CN111411060A - Selenium-resistant bacterium screening method and application - Google Patents
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Abstract
The invention belongs to the technical field of bacterial screening, and discloses a selenium-resistant bacterial screening method and application, wherein prepared selenium-rich soil suspension is added into a beef extract peptone liquid culture medium of sodium selenite, and the beef extract peptone liquid culture medium is placed on a shaking table for shake culture; inoculating the shake-cultured bacterial liquid into a beef extract peptone liquid culture medium of sodium selenite, and placing the beef extract peptone liquid culture medium in a shaking table for shake culture; inoculating the cultured bacterial liquid into a beef extract peptone liquid culture medium, and placing the beef extract peptone liquid culture medium in a shaking table for shaking culture; diluting the obtained bacterial liquid with sterile water, coating the diluted bacterial liquid on a beef extract peptone agar culture medium, and placing the beef extract peptone agar culture medium in a biochemical incubator for constant-temperature culture; streaking and separating the grown single colony, and then placing the single colony in a biochemical incubator for constant-temperature culture to obtain a pure strain; and taking the grown bacterial colony to inoculate in a beef extract peptone liquid culture medium, and then placing in a shaking table for shake culture. The invention has low material cost, simple experimental method and no pollution to the environment.
Description
Technical Field
The invention belongs to the technical field of bacterial screening, and particularly relates to a selenium-resistant bacterial screening method and application.
Background
At present, a microbial method which is super-resistant to selenium and can reduce the selenium is searched in the natural environment of a high-selenium area: 10 samples collected from selenium ore beds of new pond and rural pond dams in Enshi City of Hubei province are respectively enriched, separated, screened and purified to culture 3 strains with super selenium resistance, and the strains are respectively identified as acinetobacter baumannii, bacillus licheniformis and bacillus subtilis. The culture characteristics, morphological characteristics, biochemistry and molecular biology characteristics of the 3 strains are preliminarily studied. As a result, the Bacillus licheniformis and the Bacillus subtilis can grow normally in a nutrient agar culture medium with the selenium content of 33000 mu g/ml and can reduce inorganic selenium into red elemental selenium, and can still grow slowly when the selenium content is 46500 mu g/ml. And (4) conclusion: the discovery of the microorganisms with super selenium resistance has important significance in the aspects of treating environmental selenium pollution, developing selenium-rich agricultural products and the like.
In summary, the problems of the prior art are as follows: the existing screening method of selenium-resistant bacteria has the disadvantages of complex operation and low efficiency.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a selenium-resistant bacterium screening method and application.
The invention is realized in such a way that the screening method of the selenium-resistant bacteria comprises the following steps:
step one, adding the prepared selenium-rich soil suspension into a beef extract peptone liquid culture medium of sodium selenite, and placing the mixture on a shaking bed for shake culture;
secondly, inoculating the shake-cultured bacterial liquid into a beef extract peptone liquid culture medium of sodium selenite, and placing the beef extract peptone liquid culture medium in a shaking table for shake culture;
thirdly, inoculating the cultured bacterial liquid into a beef extract peptone liquid culture medium, and placing the beef extract peptone liquid culture medium in a shaking table for shaking culture;
fourthly, diluting the obtained bacterial liquid with sterile water 102-107Taking out and diluting 105、106、107Coating the bacterial liquid on a beef extract peptone agar culture medium, and placing the beef extract peptone agar culture medium in a biochemical incubator for constant-temperature culture;
fifthly, streaking and separating the grown single colony, and then placing the single colony in a biochemical incubator for constant-temperature culture to obtain a pure strain;
and sixthly, inoculating the grown bacterial colony in a beef extract peptone liquid culture medium, and then placing the bacterial colony in a shaking table for shake culture.
Further, the preparation method of the selenium-enriched soil suspension comprises the following steps: weighing 10g of selenium-enriched soil sample, adding the selenium-enriched soil sample into a sterilized conical flask of deionized water containing 10 glass beads, placing the conical flask in a shaking table for shaking at the temperature of 28 ℃, the rotation speed of 180r/min and the shaking time of 30min, and standing for 30 min;
and in the first step, the prepared selenium-enriched soil suspension is added into a beef extract peptone liquid culture medium containing sodium selenite with the concentration of 100-.
Further, the prepared supernatant of the selenium-enriched soil is added into a conical flask containing beef extract peptone liquid medium containing sodium selenite with the concentration of 100 mug/ml, the amount of the liquid medium is 50ml, the mixture is placed into a shaking table for shaking culture, the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the culture time is 18 h.
Further, the second step is to inoculate the shake-cultured bacterial liquid into a beef extract peptone liquid culture medium containing sodium selenite with the concentration of 100-.
Further, inoculating the shake-cultured bacterial liquid into a conical flask containing a beef extract peptone liquid medium containing sodium selenite with the concentration of 100 mu g/ml, wherein the volume fraction of the bacterial liquid inoculation is 5%, the amount of the liquid medium is 50ml, placing the bacterial liquid into a shaking table for shake culture, the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, the culture time is 18h, and repeating for 4 times.
Further, inoculating the cultured bacterial liquid into a beef extract peptone liquid culture medium containing sodium selenite in an amount of 0-20000 mug/ml, wherein the inoculation amount of the bacterial liquid is 0.5% -2% of the total volume of the beef extract peptone liquid culture medium, and then placing the bacterial liquid in a shaking table for shaking culture.
Further, inoculating the cultured bacterial liquid into a conical flask in which a beef extract peptone liquid medium containing sodium selenite is placed, and then placing the conical flask after inoculation into a shaking table for culture, wherein the liquid medium contains sodium selenite in an amount of 0, 2000, 5000, 8000, 10000, 12000, 15000, 18000, 20000, 22000 and 25000 mu g/ml, the volume fraction of the inoculated bacterial liquid is 2%, the amount of the liquid medium is 50ml, placing the conical flask in the shaking table for shaking culture, the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the culture time is 18 h.
Further, the fourth step of taking bacteria liquid and diluting with sterile water 102-107Take 105、106、107Coating the bacterial liquid on a beef extract peptone agar culture medium containing sodium selenite, and placing in a biochemical incubator for constant-temperature culture, wherein the volume of the coated bacterial liquid is 100 μ l, the temperature of the biochemical incubator is 30 ℃, and the culture time is 72 h;
the fifth step is that the single colony grown by the inoculating loop is picked up and inoculated in a conical flask in which a beef extract peptone liquid medium is placed, and then the conical flask after inoculation is placed in a shaking table for cultivation, wherein the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the cultivation time is 18 h;
the sixth step, selecting a grown single colony by using an inoculating loop, inoculating the single colony to a beef extract peptone solid culture medium by combining a plate-streaking method, placing the beef extract peptone solid culture medium in a biochemical incubator for constant-temperature culture, and selecting the single colony for gram staining;
picking out the grown single colony by using an inoculating loop, inoculating the single colony into a conical flask with a beef extract peptone liquid culture medium, inoculating 1ml of the single colony into 99ml of beef extract peptone liquid culture medium with different pH values or different salt concentrations, and placing the beef extract peptone liquid culture medium into a shaking table for shaking culture, wherein the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the culture time is 48 h;
after the seventh step it is necessary:
(1) extracting total DNA;
(2) and (3) extracting total DNA from the purified bacteria, amplifying the total DNA by using a bacterial 16S rDNA universal primer, and performing sequencing identification.
The invention also aims to provide a method for treating environmental selenium pollution, which uses the screening method of the selenium-resistant bacteria.
Another object of the present invention is to provide a method for developing selenium-rich agricultural products using the method for screening selenium-resistant bacteria.
In summary, the advantages and positive effects of the invention are: the screening method of the selenium-resistant bacteria has the advantages of low material cost, simple experimental method and no pollution to the environment. The screening method of the selenium-resistant bacteria has potential application values in the biotransformation of selenium, the biosynthesis of nano-selenium and the restoration of selenium-polluted environment.
Drawings
FIG. 1 is a flow chart of a method for screening selenium-resistant bacteria according to an embodiment of the present invention.
FIG. 2 is a growth curve for a selenium-tolerant strain provided by practice of the present invention.
FIG. 3 is a graph of the effect of liquid media of varying pH on the growth of selenium-tolerant strains provided by the practice of the invention.
FIG. 4 is a graph of the effect of liquid media of varying salt concentrations on the growth of selenium-tolerant strains provided by practice of the invention.
FIG. 5 is a graph of the colony counts of selenium-tolerant strains according to the invention at different concentrations of sodium selenite.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a screening method and application of selenium-resistant bacteria, and the invention is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for screening selenium-resistant bacteria provided by the embodiment of the present invention includes the following steps:
s101: preparing selenium-rich soil suspension;
s102: enrichment culture: adding the prepared selenium-rich soil suspension into a beef extract peptone liquid culture medium containing sodium selenite with the concentration of 100-;
s103: subculturing: inoculating the shake-cultured bacterial liquid into a beef extract peptone liquid culture medium containing sodium selenite with the concentration of 100-;
s104: gradient stress culture: inoculating the cultured bacterial liquid into a beef extract peptone liquid culture medium containing sodium selenite in an amount of 0-20000 mug/ml, wherein the inoculation amount of the bacterial liquid is 0.5% -2% of the total volume of the beef extract peptone liquid culture medium, and then placing the mixture into a shaking table for shaking culture;
s105: dilution coating of the flat plate: diluting the obtained bacterial liquid with sterile water 102-107Taking out and diluting 105、106、107Coating the bacterial liquid on a beef extract peptone agar culture medium, and placing the beef extract peptone agar culture medium in a biochemical incubator for constant-temperature culture;
s106: plate scribing: streaking and separating the grown single colony, and then placing the single colony in a biochemical incubator for constant-temperature culture to obtain a pure strain;
s107: and (3) activation: taking the grown bacterial colony to inoculate in a beef extract peptone liquid culture medium, and then placing in a shaking table for shake culture;
s108: extracting total DNA;
s109: 16SrDNA gene amplification is carried out for identification.
The screening method of the selenium-resistant bacteria provided by the embodiment of the invention specifically comprises the following steps:
(1) preparing a selenium-rich soil suspension: weighing 10g of selenium-enriched soil sample, adding into a sterilized conical flask of deionized water containing 10 glass beads, placing in a shaking table, oscillating at 28 deg.C and 180r/min for 30min, and standing for 30 min.
(2) Enrichment culture: and (2) adding the soil supernatant prepared in the step (1) into a conical flask containing a beef extract peptone liquid medium containing sodium selenite with the concentration of 100 mu g/ml, wherein the amount of the liquid medium is 50ml, placing the conical flask in a shaking table for shaking culture, and the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the culture time is 18 h.
(3) Subculturing; inoculating the bacterial liquid obtained in the step (2) into a conical flask containing a beef extract peptone liquid culture medium containing sodium selenite with the concentration of 100 mu g/ml, wherein the inoculation amount of the bacterial liquid is 5 percent (volume fraction), the amount of the liquid culture medium is 50ml, placing the bacterial liquid into a shaking table for shaking culture, the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, the culture time is 18h, and repeating the steps for 4 times.
(4) Gradient stress culture: inoculating the bacterial liquid cultured in the step (3) into a conical flask in which a beef extract peptone liquid medium containing sodium selenite is placed, and then placing the conical flask after inoculation into a shaking table for culturing, wherein the amount of the sodium selenite contained in the liquid medium is 0, 2000, 5000, 8000, 10000, 12000, 15000, 18000, 20000, 22000 and 25000 mu g/ml, the bacterial liquid inoculation amount is 2% (volume fraction), the amount of the liquid medium is 50ml, placing the conical flask into the shaking table for shaking culture, the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the culturing time is 18 h.
(5) Dilution coating of the flat plate: diluting the bacterial liquid obtained in the step (4) with sterile water 102-107Take 105、106、107The bacterial liquid is coated on a beef extract peptone agar culture medium containing sodium selenite, and is placed in a biochemical incubator for constant-temperature culture, wherein the volume of the coated bacterial liquid is 100 mu l, the temperature of the biochemical incubator is 30 ℃, and the culture time is 72 h.
(6) Plate scribing: and (3) selecting the single colony grown in the step (5) by using an inoculating loop, inoculating the single colony on a beef extract peptone solid medium containing sodium selenite by combining a plate-streaking method, and placing the single colony in a biochemical incubator for constant-temperature culture.
(7) And (3) activation: and (3) picking the single bacterial colony grown in the step (6) by using an inoculating ring, inoculating the single bacterial colony into a conical flask containing a beef extract peptone liquid culture medium, and then placing the inoculated conical flask into a shaking table for culturing, wherein the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the culturing time is 18 h.
(8) Gram staining: and (3) selecting the single bacterial colony grown in the step (6) by using an inoculating loop, inoculating the single bacterial colony to a beef extract peptone solid culture medium by combining a plate-streaking method, placing the beef extract peptone solid culture medium in a biochemical incubator for constant-temperature culture, and selecting the single bacterial colony for gram staining.
(9) Environmental factor influence experiment: and (3) picking the single bacterial colony grown in the step (6) by using an inoculating loop, inoculating the single bacterial colony into a conical flask with a beef extract peptone liquid culture medium, inoculating 1ml of the single bacterial colony into 99ml of beef extract peptone liquid culture medium with different pH values or different salt concentrations, and placing the beef extract peptone liquid culture medium into a shaking table for shaking culture, wherein the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the culture time is 48 h.
(10)16S rDNA gene amplification for identification: and (4) amplifying the total DNA extracted from the bacteria purified in the step (6) by using a bacteria 16SrDNA universal primer, and further performing sequencing identification.
The screening method of selenium-tolerant bacteria provided by the invention adopts natural selenium-rich soil as a material, can separate and screen a super-bacterial stenotrophomonas maltophilia (stenotrophomonas maltophilia) from the natural selenium-rich soil as a material, and has the advantages of super-strong selenium-tolerant capability through experimental verification, low material cost, simple experimental method and no pollution to the environment as shown in figures 2-5.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for screening selenium-resistant bacteria, which is characterized by comprising the following steps:
step one, adding the prepared selenium-rich soil suspension into a beef extract peptone liquid culture medium of sodium selenite, and placing the mixture on a shaking bed for shake culture;
secondly, inoculating the shake-cultured bacterial liquid into a beef extract peptone liquid culture medium of sodium selenite, and placing the beef extract peptone liquid culture medium in a shaking table for shake culture;
thirdly, inoculating the cultured bacterial liquid into a beef extract peptone liquid culture medium, and placing the beef extract peptone liquid culture medium in a shaking table for shaking culture;
fourthly, diluting the obtained bacterial liquid with sterile water, coating the diluted bacterial liquid on a beef extract peptone agar culture medium, and placing the beef extract peptone agar culture medium in a biochemical incubator for constant-temperature culture;
fifthly, streaking and separating the grown single colony, and then placing the single colony in a biochemical incubator for constant-temperature culture to obtain a pure strain;
and sixthly, inoculating the grown bacterial colony in a beef extract peptone liquid culture medium, and then placing the bacterial colony in a shaking table for shake culture.
2. The method for screening selenium-tolerant bacteria according to claim 1, wherein the selenium-rich soil suspension is prepared by a method comprising: weighing 10g of selenium-enriched soil sample, adding the selenium-enriched soil sample into a sterilized conical flask of deionized water containing 10 glass beads, placing the conical flask in a shaking table for shaking at the temperature of 28 ℃, the rotation speed of 180r/min and the shaking time of 30min, and standing for 30 min;
and in the first step, the prepared selenium-enriched soil suspension is added into a beef extract peptone liquid culture medium containing sodium selenite with the concentration of 100-.
3. The method for screening selenium-resistant bacteria according to claim 2, wherein the prepared supernatant of the selenium-rich soil is added into a conical flask containing a beef extract peptone liquid medium containing sodium selenite at a concentration of 100 μ g/ml, the amount of the liquid medium is 50ml, and the mixture is placed in a shaking table for shaking culture at a temperature of 30 ℃ and a rotation speed of 180r/min for 18 hours.
4. The method for screening selenium-resistant bacteria as claimed in claim 1, wherein the second step comprises inoculating the shake-cultured broth into a beef extract peptone liquid medium containing sodium selenite at a concentration of 100-200 μ g/ml, in an amount of 5% -10% of the total volume of the beef extract peptone liquid medium, and then placing the mixture in a shaking table for shake culture for 3-5 times.
5. The method for screening selenium-resistant bacteria according to claim 4, wherein the shake-cultured bacterial solution is inoculated into a conical flask containing beef extract peptone broth with a sodium selenite concentration of 100 μ g/ml, the inoculum size is 5% by volume and the broth size is 50ml, and the flask is shake-cultured in a shaker at a shaker temperature of 30 ℃ and a rotation speed of 180r/min for 18 hours, and the process is repeated 4 times.
6. The method for screening selenium-resistant bacteria according to claim 1, wherein the third step comprises inoculating the cultured broth into a beef extract peptone broth containing sodium selenite in an amount of 0-20000 μ g/ml, in an amount of 0.5% -2% of the total volume of the beef extract peptone broth, and then culturing the broth in a shaker.
7. The method for screening selenium-resistant bacteria according to claim 6, wherein the cultured bacterial solution is inoculated into a conical flask containing a beef extract peptone broth containing sodium selenite, and the inoculated conical flask is then placed in a shaker for culture, wherein the amount of sodium selenite contained in the broth is 1, 2000, 5000, 8000, 10000, 12000, 15000, 18000, 20000, 22000, 25000 μ g/ml, the volume fraction of the inoculated bacterial solution is 2%, the amount of the broth is 50ml, the liquid medium is placed in a shaker for shaking culture, the temperature of the shaker is 30 ℃, the rotation speed is 180r/min, and the culture time is 18 h.
8. The method for screening selenium-resistant bacteria according to claim 1, wherein the fourth step of taking the bacterial liquid is diluted 10 with sterile water2-107Take 105、106、107Coating the bacterial liquid on a beef extract peptone agar culture medium containing sodium selenite, and placing in a biochemical incubator for constant-temperature culture, wherein the volume of the coated bacterial liquid is 100 μ l, the temperature of the biochemical incubator is 30 ℃, and the culture time is 72 h;
the fifth step is that the single colony grown by the inoculating loop is picked up and inoculated in a conical flask in which a beef extract peptone liquid medium is placed, and then the conical flask after inoculation is placed in a shaking table for cultivation, wherein the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the cultivation time is 18 h;
the sixth step, selecting a grown single colony by using an inoculating loop, inoculating the single colony to a beef extract peptone solid culture medium by combining a plate-streaking method, placing the beef extract peptone solid culture medium in a biochemical incubator for constant-temperature culture, and selecting the single colony for gram staining;
picking out the grown single colony by using an inoculating loop, inoculating the single colony into a conical flask with a beef extract peptone liquid culture medium, inoculating 1ml of the single colony into 99ml of beef extract peptone liquid culture medium with different pH values or different salt concentrations, and placing the beef extract peptone liquid culture medium into a shaking table for shaking culture, wherein the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, and the culture time is 48 h;
after the seventh step it is necessary:
(1) extracting total DNA;
(2) and (3) extracting total DNA from the purified bacteria, amplifying the total DNA by using a bacterial 16S rDNA universal primer, and performing sequencing identification.
9. A method for treating environmental selenium pollution, which is characterized in that the method for treating environmental selenium pollution uses the screening method of selenium-resistant bacteria as claimed in any one of claims 1 to 8.
10. A method for developing selenium-rich agricultural products, which comprises using the method for screening the selenium-resistant bacteria according to any one of claims 1 to 8.
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| CN113930353A (en) * | 2021-08-31 | 2022-01-14 | 广西大学 | Selenite-resistant serratia marcescens and reduction characteristic identification method thereof |
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Application publication date: 20200714 |