CN114317369A - 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 amyloliquefaciens SM01, which is preserved in China center for type culture collection with a preservation number of CCTCC NO: m20211038. The bacillus Bacullus amyloliquefaciens SM01 has higher tolerance to vanadium and chromium in the environment and can resist vanadiumAnd chromium in tolerable concentrations of up to 1200 mg.L‑1And 1000 mg. L‑1The bacterium can adapt to the living conditions of high background pollution areas, can completely reduce hexavalent chromium, realizes the reduction rate of 98.42 +/-0.11% on pentavalent vanadium, has high reduction rate and simple operation process, is convenient and efficient, and lays a foundation for the bioremediation technology of vanadium and chromium pollution.
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
Mineral resources in China are abundant, and although mineral resources play an important role in promoting the development of socioeconomic 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 the soil, affecting on the one hand plant growth and on the other hand microbial community diversity, metabolic potential and functional cycling of the relevant ecosystem. Heavy metals exist in the environment for a long time, and the heavy metals are enriched into human bodies through food chains and finally threaten the health of human bodies. Acute inhalation of vanadium, for example, may cause bronchopneumonia, and chronic inhalation may cause damage to the nervous system and respiratory system, and may lead to toxic nephropathy and abnormal protein metabolism. Chromium exposure 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 earth crust. Because of its excellent properties of high melting point, hardness, toughness, etc., it is widely used in industrial production, such as metallurgy, atomic energy industry, chemistry and textile. With the development of scientific technology, the demand for vanadium products is increasing, and the combustion of vanadium-containing fuels and the extraction and smelting of vanadium-containing minerals produce large amounts of vanadium, which migrates to the environment in the form of atmosphere and solutions. Vanadium exists in natural waters in oxidation states of +3, +4, and + 5. The vanadate species pentavalent vanadium is thermodynamically stable under oxidizing conditions, whereas tetravalent vanadium is stable under sub-oxidizing conditions, and trivalent vanadium is found in anoxic environments. The toxicity of vanadium increases with the increase of valence, and the pentavalent vanadium has the highest toxicity.
Chromium, a common contaminant in soil, has several oxidation states ranging from-2 to + 6. Among them, the element chromium Cr (0) is inert in biological materials and does not occur naturally in soil. Chromium pollution is mainly caused by human activities, and besides mineral mining and smelting, the discharge of industrial wastewater of tanneries, paper mills and the like also causes serious chromium pollution. Chromium is usually present in nature in both trivalent chromium cr (iii) and hexavalent chromium cr (vi) forms and can be interconverted under specific conditions. Trivalent chromium is relatively stable, is one of essential trace elements in organisms, is easy to form precipitate in water, and realizes solid-liquid separation to achieve the aim of removing; hexavalent chromium is toxic, strong in migration capability and strong in oxidation, is one of accepted carcinogens, and has the toxicity about 100 times that of trivalent chromium. Therefore, the reduction of Cr (VI) with strong toxicity into Cr (III) is the main way to reduce the chromium pollution in the environment.
Since vanadium and chromium both belong to valence-changing elements and are located in the same main group of the periodic table of elements. The toxicity of the high-valence metal is far greater than that of the low-valence metal, so that the groundwater polluted by vanadium and chromium is generally treated by a reduction method to reduce the harm of the two to the environment. But the currently adopted physical and chemical remediation techniques are costly 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 find whether the ideal restoration effect can be achieved in the actual environment.
Bioremediation techniques are mainly divided into microbial and phytoremediation, and because of its simple operation and environmental friendliness, have received extensive attention in the field of environmental remediation. Microorganisms and plants metabolize and absorb or reduce high-valence metals through self-growing functions, so that the heavy metals in soil and water are repaired. Certain microorganisms are capable of reducing the levels of heavy metals in the environment by adsorption, reduction and intracellular biological accumulation on the cell surface. Exopolysaccharide existing on the cell surface has specific functional groups, such as amino, carboxyl, hydroxyl and the like, and the functional groups can absorb metal cations or oxygen anions through electrostatic force; under the action of an external electron donor, the microorganism conducts electrons through enzyme of the microorganism, and the heavy metal is used as a final electron acceptor and is reduced into metal cations with low valence states; microorganisms absorb heavy metal ions by active transport, and these ions are bound to lipids in cells and thus retained in the cells. Through the three processes, the microorganism can effectively realize the restoration of heavy metals in the environment. In addition, the microorganisms come from polluted areas, the microorganisms screened in situ can better adapt to local environments, and the addition of the microorganisms can coordinate the in situ microbial community structure and can better realize in situ remediation of heavy metals.
The Chinese invention patent with application number of 201910681909.0 discloses a strain of albobacter and application thereof in reducing hexavalent chromium, the tolerance capacity of the strain to hexavalent chromium reaches 30g/L, and the processes of culturing the strain LYC-2 and reducing hexavalent chromium can be carried out under the conditions of aerobism, normal pressure, a wider temperature range (25-35 ℃) and a wider pH range (pH7-11), so that the hexavalent chromium with high toxicity can be effectively reduced into trivalent chromium with low toxicity.
Chinese patent application No. 201610723745.X discloses a vanadium-tolerant bacillus having tolerance to Cd, Cr, Pb, Ni, Co, Zn and other heavy metals as high as 2000mg/L in addition to high vanadium tolerance and its use. 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 high-valent vanadium in the solution can be achieved, the strain can be particularly developed and used for removing the toxicity of the vanadium in a high-vanadium-toxicity liquid environment containing other heavy metal pollutants, and the strain has a good application prospect in bioremediation of heavy metal polluted environments.
Therefore, at present, no good practical scheme exists for the research on the biological reduction of two variable valence elements, namely vanadium and chromium. The currently disclosed bacteria rarely can simultaneously reduce vanadium and chromium, and the reduction rate of vanadium is not high, so that further improvement is needed.
Disclosure of Invention
In view of the above drawbacks, the first technical problem solved by the present invention is to provide a bacterium, i.e., Bacillus amyloliquefaciens SM01, capable of simultaneously reducing vanadium and chromium.
The Bacillus amyloliquefaciens SM01 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m20211038, preservation date 2021, 8 months and 16 days.
In one embodiment of the invention, the nucleotide sequence of 16s rDNA of Bacillus amyloliquefaciens SM01 is shown as SEQ ID No. 1.
The second technical problem to be solved by the invention is to provide the application of the Bacillus amyloliquefaciens SM01 in the reduction of pentavalent vanadium or hexavalent chromium.
The Bacillus amyloliquefaciens SM01 can reduce pentavalent vanadium into tetravalent vanadium and hexavalent chromium into trivalent chromium, and has good functions of reducing pentavalent vanadium and hexavalent chromium, wherein the reduction rate of pentavalent vanadium can reach 98.42 +/-0.11%, the reduction rate of 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 the Bacillus amyloliquefaciens SM 01.
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 amyloliquefaciens SM01 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 Bacullus sp SM01 has high tolerance to vanadium and chromium in the environment, and the tolerance concentration of the bacillus Bacullus sp SM01 to the vanadium and the chromium is up to 1200 mg.L-1And 1000 mg. L-1And the method can adapt to the survival conditions of high background pollution areas. And it is reported for the first time that Bacillus amyloliquefaciens SM01 can reduce pentavalent vanadium to tetravalent vanadium and hexavalent chromium to trivalent chromium simultaneously, and provide bioremediation technology for vanadium and chromium pollution.
2. According to the invention, Bacillus amyloliquefaciens SM01 is cultured for 16-20h to grow to a logarithmic phase by simply adopting an LB liquid culture medium, and after washing, citric acid is added as a nutrient substance and an electron donor for reducing pentavalent vanadium and hexavalent chromium by bacteria, so that the operation process is simple.
3. The vanadium-chromium toxicity remover can completely reduce hexavalent chromium, realizes the reduction rate of 98.42 +/-0.11% on pentavalent vanadium, has the reduction rate far higher than that of bacteria on pentavalent vanadium and hexavalent chromium reported before, and has the advantages of convenience and high efficiency.
Detailed Description
The method comprises the steps of collecting soil in a region with severe vanadium and chromium pollution near a Panzhihua vanadium-titanium magnetite smelting plant, separating and purifying to obtain a strain of bacteria, identifying through morphology and 16S rDNA, wherein the bacteria and Bacillus amyloliquefaciens have 100% homology and are named as Bacillus amyloliquefaciens SM 01. The bacterium has submitted biological preservation to China center for type culture Collection in 2021, 8 months and 16 days, and the preservation number is CCTCC NO: m20211038. The China center for type culture Collection is addressed to the university of Wuhan, China.
Researches show that the bacteria have high tolerance to vanadium and chromium in the environment, and the tolerance concentration of the bacteria to the vanadium and the chromium is up to 1200 mg-L-1And 1000 mg. L-1Survival strip capable of adapting to high background pollution areaAnd (3) a component. The bacteria can reduce pentavalent vanadium into tetravalent vanadium and hexavalent chromium into trivalent chromium, the reduction rate is high, and a bioremediation technology is provided for vanadium and chromium pollution.
Suitable culture conditions for the bacterium are: tryptone 10g/L, yeast 5g/L, sodium chloride 10g/L, pH 7.2.
In one embodiment of the invention, the nucleotide sequence of 16s rDNA of Bacillus amyloliquefaciens SM01 is shown as SEQ ID No. 1. After 16S rDNA of the bacterium is extracted, PCR amplification sequencing is carried out, and the nucleotide sequence of the 16S rDNA of the bacterium is shown in SEQ ID No.1, and the bacterium has 100% homology with Bacillus amyloliquefaciens, so that the strain is named as Bacillus amyloliquefaciens SM 01.
The Bacillus amyloliquefaciens SM01 can reduce pentavalent vanadium into tetravalent vanadium and hexavalent chromium into trivalent chromium, and has good functions of reducing pentavalent vanadium and hexavalent chromium, wherein the reduction rate of pentavalent vanadium can reach 98.42 +/-0.11%, the reduction rate of hexavalent chromium can reach 100%, and the reduction rate is high.
The active ingredient of the vanadium-chromium toxicity remover comprises the Bacillus amyloliquefaciens SM 01.
The method for simultaneously reducing vanadium and chromium comprises the following steps: the Bacillus amyloliquefaciens SM01 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 grown to log phase is added in an inoculum size of 1%.
The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.
Example 1 isolation, purification and characterization of Bacillus amyloliquefaciens SM01
Vanadium-titanium magnet for selecting PanzhihuaIn areas with severe vanadium and chromium pollution near iron ore smelting plants, the content of vanadium and chromium in local soil can reach 4156.47 mg-kg at most according to test results-1And 1377.15mg kg-1。
1. Isolation and purification of bacteria
(1) Selecting 1g of soil in a polluted area, adding the soil into a conical flask containing 99mL of sterilized deionized water, shaking the soil at 38 ℃ and 150rpm for two hours, adding 1mL of supernatant into 9mL of sterile water, uniformly mixing the supernatant and the sterile water, and repeating the steps to respectively obtain 10 percent of stock solution with concentration-1，10-2，10-3，10-4，10-5Take 10-3，10-4，10-5Coating the stock solution with the concentration of the stock solution into an LB solid culture medium, and culturing at the constant temperature of 30 ℃ for 24 hours.
(2) Respectively selecting individual bacteria in the solid culture medium in the step (1), and inoculating to the culture medium containing vanadium and chromium at a concentration of 100 mg.L-1Culturing in solid culture medium at 30 deg.C for 24 hr.
(3) Inoculating the bacteria with good growth in the step (2) to the vanadium and chromium with the concentration of 300 mg.L respectively-1And 100 mg. L-1The solid medium of (3) was incubated at 30 ℃ for 24 hours.
(4) Inoculating the bacteria with good growth vigor in the step (3) to the vanadium and chromium with the concentration of 500 mg.L respectively-1And 300 mg. L-1The solid medium of (3) was incubated at 30 ℃ for 24 hours.
(5) Inoculating the bacteria with good growth vigor in the step (4) to the vanadium and chromium with the concentration of 700 mg.L respectively-1And 500 mg. L-1The solid medium of (3) was incubated at 30 ℃ for 24 hours.
(6) Inoculating the bacteria with good growth vigor in the step (5) to the vanadium and chromium with the concentration of 900 mg.L respectively-1And 700 mg. L-1The solid medium of (3) was incubated at 30 ℃ for 24 hours.
(7) With increasing inoculation concentration, a tolerant bacterium, designated SM01, was finally obtained. The bacteria are streaked and purified on a heavy metal-free plate, and a single colony is selected and inoculated into an LB liquid culture medium and cultured for 20h at 30 ℃ and 150 rpm.
The LB solid culture medium comprises the following components in percentage by weight: to 1L of water were 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 were added 10g of tryptone, 5g of yeast and 10g of sodium chloride, and the pH was adjusted to 7.2.
2. Bacterial identification
The SM01 colony is round, milky white, smooth and moist in surface and sticky. The bacteria were gram-stained purple and in short rod-like shape, gram-positive.
After extracting 16S rDNA of bacteria, PCR amplification sequencing is carried out, the universal 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 program is: firstly, pre-denaturation is carried out for 5min at 94 ℃; ② denaturation at 94 ℃ for 30 s; ③ annealing at 54 ℃ for 30 s; extension for 1min 30s at 72 ℃; repeating the steps II, III and IV 39 times, and extending for 10min at 72 ℃; storing at 4 ℃. The amplified PCR products were subjected to agarose gel electrophoresis (2uL sample +6uL bromophenol blue) at 300V for 12 min. Sending the PCR product to Beijing Optimalaceae Biotechnology Limited company for sequencing, splicing the sequencing result by using contigExpress, removing the part with inaccurate two ends, comparing the spliced sequence in NCBI database (blast. NCBI. nlm. nih. gov), and performing phylogenetic construction by adopting MEGA software.
SM01 has 100% homology with Bacillus amyloliquefaciens, which was named Bacillus amyloliquefaciens SM 01.
Example 2 tolerated concentration test
Inoculating Bacillus amyloliquefaciens SM01 to a vanadium-containing solution with a concentration of 1000 mg.L by continuous streaking-1，1100mg·L-1，1200mg·L-1，1300mg·L-1The concentration of chromium is 900 mg.L-1，1000mg·L-1，1100mg·L-1，1200mg·L-1The solid medium of (3) was incubated at 30 ℃ for 24 hours. Finally at a vanadium and chromium concentration of 1200 mg.L-1And 1000 mg. L-1The growth of bacteria gradually weakens. Shows that the tolerant concentration of the strain to vanadium and chromium respectively reaches 1200 mg.L-1And 1000 mg. L-1。
Example 3 reduction Effect test
Inoculating Bacillus amyloliquefaciens SM01 into LB liquid culture medium, performing amplification culture at 30 deg.C and 150rpm for 20 hr, washing with sterile water twice, and adjusting OD600Is 1. Adding into a container containing 50 mg.L according to an inoculation amount of 1%-1And V (V) and 50 mg.L-1Adding citric acid as an electron donor into the Cr (VI) solution, adding 6mM of citric acid, culturing at 38 ℃ for 24h at a constant temperature of 150rpm, centrifuging at 4000rpm, filtering supernatant, measuring the concentration of pentavalent vanadium and hexavalent chromium in the solution by adopting an ultraviolet spectrophotometry, and measuring the content of the total vanadium and the total chromium by adopting ICP-OES. Meanwhile, an inactivated bacteria group control is set to determine whether the bacteria have the adsorption effect. The results are shown in table 1:
TABLE 1
Where "-" indicates no data, the concentration measured spectrophotometrically is inaccurate due to too high a concentration, and the data measured after dilution is also very different.
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: re＝(c(0)-c)/c(0)
c is the concentration of pentavalent vanadium and hexavalent chromium in the solution after reaction; c. C(0)The initial pentavalent vanadium and hexavalent chromium concentrations in the solution.
Therefore, the 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 (6)
1. Bacillus amyloliquefaciens SM01, characterized in that: is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m20211038.
2. The Bacillus amyloliquefaciens SM01 of claim 1, wherein: the nucleotide sequence of the 16s rDNA is shown in SEQ ID No. 1.
3. Use of the Bacillus amyloliquefaciens SM01 according to claim 1 or 2 for reducing pentavalent vanadium or hexavalent chromium.
4. The vanadium-chromium toxicity remover is characterized in that: the active ingredient thereof comprises the Bacillus amyloliquefaciens SM01 as claimed in claim 1 or 2.
5. A method for simultaneously reducing vanadium and chromium is characterized by comprising the following steps: the Bacillus amyloliquefaciens SM01 of claim 1 or 2 added to a water body or soil containing pentavalent vanadium and hexavalent chromium.
6. The method for simultaneously reducing vanadium chromium according to claim 5, characterized in that: citric acid is added into water or soil containing pentavalent vanadium and hexavalent chromium to serve 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" * |
| 王洪婷: "两株耐钒细菌的V(V)还原特性及其基因组学研究" * |
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