CN111500493A - Bacillus and method for synthesizing nano-selenium by using same - Google Patents
Bacillus and method for synthesizing nano-selenium by using same Download PDFInfo
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Abstract
The invention discloses a bacillus, which is characterized in that soil samples of selenium-enriched tea plants in Kaiyang county of Guizhou province in 2018 are coated on L B culture medium by a 7-time flat plate dilution coating method, the soil samples are cultured under the conditions of 30 +/-0.5 ℃ and 150 +/-5 r/min to obtain bacterial liquid, the bacterial strain of the bacillus is rod-shaped and 3-5 mu m in size, the bacillus belongs to gram-positive bacteria, the bacterial strain is rod-shaped, the Genbank accession number of the 16SrRNA gene sequence of the bacillus is MN371282, and the bacillus can tolerate SeO with higher concentration3 2‑And has the capability of reducing the selenium into elemental nano-selenium particles with different particle sizes.
Description
Technical Field
The invention relates to the technical field of microorganism cultivation and application, in particular to bacillus and a method for synthesizing nano-selenium by using the same.
Background
In nature, the element selenium usually exists in two forms of organic selenium and inorganic selenium, the organic selenium usually exists in organisms in the form of seleno amino acid and participates in various life activities in organisms, the inorganic selenium usually exists in four forms of selenate (VI), selenite (IV), elemental selenium (0) and metal selenium compound (-II) in environmental media such as rocks, soil, natural water body and selenium-containing wastewater, but the selenate and the selenite in the inorganic selenium usually have larger biological toxicity to the organisms, and relevant experiments prove that the sodium selenite has great biological toxicity to L D selenate of rats50L D of sodium selenate in rat at 7mg/kg50It was 1.6 mg/kg. However, over the past decades, SeO's have been produced by coal mining, chemical manufacturing, and other industrial activities3 2-And SeO4 2-The water is heavily enriched in water, which causes serious damage to the ecological environment.
For the treatment of selenium-containing wastewater, the traditional treatment methods are mainly physical treatment and chemical treatment. Common technologies in the physical method are nanofiltration, ion exchange, reverse osmosis and the like, and although the method is applied to treatment of urban drinking water and wastewater to a certain extent, the membrane system has certain limitations in practical application due to the problems of high energy consumption, high operation and maintenance cost and the like in the use process. The chemical method mainly uses chemical reagents such as iron oxide to reduce Se (IV/VI) into elemental selenium precipitate, and then further uses chemical adsorbent to adsorb to achieve the purpose of completely removing the elemental selenium, but the method may use toxic chemical reagents to cause secondary pollution to the environment. In recent years, it has been reported in the literature that certain microorganisms have SeO3 2-And SeO4 2-The ability to reduce elemental selenium. Zhang et al use Pseudomonas alcalophila to transform SeO3 2-Reducing the selenium into monoclinic selenium nano particles with the particle size of 200 nm; dwivedi et al use Pseudomonas aeruginosa JS-11 to transform SeO3 2-Reduced to granulesSpherical selenium nanoparticles with a diameter of 21 nm. In the process, the microorganisms can remove SeO which has high toxicity and serious environmental pollution3 2-And SeO4 2-Reduction to less toxic and more environmentally friendly Se0. Meanwhile, the biosynthesized nano selenium has good photoelectric characteristics and can be widely applied to medical treatment, electrical and manufacturing industries and the like. However, most of the strains found so far can only reduce SeO at a lower concentration3 2-For SeO which can be reduced under high concentration conditions3 2-The strain of (a) is yet to be developed.
Disclosure of Invention
The object of the present invention is to develop a process for reducing SeO under high concentration conditions3 2-The strain of (1).
In order to achieve the aim, the invention provides a Bacillus, namely the Bacillus (named Bacillus sp.S L by Latin classification), wherein the registration accession number of the strain is CGMCC No.19051, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the address is No. 3 of Beijing Shang Yang-ward West Lu No.1, the institute of microbiology of China academy of sciences, and the preservation date of 100101 is 11 months and 28 days in 2019.
According to the bacillus, soil samples of selenium-enriched tea plants in Kaiyang county of Guizhou province in 2018 are coated on L B culture medium through a 7-time flat plate dilution coating method, the soil samples are cultured at 30 +/-0.5 ℃ under the condition of 150 +/-5 r/min to obtain bacterial liquid, the cell morphology of the bacillus strain is rod-shaped, and the size of the bacillus strain is 3-5 mu m, and the L B culture medium comprises NaCl10 g/L, tryptone 10 g/L, yeast powder 5 g/L and deionized water.
The bacillus belongs to gram-positive bacteria, the bacteria are rod-shaped, and the Genbank accession number of the 16SrRNA gene sequence is MN 371282.
A method for synthesizing nano-selenium by bacillus comprises the following specific steps:
s1, processing L B culture medium;
sterilizing the L B culture medium at 121 deg.C under 0.15MPa for 20min, adding alkaline solution or acid solution to adjust the pH value of L B culture medium to 3.0-10.0;
s12, inoculating bacillus to synthesize nano selenium;
adding Na to the L B medium described in S11 at a final concentration of 1 mmol/L-50 mmol/L, respectively2SeO3Inoculating the bacillus with 2% (V/V) of inoculation amount after the solution is dissolved, and culturing for 2-11 h under the conditions of 30 +/-0.5 ℃ and 150 +/-5 r/min;
preferably, the L B medium has a pH of 5.0-8.0.
Preferably, the Na is2SeO3The final concentration of the solution is 5 mmol/L-20 mmol/L.
Preferably, the culture time for synthesizing the nano-selenium by inoculating the bacillus is 2-8 h.
The bacillus of the invention can tolerate SeO with higher concentration3 2-And has the capability of reducing the selenium into elemental nano-selenium particles with different particle sizes.
Drawings
FIG. 1 SEM picture of strain S L.
FIG. 2 phylogenetic tree of strain S L.
FIG. 3 growth curve of strain S L in L B medium.
FIG. 4 different SeOs3 2-SeO pair of strains S L at concentration3 2-The reduction rate of (3).
FIG. 5 Strain S L vs SeO at different pH3 2-The reduction rate of (3).
FIG. 6 TEM image of strain S L synthesized selenium nanoseles at a scale bar of 100 nm.
FIG. 7 TEM image of strain S L synthesized selenium nanoseles at a scale bar of 500 nm.
FIG. 8 is a graph of extracellular EPS as a function of time during the synthesis of nano-selenium by strain S L.
Detailed Description
The invention provides a bacillus, wherein the strain registration number of the bacillus is CGMCC No.19051, the bacillus is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation date is 11 months and 28 days in 2019.
According to the bacillus, soil samples of selenium-enriched tea plants in Kaiyang county of Guizhou province in 2018 are coated on L B culture medium through a 7-time flat plate dilution coating method, the soil samples are cultured at 30 +/-0.5 ℃ under the condition of 150 +/-5 r/min to obtain bacterial liquid, the cell morphology of the bacillus strain is rod-shaped, and the size of the bacillus strain is 3-5 mu m, and the L B culture medium comprises NaCl10 g/L, tryptone 10 g/L, yeast powder 5 g/L and deionized water.
The bacillus belongs to gram-positive bacteria, the bacteria are rod-shaped, and the Genbank accession number of the 16SrRNA gene sequence is MN 371282.
The invention also provides a method for synthesizing nano-selenium by using bacillus, which comprises the following steps:
s1, processing L B culture medium;
sterilizing L B culture medium at 121 deg.C under 0.15MPa for 20min, adding alkaline solution or acid solution to adjust pH of L B culture medium to 3.0-10.0, adjusting pH of L B culture medium to 5.0-8.0, and reacting to obtain SeO3 2-The reduction rate of the catalyst is more than 98 percent;
s12, inoculating bacillus to synthesize nano selenium;
adding Na to the L B medium described in S11 at a final concentration of 1 mmol/L-50 mmol/L, respectively2SeO3Inoculating the bacillus with 2% (V/V) of inoculation amount after the solution is dissolved, and culturing for 2-11 h under the conditions of 30 +/-0.5 ℃ and 150 +/-5 r/min; the Na is2SeO3The final concentration of the solution is 5 mmol/L-20 mmol/L, Na2SeO3The final concentration of the solution was 5 mmol/L of its SeO3 2-The reduction rate of (A) is maximized to a SeO of 20 mmol/L3 2-The reduction rate of (b) is reduced. The culture time for synthesizing the nano-selenium by inoculating the bacillus is 2-8 h. When the bacillus strain is cultured for 2-8h, the bacillus strain is in logarithmic growth phase.
Example 1:
the bacillus strain S L is obtained by coating a soil sample of selenium-enriched tea factories in Kaiyang county of Guizhou province in 2018 on a L B culture medium (NaCl10 g/L, tryptone 10 g/L, yeast powder 5 g/L and deionized water) by a 7-time flat plate dilution coating method, culturing at the temperature of 30 +/-0.5 ℃ and 150 +/-5 r/min to obtain a bacterial liquid, wherein the bacterial strain S L is rod-shaped in cell form and 3-5 mu m in size, and a scanning electron microscope picture of the bacillus strain S L is shown in figure 1.
The bacillus strain is preserved in the general microbiological culture collection center of China Committee for culture Collection of microorganisms (No. 3 of West Lu No.1 of Beijing city Korean-Yang district, microbiological research institute of Chinese academy of sciences, 100101) in 2019, at 11 and 28 days, and the preservation number is as follows: CGMCC No. 19051.
Example 2: molecular identification of 16SrRNA gene of bacillus strain
Extracting genome DNA of bacillus S L, amplifying 16SrRNA gene sequence by PCR, carrying out homology comparison on the sequence by using B L AST program, obtaining bacillus as the most similar genus, wherein the similarity of the 16SrRNA gene sequence is 100 percent, and therefore, S L is judged to be bacillus, as shown in FIG. 2, the 16SrRNA gene sequence of the 16SrRNA gene phylogenetic tree of the strain S L is as follows:
GGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGC GAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAA CACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGC TAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTT CGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTA ACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCC ACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGG GAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGA TGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTA GTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAA CTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAAT TATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCC CACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAG AAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGG AGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGG CGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCC GTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAG TTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACT CAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATT CGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCT AGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTG TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA CCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCG GTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTA TGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGA CCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGC TGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATG CCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACG AGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCG CCTAAGGTGGGACAGATGATTGGGGTGAAGTC
example 3 growth Curve of the Bacillus Strain S L in L B Medium
The bacterial liquid in example 1 is used, and cultured under conditions of 121 ℃, 20min high-pressure sterilization and L B culture medium (NaCl10 g/L, tryptone 10 g/L, yeast powder 5 g/L, deionized water and pH6.0), 30 +/-0.5 ℃, 150 +/-5 r/min, and the inoculation amount is 2% (V/V). As shown in FIG. 3, the growth condition of the bacillus strain S L is that the strain grows for 10 +/-1 h and enters a stationary phase, and 2-8h is a logarithmic growth phase.
Example 4: different SeO3 2-The Bacillus strain S L vs SeO at the concentration3 2-Reduction rate of (2)
The bacterial solution of example 1 was autoclaved at 121 ℃ for 20min in L B medium (NaCl10 g/L, tryptone 10 g/L, yeast powder 5 g/L, deionized water, pH6.0) and Na was added to the medium to a final concentration of 1, 5, 10, 20, 50 mmol/L2SeO3Culturing the solution with inoculum size of 2% (V/V) for 12h, centrifuging the obtained bacterial solution at 10000 + -500 r/min and 5 + -1 min respectively, and measuring the residual SeO in the reaction system with 1m L supernatant3 2-And (4) concentration. SeO3 2-The concentration is determined by the reduction method of ascorbic acid and hydrochloric acid. SeO3 2-Can be reduced into elemental selenium by ascorbic acid in strong acid solution, the solution is orange red, and the absorbance value and SeO at 500nm3 2-In this experiment, the supernatant of 1m L obtained above was added to 0.5m L4 mol/L hydrochloric acid and 1m L1 mol/L ascorbic acid, reacted for 10min, and then the absorbance was measured at a wavelength of 500nm, according to a standard curve (y 0.0046x +0.16908, R ═ 0.0046x +0.1690820.99) calculating Se in the sampleO3 2-The concentration was calculated, and the reduction ratio was calculated, and the result is shown in FIG. 4. When Na is present2SeO3SeO at a concentration of 5 mmol/L3 2-The reduction ratio of (2) was 98.67%, indicating that at this concentration, the cells were able to react with Na2SeO3Fully contacting the solution; and with Na2SeO3When the concentration increased to 20 mmol/L, its SeO3 2-The reduction rate of (B) was decreased, but it was found from the figure that the Bacillus strain S L was resistant to Na in high concentration2SeO3Has better tolerance capability.
Example 5 the Bacillus strains S L vs SeO at different pH3 2-Reduction rate of (2)
Adjusting the pH of L B culture medium (NaCl10 g/L, tryptone 10 g/L, yeast powder 5 g/L and deionized water) to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively by using NaOH and HCl solution, autoclaving at 121 ℃ for 20min, adding Na with the final concentration of 1 mmol/L after the sterilization is finished2SeO3In the solution, the inoculum size was 2% (V/V), and the culture was continued to the end of log. Then, the obtained strain was centrifuged at 10000. + -. 500r/min for 5. + -.1 min, and 1ml of the supernatant was taken to measure SeO therein3 2-The remaining concentration. SeO as in example 43 2-The concentration is determined by the reduction method of ascorbic acid and hydrochloric acid. As shown in FIG. 5, SeO was present in the reaction system when the pH of the medium was 5.0 to 8.03 2-The reduction rate of the strain S L reaches more than 98 percent, wherein the reduction rate with the pH of 6.0 is the highest, so that the condition is the optimal pH value of the strain S L for synthesizing the nano selenium, and when the pH value is too acid or too alkali, the SeO in the system is3 2-The reduction rate of (A) is reduced.
Example 6 TEM image of selenium nanoseles synthesized by the Bacillus strain S L.
The bacterial solution of example 1 was added to L B medium (NaCl10 g/L, tryptone 10 g/L, yeast powder 5 g/L, deionized water, pH6.0) which had been autoclaved (121 ℃, 20min), and Na was added to the final concentration of 1 mmol/L2SeO3And (5) solution culture. The final synthesized product was subjected to transmission electron microscopy (Transmission Electron micro)scope, TEM) and the results are shown in fig. 6 and fig. 7. The nano-selenium particles synthesized by the strain are spherical and have good dispersibility, the average particle size is 150.47nm, the maximum particle size is 226.00nm, and the minimum particle size is 44.15 nm.
Example 7:
as shown in fig. 8, the extracellular EPS changes with time during the synthesis of nano-selenium by the bacillus strain S L.
Extracellular Polymer (EPS) is a high molecular polymer secreted by microorganisms in the extracellular space, and mainly consists of polysaccharides and proteins. For microorganisms, EPS has important physiological functions, and can not only enrich nutrient components in the environment, but also resist the harm of bactericides and toxic substances to cells.
In the experiment, SeO is used to examine the process of biosynthesizing nano-selenium3 2-And Se0The method comprises the steps of culturing the strain in the mode of example 6, sampling every 4 hours within 24 hours, centrifuging the obtained 10m L sample under the conditions of 12000 +/-500R/min and 10min, taking supernatant, and measuring the concentration of polysaccharide and protein, wherein the content of the polysaccharide is measured by adopting a concentrated sulfuric acid-phenol method, the polysaccharide is firstly hydrolyzed into monosaccharide under the action of sulfuric acid, and is rapidly dehydrated to generate a furfural derivative, then the furfural derivative and phenol are used for generating an orange yellow compound, in an experiment, 1m L supernatant is added into a 1m L phenol solution, is shaken evenly, 5m L concentrated sulfuric acid is slowly added, the mixture is heated for 15 +/-1 min and is cooled to room temperature, the mixture is kept for 20min, the absorbance is measured at 490nm wavelength, and the absorbance is compared with a drawn polysaccharide standard curve (y is 0.1112x +0.0542, R is 0.1112x +0.0542, and R is compared with the drawn polysaccharide standard curve (y is 020.9995) control, calculating polysaccharide content, measuring protein content by using a Coomassie brilliant blue method, under an acidic condition, binding Coomassie brilliant blue G-250 dye with a protein hydrophobic region to cause that the maximum absorption peak is red-shifted from 465nm to 595nm, simultaneously the color is changed from brown to blue, and the absorbance value at 595nm is in direct proportion to the protein concentration, taking 300 mu L supernatant in the experiment, adding 1.5m L Coomassie brilliant blue reagent, shaking uniformly, standing for 2min, taking deionized water as reference, measuring the absorbance at 595nm, comparing with a standard curve (y is 0.0082x +0.5228,R20.996) control, protein content was calculated and the results are shown in fig. 8. When the reaction lasts for 12 hours, the EPS content in the system is the highest, and after 12 hours, the EPS content gradually decreases. The results were consistent with the growth curve of the strain. Presumably due to the high selenite content in the medium before 12h, the strain was stimulated to secrete large amounts of EPS to avoid interference from selenite toxicity. After 12h, selenite is reduced to nano-selenium, the toxicity is reduced, and EPS secreted by the strain is reduced. Meanwhile, the protein is utilized as an end-capping reagent of the nano-selenium, and the total content of EPS in the culture medium is reduced.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (5)
1. The bacillus is characterized in that the strain registration number of the bacillus is CGMCC No.19051, the bacillus is preserved in the China general microbiological culture Collection center, and the preservation date is 2019, 11 months and 28 days;
according to the bacillus, soil samples of selenium-enriched tea plants in Kaiyang county of Guizhou province in 2018 are coated on L B culture medium by a 7-time flat plate dilution coating method, and the soil samples are cultured at 30 +/-0.5 ℃ under the condition of 150 +/-5 r/min to obtain bacterial liquid;
the bacillus strain is rod-shaped, and the size of the bacillus strain is 3-5 mu m;
the L B culture medium comprises NaCl10 g/L, tryptone 10 g/L, yeast powder 5 g/L and deionized water;
the bacillus belongs to gram-positive bacteria, the bacteria are rod-shaped, and the Genbank accession number of the 16SrRNA gene sequence is MN 371282.
2. A method for synthesizing nano selenium by bacillus is characterized by comprising the following steps:
s1, processing L B culture medium;
sterilizing the L B culture medium at 121 deg.C under 0.15MPa for 20min, adding alkaline solution or acid solution to adjust the pH value of L B culture medium to 3.0-10.0;
s12, inoculating bacillus to synthesize nano selenium;
adding Na to the L B medium described in S11 at a final concentration of 1 mmol/L-50 mmol/L, respectively2SeO3Inoculating the bacillus with 2% (V/V) of inoculation amount after the solution is dissolved, and culturing for 2-11 h under the conditions of 30 +/-0.5 ℃ and 150 +/-5 r/min.
3. The method for synthesizing nano-selenium by using bacillus as claimed in claim 2, wherein the pH value of the L B medium is 5.0-8.0.
4. The method for synthesizing nano-selenium by using bacillus as claimed in claim 2, wherein Na is added2SeO3The final concentration of the solution is 5 mmol/L-20 mmol/L.
5. The method for synthesizing nano-selenium by using bacillus as claimed in claim 2, wherein the culture time for synthesizing nano-selenium by using inoculated bacillus is 2-8 h.
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Cited By (5)
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| CN112481166A (en) * | 2020-12-04 | 2021-03-12 | 西安恒田生物科技有限公司 | Method for synthesizing biological nano-selenium by utilizing bacillus methylotrophicus LW-6 and application |
| CN112481166B (en) * | 2020-12-04 | 2024-05-28 | 西安恒田生物科技有限公司 | Method for synthesizing biological nano-selenium by using bacillus methylotrophicus LW-6 and application thereof |
| CN115678793B (en) * | 2021-07-29 | 2023-09-01 | 中国农业大学 | Bacillus subtilis natto subspecies N14 and application thereof |
| CN114058535A (en) * | 2021-10-09 | 2022-02-18 | 大连理工大学 | Paracoccus denitrificans and method for preparing nano-selenium by using same |
| CN114058535B (en) * | 2021-10-09 | 2023-08-18 | 大连理工大学 | Paracoccus denitrificans and method for preparing nano-selenium by using same |
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