CN107058108B - Method for screening and separating sulfate reducing bacteria by using improved dish-stacking plate method - Google Patents
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Abstract
The invention relates to a method for screening and separating sulfate reducing bacteria by using an improved dish-stacking plate method. The method overcomes the defects that the bacterial liquid in the prior art can not be uniformly coated and permeated on the first layer of flat plate, the bacterial liquid is not beneficial to growth after being diluted, bacterial colonies are easy to overlap up and down and even have the growth of mixed bacteria, and the like, and has the advantages of simple material, high screening efficiency, short screening period, high conversion rate of the obtained strains, and the like.
Description
Technical Field
The invention belongs to the technical field of environmental microbiology, and particularly relates to a method for screening and separating sulfate reducing bacteria by using an improved dish-stacking plate method.
Background
At present, the application of high-tech biotechnology such as biological engineering and the like to culture plant and soil microorganism super engineering bacteria capable of being used for fixing heavy metals and the application of the super engineering bacteria to soil pollution treatment is one of the most active leading-edge fields in the research of environmental science, environmental engineering, plant physiology, microbiology and the like. Microorganisms convert heavy metals by redox, methylation and demethylation, and toxic substances into forms that are not available to animals and plants.
Sulfate-Reducing bacteria (SRB) are capable of Reducing sulfur oxides such as Sulfate, sulfite, thiosulfate, and elemental sulfur to form sulfide ions in nature, and because the solubility product of heavy metal sulfides is generally small, sulfide ions can be immediately combined with trace heavy metal ions to form a form that cannot be utilized by organisms, so that Sulfate-Reducing bacteria can be used for treating heavy metal ions in a fixed environment. SRB bacteria play an important role in the corrosion of industrial pipelines, the environmental remediation of wastewater polluted by heavy metals and acidic mine wastewater.
SRB is strict anaerobe, but with the intensive research, the research results show that the SRB is not strictly absolute anaerobe but facultative anaerobe, but the SRB is extremely sensitive to oxygen, so that strict anaerobe technology is used for culturing and separating the SRB. There are many culture methods available for SRB, such as sandwich dish method, dilution tube method, Hungate tube technique, and the use of anaerobic bags, anaerobic tanks, anaerobic glove boxes, etc. The dilution shaking tube method has the defects that the observation and the selection of bacterial colonies are difficult, the Hungate rolling tube method and the method using anaerobic equipment are limited by the technical method and the equipment material cost, and the dish-stacking interlayer method is essentially to sandwich bacteria between an upper layer of culture medium and a lower layer of culture medium to cause a relatively oxygen-free environment, so that SRB can grow in a gap. The laminated cell culture method has the advantages that cultures are coated and grown in the nutrient agar interlayer, fixed-point bacteria taking can be conveniently achieved when bacterial colonies are taken, meanwhile, an anaerobic environment does not need to be additionally created, time and labor are saved, and the method has the advantages of all aerobic and anaerobic separation methods while being easy to achieve.
However, the conventional sandwich separation method cannot uniformly coat and permeate the bacteria on the first flat plate, and the oxidation-reduction potential of the diluted bacteria liquid is increased along with the increase of the dilution factor, which is very unfavorable for the growth of sulfate reducing bacteria. In addition, the diluent can be diffused into the second layer along with the pouring of the second layer agar layer, so that the phenomenon that the bacterial colony is overlapped up and down and even the surface of the second layer where the mixed bacteria grow is caused, a single bacterial colony is not easy to obtain, and certain difficulty is brought to separation and bacterial colony screening. In order to inherit the advantages of the traditional anaerobic culture separation method and overcome the defects of the traditional anaerobic culture separation method, the invention improves the dilution coating-dish folding sandwich method separation culture method of Sulfate Reducing Bacteria (SRB) according to the actual needs of scientific research.
Disclosure of Invention
Aiming at the problems of the existing sulfate reducing bacteria screening technology, the invention provides a method for screening, separating and purifying sulfate reducing bacteria with higher heavy metal ion tolerance from soil by using an improved dish-stacking plate method. The method is simple, and has improved screening and separating efficiency and shortened screening and separating period. In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for screening and separating sulfate reducing bacteria by using an improved dish stacking plate method comprises the following steps: (a) adding the pretreated soil into a sulfate reducing bacteria liquid enrichment culture medium for anaerobic culture, inoculating the obtained enrichment liquid into a new sulfate reducing bacteria liquid enrichment culture medium for anaerobic culture again, and performing transfer culture for multiple times to obtain enrichment bacterial liquid; (b) diluting the enriched bacterial liquid into different concentrations, adding L-cysteine, ascorbic acid and ammonium ferrous sulfate into the enriched bacterial liquid for later use, pouring the screening culture medium into a culture dish, respectively coating the dilution liquid with different concentrations on a flat plate after the dilution liquid is condensed, pouring the dilution liquid into the screening culture medium twice after the dilution liquid is permeated, sequentially forming a second layer and a third layer after the dilution liquid is condensed, and performing constant-temperature culture after the culture dish is sealed; (c) pouring the sterilized screening culture medium into a culture dish for condensation for later use, selecting the black single bacteria which obviously grow in the second layer of the strain growth layer in the step (b), carrying out streak separation on a newly prepared plate, pouring the screening culture medium onto the streaked and separated plate, sealing, and repeatedly carrying out separation culture for multiple times; (d) inoculating the single colony obtained by streaking separation into a preservation culture medium, and culturing at constant temperature to obtain the final product.
According to the scheme, the components of the sulfate reducing bacteria liquid enrichment culture medium comprise: k of 0.4-0.6g/2HPO41-2g/L of MgSO4·7H2O, 0.4-0.6g/L Na2SO40.1-0.2g/L of CaCl21-2g/L NaCl, 0.8-1.0g/L NH4Cl and yeast extract of 1.0-2.0g/L, sterilizing at 121 deg.C for 20min, and adjusting pH to 8.0-8.5.
According to the scheme, the screening medium comprises the following components: k of 0.4-0.6g/L2HPO40.1-0.2g/L of CaCl20.8-1.0g/L NH4Cl, 1-2g/L MgSO4·7H2O, 1-2g/L NaCl, 0.4-0.6g/L Na2SO42.0-3.0g/L of C3H5O3Na,0.5-0.6 g/L-cysteine, 0.5-0.6g/L ascorbic acid, 0.5-0.6g/L Fe (NH)4)2(SO)2And agar 2wt%, and is prepared by mixing other raw materials, sterilizing at 121 deg.C for 20min, cooling to 55 deg.C, adding L-cysteine, ascorbic acid, and Fe (NH)4)2(SO4)2And adjusting the pH value to 8.0-8.5.
According to the scheme, the components of the preservation medium comprise: k of 0.5-0.7g/L2HPO40.1-0.2g/L of CaCl21-1.2g/L of NH4Cl, 1-2g/L MgSO4·7H2O, 2-3g/L NaCl, 0.4-0.6g/L Na2SO43.0-4.0g/L of C3H5O3Na,0.5-0.6 g/L-cysteine, 0.5-0.6g/L ascorbic acid, mixing other raw materials, sterilizing at 121 deg.C for 20min, cooling to 55 deg.C, adding L-cysteine and ascorbic acid, and adjusting pH to 8.0-8.5.
According to the scheme, in the step (a), firstly, a soil sample is subjected to screening pretreatment by using a screen, and then, the soil sample is subjected to screening pretreatment according to the weight ratio of 1g:200-220mL, adding soil into the sterilized sulfate reducing bacteria liquid enrichment medium, covering a layer of liquid paraffin on the surface, filling nitrogen, and placing the mixture into a constant-temperature shaking table at 30 ℃ for anaerobic culture at the rotating speed of 100-120 r/min.
According to the scheme, the primary enrichment time in the step (a) is 7d, and then the primary enrichment time is inoculated for 2-3 times in a new sulfate reducing bacteria liquid enrichment culture medium by 5 per mill, wherein the time length of each time is 4 d.
According to the scheme, diluting the enriched bacterial liquid to 10 in the step (b)-4、10-5、10-6Three concentrations, followed by 0.5wt% L-cysteine and 0.5wt% ascorbic acid and 0.5% ferrous ammonium sulfate to the dilution.
According to the scheme, in the step (b), 55 ℃ screening culture medium with the height of about 1/2 is poured into a plurality of culture dishes, after condensation, 0.2-0.3mL of diluent with different concentrations is sucked and quickly coated on each flat plate, after the diluent permeates for a period of time, 1/3 height of the same screening culture medium is poured into the culture dishes to form a second strain growth layer, the same culture medium is poured into the culture dishes until overflowing to form a third agar layer, the culture dishes are covered and sealed, and then the culture dishes are placed at the constant temperature of 30 ℃ for 2-3d until obvious black colonies appear.
According to the scheme, the streak isolation culture temperature in the step (c) is 30 ℃, and the streak isolation culture is carried out for 3-4d and 2-3 times each time.
According to the scheme, in the step (d), a single colony obtained by streaking separation is inoculated into a storage medium by adopting a needle punching method, and is cultured at a constant temperature of 30 ℃ until obvious filamentous fungi appear, and then is placed in a low-temperature environment for refrigeration.
Compared with the prior art, the invention has the beneficial effects that:
1) the bacteria liquid coated on the flat plate can be well and uniformly dispersed in the second thin flat plate layer, and the problems that bacterial colonies are overlapped up and down and even mixed bacteria grow on the surface of the second agar layer due to the fact that the bacteria liquid coated on the first layer is easily mixed into the second agar layer poured later when the bacteria liquid is poured into the second agar layer by a traditional dish stacking flat plate method are solved;
2) compared with other improved methods, the laminated cell sandwich method provided by the invention is simpler and more convenient to operate, and simultaneously, the upper agar layer and the lower agar layer are seamless and bubble-free to clamp the intermediate strain growth layer, so that a better anaerobic environment is provided for the growth of sulfate reducing bacteria;
3) compared with the existing screening and separating method of sulfate reducing bacteria by a dish-folding sandwich method, the improved method can screen 50% more sulfate reducing bacteria single colonies under the same concentration gradient coating compared with the traditional method, and improves the screening and separating efficiency;
4) according to the invention, 0.5wt% of L-cysteine, 0.5wt% of ascorbic acid and 0.5wt% of ammonium ferrous sulfate are added into the diluted coating liquid, so that the screening and separating period of the screened and separated sulfate reducing bacteria is shortened by 2-3d compared with that of the sulfate reducing bacteria prepared by a traditional method.
Detailed Description
In order to make those skilled in the art fully understand the technical solutions and advantages of the present invention, the following embodiments are further described.
Example 1
The first step is as follows: enrichment of sulfate-reducing bacteria
Firstly, large-particle stones, plant roots and stems and the like of the sampled soil are removed by a screen for standby. Preparing a sulfate reducing bacteria liquid enrichment culture medium in a beaker, pouring 200-220ml of sulfate reducing bacteria liquid enrichment culture medium into a conical flask, and sterilizing for 20min at 121 ℃ in a high-temperature sterilization pot. After the soil is cooled, weighing 1g of the treated soil, adding the soil into a conical flask cooled to normal temperature, covering a layer of liquid paraffin on the surface layer of the conical flask culture solution, simultaneously filling nitrogen, sealing to keep an anaerobic environment, and putting the conical flask into a 30 ℃ constant-temperature shaking table for culture at a rotating speed of 100-120 r/min. The primary enrichment time is 7d, successful enrichment is shown when the color of the enrichment solution reaches the ink color, and then inoculation culture is carried out for 2-3 times in a new sulfate reducing bacteria liquid enrichment culture medium by 5 per mill of inoculation amount, wherein the time duration of each time is 4 d.
The sulfate reducing bacteria liquid enrichment culture medium comprises the following components: k of 0.4-0.6g/2HPO41-2g/L of MgSO4·7H2O, 0.4-0.6g/L Na2SO40.1-0.2g/L of CaCl21-2g/L NaCl, 0.8-1.0g/L NH4Cl and 1.0-2.0g/L yeast extract, sterilizing at 121 deg.C for 20min, adjusting pH to 8.0-8.5。
The second step is that: dilution coating-dish-stacking sandwich method screening of bacterial strains
Irradiating the bacterium solution subjected to enrichment and domestication for 2-3 times for more than 15min by using ultraviolet rays, and diluting the bacterium solution into 10 parts by using a liquid transfer gun on a sterile operating platform-4、10-5、10-6Three concentration gradients, and 0.5wt% L-cysteine, 0.5wt% ascorbic acid and 0.5wt% ferrous ammonium sulfate were added to the dilution. And then, sterilizing the screening culture medium at high temperature, keeping the temperature at about 55 ℃, pouring the screening culture medium into the bottoms of the culture dishes to a height of about 1/2, and condensing the screening culture medium for later use. Sucking 0.2-0.3mL of 10-4、10-5、10-6And (3) rapidly coating the dilution liquid with the concentration gradient on a plurality of condensed flat plates respectively. After the dilution solution permeates for about 10min on the flat plate, slowly pouring the same screening culture medium to the height of 1/3 at the middle position of the bottom of the culture dish, fixing the bacterial solution in a second strain growth layer formed in the culture dish, and pouring the same screening culture medium until the bacterial solution overflows and does not overflow to form a third agar layer. Quickly covering the culture dish cover without leaving air bubbles as much as possible, removing redundant agar between the inner and outer side walls of the culture dish, and pouring a proper amount of molten paraffin into the side seams to seal the side wall seams of the culture dish by the paraffin and avoid leaving air bubbles as much as possible. Finally, putting the culture dish into a 30 ℃ incubator for culturing for 2-3d, and culturing at 10 DEG C-520-30 black round single colonies appeared in the culture dish under the concentration gradient, 10-62-4 black round single colonies appeared in the dish under the concentration gradient.
Wherein, the screening culture medium comprises the following components: k2HPO40.4-0.6g/L，CaCl20.1-0.2g/L，NH4Cl 0.8-1.0g/L，MgSO4·7H2O 1-2g/L，NaCl 1-2g/L，Na2SO40.4-0.6g/L，C3H5O3Na2.0-3.0g/L, L-cysteine 0.5-0.6g/L, ascorbic acid 0.5-0.6g/L, Fe (NH)4)2(SO)20.5-0.6g/L and 2wt% agar. The preparation method comprises mixing other raw materials, sterilizing at 121 deg.C for 20min, cooling to 55 deg.C, adding L-cysteine, ascorbic acid, and Fe (NH)4)2(SO4)2And adjusting the pH value to 8.0-8.5.
The third step: separation and purification of bacterial strains
On a sterile operating table, the screening medium was sterilized at high temperature, kept at about 55 ℃, and poured into a plurality of petri dishes at a height of about 1/2. After the culture medium is condensed, the culture medium for screening and culturing the plate is unsealed, agar plate layers solidified in different cooling time are carefully pried, obvious black single bacterial colonies are picked and are transversely scribed and separated in the newly prepared plate. Pouring the screened culture medium which is sterilized at high temperature and kept at about 55 ℃ onto the flat plate after the lineation separation until the screened culture medium overflows and does not overflow, quickly covering the culture dish, keeping air bubbles as little as possible, removing the redundant agar between the inner and outer side walls of the culture dish, and sealing the gap of the culture dish by using an adhesive tape. The culture dish is put into a thermostat at 30 ℃ for culture until obvious black original-point-shaped single colonies appear. Performing streaking separation culture for 3-4d for 2-3 times.
The fourth step: preservation of the Strain
The preservation medium and 10mL cuvette were sterilized at 121 ℃ for 20min, poured into the 10mL cuvette on a sterile bench, and left in a 1-2mm gap. After the solid culture medium is cooled and solidified, the black single colony after lineation and separation is picked by a needle method and then is punctured into the solidified solid storage culture medium, the small test tube is put into a constant temperature oven at 30 ℃ for culture until obvious filamentous colony appears, and then the small test tube is refrigerated at low temperature, and the storage life can reach more than half a year.
Wherein, the preservation medium comprises the following components: k2HPO40.5-0.7g/L，CaCl20.1-0.2g/L，NH4Cl1-1.2g/L，MgSO4·7H2O 1-2g/L，NaCl2-3g/L，Na2SO40.4-0.6g/L，C3H5O33.0-4.0g/L of Na3, 0.5-0.6g/L of L-cysteine and 0.5-0.6g/L of ascorbic acid. The preparation method comprises mixing other raw materials, sterilizing at 121 deg.C for 20min, cooling to 55 deg.C, adding L-cysteine and ascorbic acid, and adjusting pH to 8.0-8.5.
Determination of sulfate-reducing ability of sulfate-reducing bacteria obtained by screening
Picking from preservation MediumThe sulfate reducing bacteria are inoculated into a liquid culture medium, and the strains are activated under anaerobic conditions. Activating in a constant temperature shaking table at 30 ℃ at the rotating speed of 100-120r/min for more than 4d, and measuring the OD600Transferring to new liquid culture medium after more than 0.5% according to inoculum size of 5 ‰, culturing under anaerobic condition, and measuring S in bacterial liquid by methylene blue spectrophotometry2-The content of SO is calculated4 2-Conversion to S2-The conversion rate of the ions is 70-80%. Wherein, the anaerobic condition is as follows: sealing with liquid paraffin, and introducing nitrogen to maintain anaerobic state; the liquid medium used consisted of: k2HPO40.4-0.6g/L，NH4Cl0.8-1g/L，MgSO4·7H2O 1-2g/L，NaCl2-3g/L，Na2SO40.4-0.6g/L，C3H5O3Na2.0-3.0g/L, L-cysteine 0.5-0.6g/L and ascorbic acid 0.5-0.6 g/L. The preparation method comprises mixing other raw materials, sterilizing at 121 deg.C for 20min, cooling to 55 deg.C, adding L-cysteine and ascorbic acid, and adjusting pH to 8.0-8.5.
Through the comparison test of the improved three-layer laminated dish plate method and the traditional double-layer laminated dish plate method, the number of single bacterial colonies of the sulfate reducing bacteria obtained under the same concentration gradient is increased by about 50 percent; and because 0.5wt% of L-cysteine, 0.5wt% of ascorbic acid and 0.5wt% of ammonium ferrous sulfate are added into the diluent, a good anaerobic environment is created for the growth of sulfate reducing bacteria, so that the screening and separating period is shortened by 2-3 d; at the same time, the final determination shows that the SO is obtained by screening the obtained strain4 2-Conversion to S2-The conversion rate of the ions is as high as 70-80%, which is 10-20% higher than that of the traditional screening method.
Claims (9)
1. A method for screening and separating sulfate reducing bacteria by using an improved dish stacking plate method is characterized by comprising the following steps:
(a) adding the pretreated soil into a sulfate reducing bacteria liquid enrichment culture medium for anaerobic culture, inoculating the obtained enrichment liquid into a new sulfate reducing bacteria liquid enrichment culture medium for anaerobic culture again, and performing transfer culture for multiple times to obtain enrichment bacterial liquid;
(b) diluting the enriched bacterial liquid into different concentrations, adding L-cysteine, ascorbic acid and ammonium ferrous sulfate into the enriched bacterial liquid for later use, pouring the screening culture medium into a culture dish, respectively coating the dilution liquid with different concentrations on a flat plate after the dilution liquid is condensed, pouring the dilution liquid into the screening culture medium twice after the dilution liquid is permeated, sequentially forming a second layer and a third layer after the dilution liquid is condensed, and performing constant-temperature culture after the culture dish is sealed;
(c) pouring the sterilized screening culture medium into a culture dish for condensation for later use, selecting the black single bacteria which obviously grow in the second layer of the strain growth layer in the step (b), carrying out streak separation on a newly prepared plate, pouring the screening culture medium onto the streaked and separated plate, sealing, and repeatedly carrying out separation culture for multiple times;
(d) inoculating the single colony obtained by streaking separation into a preservation culture medium, and carrying out constant-temperature culture to obtain the bacterial strain;
the components of the screening medium comprise: k of 0.4-0.6g/L2HPO40.1-0.2g/L of CaCl20.8-1.0g/L NH4Cl, 1-2g/L MgSO4·7H2O, 1-2g/L NaCl, 0.4-0.6g/L Na2SO42.0-3.0g/L of C3H5O3Na,0.5-0.6 g/L-cysteine, 0.5-0.6g/L ascorbic acid, 0.5-0.6g/L Fe (NH)4)2(SO)2And 2wt% agar; the preparation method comprises mixing other raw materials, sterilizing at 121 deg.C for 20min, cooling to 55 deg.C, adding L-cysteine, ascorbic acid, and Fe (NH)4)2(SO4)2And adjusting the pH value to 8.0-8.5.
2. The method for screening and separating sulfate-reducing bacteria by using the improved dish-stacking plate method according to claim 1, wherein the components of the sulfate-reducing bacteria liquid enrichment medium comprise: k of 0.4-0.6g/2HPO41-2g/L of MgSO4·7H2O, 0.4-0.6g/L Na2SO40.1-0.2g/L of CaCl21-2g/L NaCl, 0.8-1.0g/L NH4Cl and yeast extract of 1.0-2.0g/L, sterilizing at 121 deg.C for 20min, and adjusting pH to 8.0-8.5.
3. The method of claim 1, wherein the preservation medium comprises the following components: k of 0.5-0.7g/L2HPO40.1-0.2g/L of CaCl21-1.2g/L of NH4Cl, 1-2g/L MgSO4·7H2O, 2-3g/L NaCl, 0.4-0.6g/L Na2SO43.0-4.0g/L of C3H5O3Na,0.5-0.6 g/L-cysteine, 0.5-0.6g/L ascorbic acid, mixing other raw materials, sterilizing at 121 deg.C for 20min, cooling to 55 deg.C, adding L-cysteine and ascorbic acid, and adjusting pH to 8.0-8.5.
4. The method for screening and separating sulfate-reducing bacteria by using the improved dish-stacking plate method according to claim 1, wherein the method comprises the following steps: in the step (a), firstly, a soil sample is subjected to screening pretreatment by using a screen, then, the soil is added into a sterilized sulfate reducing bacteria liquid enrichment culture medium according to the proportion of 1g: 200-.
5. The method for screening and separating sulfate-reducing bacteria by using the improved dish-stacking plate method according to claim 1, wherein the method comprises the following steps: the primary enrichment time in the step (a) is 7d, and then the inoculation quantity of 5 per mill is used for carrying out the transfer culture for 2 to 3 times in a new sulfate reducing bacteria liquid enrichment culture medium, wherein the time duration of each time is 4 d.
6. The method for screening and separating sulfate-reducing bacteria by using the improved dish-stacking plate method according to claim 1, wherein the method comprises the following steps: diluting the enriched bacterial liquid to 10 in the step (b)-4、10-5、10-6Three concentrations, followed by addition of 0.5wt% L to the dilutionCysteine and 0.5wt% ascorbic acid and 0.5wt% ferrous ammonium sulphate.
7. The method for screening and separating sulfate-reducing bacteria by using the improved dish-stacking plate method according to claim 1, wherein the method comprises the following steps: pouring 1/2-high 55-DEG C screening culture medium into a plurality of culture dishes in the step (b), condensing, sucking 0.2-0.3mL of diluent with different concentrations, quickly coating the diluent on each flat plate, pouring 1/3-high homogeneous screening culture medium into the culture dishes after the diluent permeates for a period of time to form a second strain growth layer, pouring the homogeneous culture medium until the overflowing form forms a third agar layer, covering the culture dishes, sealing, and placing at 30-DEG C for constant-temperature culture for 2-3d until obvious black colonies appear.
8. The method for screening and separating sulfate-reducing bacteria by using the improved dish-stacking plate method according to claim 1, wherein the method comprises the following steps: in the step (c), the streak isolation culture temperature is 30 ℃, and the streak isolation culture is performed for 3-4d and 2-3 times each time.
9. The method for screening and separating sulfate-reducing bacteria by using the improved dish-stacking plate method according to claim 1, wherein the method comprises the following steps: and (d) inoculating the single colony obtained by streaking separation into a preservation culture medium by adopting a needle punching method, and culturing at a constant temperature of 30 ℃ until obvious filamentous fungi appear, and then placing in a low-temperature environment for refrigeration.
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