | 武汉科技大学 | Antibiotic-resistant hexavalent chromium reducing bacterium and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Xue Tang等.The response of bacterial communities to V and Cr and novel reducing bacteria near a vanadium‑titanium magnetite refinery.《Science of the Total Environment》.2021,第806卷第1-23页. * |
| 王洪婷.两株耐钒细菌的V(V)还原特性及其基因组学研究.《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》.2021,第1-90页. * |
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