CN110699293A - Method for screening bacteria with phosphate solubilizing function - Google Patents
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Abstract
The invention discloses a method for screening bacteria with a phosphate solubilizing function, which comprises the steps of preparing an inorganic phosphate bacteria culture medium, preparing a phosphate standard stock solution, preparing a phosphate standard cone working solution, measuring absorbance, inoculating strains and detecting the content of phosphorus in the liquid inorganic phosphate culture medium by adopting a molybdenum-antimony anti-spectrophotometry method. The invention can screen out bacteria with different phosphate-solubilizing functions from microbial bacteria, proves that the bacillus and the klebsiella are stronger in phosphate-solubilizing than acinetobacter and proteus mirabilis, and have species and individual difference, thereby providing favorable guarantee for the subsequent research of the microbial bacteria.
Description
Technical Field
The invention relates to the technical field of microbial bacteria screening, in particular to a bacterial screening method with a phosphate solubilizing function.
Background
In the field of microbial bacteria, bacillus is widely distributed and has strong adaptability, wherein bacillus subtilis and bacillus licheniformis are common probiotics and have multiple biological functions. The closest prior art to the present invention:
li Wen, Mo gang ao, Sunxin, Wang pottery, Li Tong Xiang, Liu, Wang SAMING. Acinetobacter JL-1 Performance on degradation of phosphorus research [ J ] Jiangsu agricultural science, 2019,47(12): 311) 315. Acinetobacter (Acinetobacter indicus) JL-1 was used as a strain, and the phosphorus-solubilizing performance was determined by using an inorganic phosphorus liquid medium. Under the optimal condition, the phosphorus dissolving amount is highest at 48h and is 61.02 mu g/mL. However, the phosphorus-solubilizing ability of proteus mirabilis is not confirmed in this document, and there is no report on the phosphorus-solubilizing ability of proteus mirabilis.
Disclosure of Invention
The invention aims to provide a bacteria screening method with a phosphate solubilizing function, which can carry out preliminary screening on the phosphate solubilizing function of bacillus subtilis, klebsiella, bacillus megaterium, bacillus licheniformis, waxy, brevibacterium paradoxum, bacillus alopecuroides, acinetobacter and proteus mirabilis in strains so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for screening bacteria with phosphate solubilizing function comprises the following steps:
s1: preparing an inorganic phosphorus bacteria culture medium, comprising: (NH)4)2SO4：0.5g/L；FeSO4·7H2O：0.03g/L；MnCl2：0.03g/L；KCl：0.3g/L；MgSO4·7H20: 0.3 g/L; glucose: 10 g/L; NaCl: 0.3 g/L; agar: 15 g/L; ca3(PO4)2：5g/L。
S2: preparing a phosphorus standard stock solution, wherein rho P is 100 mu g/mL; weighing 0.439g of monopotassium phosphate, drying in an oven, dissolving in 200mL of water, adding 5mL of concentrated sulfuric acid, transferring into a 1000mL volumetric flask, and diluting to a marked line;
s3: preparing a phosphorus standard vertebral working solution, wherein rho N is 5 mu g/mL, sucking 5.00mL of phosphorus standard stock solution into a 100mL volumetric flask, and diluting to a scale;
s4: respectively adding 0.00, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20, 1.40 and 1.60mL of phosphorus standard working solution into 6 10mL colorimetric tubes, adding 1mL of molybdenum-antimony color-resisting agent, adding water to the marked line, and shaking up; standing at 25 deg.C for 30min, measuring absorbance with 10mm cuvette and water as reference at wavelength of 700 nm;
s5: inoculating the strain: sucking 1-1.5 mL of bacteria liquid into a sterile centrifugal tube, centrifuging for 5min after 5000-5 mL, discarding the supernatant, resuspending with sterile water, repeating for three times, washing off the culture medium with thallus residue, inoculating into a phosphorus liquid culture medium according to the inoculum size of 10%, simultaneously establishing a group of control groups, and culturing for 6 days at 34.5 ℃ after 180 revolutions;
s6: detecting the phosphorus content of the liquid inorganic phosphorus culture medium by adopting a molybdenum-antimony anti-spectrophotometry method: sucking 5 mu L of bacterial liquid, dropping the bacterial liquid on an inorganic phosphorus bacteria culture medium for 3 times, culturing for 6 days, observing the growth condition of the bacteria and whether a phosphorus-dissolving ring exists in 6 days, and selecting the bacterial strain with the solubility index larger than 2 according to the solubility index EDI (solubility index EDI) which is the diameter of the phosphorus-dissolving ring/the diameter of a bacterial colony.
Further, no agar was added to the liquid inorganic phosphorus medium in S1.
Further, the phosphorus concentrations in the standard series after shaking up in S4 were 0.00,0.10,0.20,0.30,0.40,0.50,0.60,0.70,0.80mg/L in order.
Further, after the absorbance measurement in S4, a standard curve is drawn with the blank corrected absorbance of the reagent as the ordinate and the corresponding phosphorus concentration mg/L as the abscissa.
Compared with the prior art, the invention has the beneficial effects that:
the method for screening bacteria with the phosphate solubilizing function can screen bacteria with different phosphate solubilizing functions from microbial bacteria, proves that bacillus and klebsiella are stronger in phosphate solubilizing than acinetobacter and proteus mirabilis and have species and individual difference, and provides favorable guarantee for the subsequent research of the microbial bacteria.
Drawings
FIG. 1 is a graph showing the results of a soluble phosphorus content standard curve according to the present invention;
FIG. 2 is a bar chart of the screening results of the phosphate solubilizing bacteria of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the embodiment of the invention: provides a method for screening bacteria with phosphate solubilizing function, which comprises the following steps:
the first step is as follows: preparing an inorganic phosphorus bacteria culture medium, comprising: (NH)4)2SO4：0.5g/L；FeSO4·7H2O：0.03g/L；MnCl2：0.03g/L；KCl：0.3g/L；MgSO4·7H20: 0.3 g/L; glucose: 10 g/L; NaCl: 0.3 g/L; agar: 15 g/L; ca3(PO4)2: 5 g/L; see table 1 below:
table 1: inorganic phosphorus bacteria culture medium component
Wherein agar is not added in the liquid inorganic phosphorus culture medium.
The second step is that: preparing a phosphorus standard stock solution, wherein rho P is 100 mu g/mL; weighing 0.439g of monopotassium phosphate, drying in an oven, dissolving in 200mL of water, adding 5mL of concentrated sulfuric acid, transferring into a 1000mL volumetric flask, and diluting to a marked line;
the third step: preparing a phosphorus standard vertebral working solution, wherein rho N is 5 mu g/mL, sucking 5.00mL of phosphorus standard stock solution into a 100mL volumetric flask, and diluting to a scale;
the fourth step: respectively adding 0.00, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20, 1.40 and 1.60mL of phosphorus standard working solution into 6 10mL colorimetric tubes, adding 1mL of molybdenum-antimony color-resisting agent, adding water to the marked line, and shaking up; standing at 25 deg.C for 30min, measuring absorbance with 10mm cuvette and water as reference at wavelength of 700 nm;
the fifth step: inoculating the strain: sucking 1-1.5 mL of bacteria liquid into a sterile centrifugal tube, centrifuging for 5min after 5000-5 mL, discarding the supernatant, resuspending with sterile water, repeating for three times, washing off the culture medium with thallus residue, inoculating into a phosphorus liquid culture medium according to the inoculum size of 10%, simultaneously establishing a group of control groups, and culturing for 6 days at 34.5 ℃ after 180 revolutions;
and a sixth step: detecting the phosphorus content of the liquid inorganic phosphorus culture medium by adopting a molybdenum-antimony anti-spectrophotometry method: sucking 5 mu L of bacterial liquid, dropping the bacterial liquid on an inorganic phosphorus bacteria culture medium for 3 times, culturing for 6 days, observing the growth condition of the bacteria and whether a phosphorus-dissolving ring exists in 6 days, and selecting the bacterial strain with the solubility index larger than 2 according to the solubility index EDI (solubility index EDI) which is the diameter of the phosphorus-dissolving ring/the diameter of a bacterial colony.
In the above examples, the phosphorus concentrations in the standard series after shaking in step four were 0.00,0.10,0.20,0.30,0.40,0.50,0.60,0.70,0.80mg/L in order.
In the above examples, after the absorbance measurement, the absorbance of the reagent blank correction was plotted as the ordinate and the corresponding phosphorus concentration mg/L was plotted as the abscissa, thereby drawing a standard curve.
The experimental results are as follows:
referring to FIG. 1, the results of the soluble phosphorus content standard curve are shown.
See table 2 below:
table 2: phosphate solubilizing bacteria screening result table
Referring to fig. 2, a histogram of the phosphate solubilizing bacteria screening results is shown, wherein Bs: b, bacillus subtilis; bv: bacillus belgii; kq: klebsiella pneumoniae pseudo; kp: klebsiella pneumoniae; kv: klebsiella variicola; bm: bacillus megaterium; bacillus licheniformis; bp: brevibacillus parabrevis Bc: bacillus cereus; bal: high-altitude bacillus; ab: acinetobacter baumannii; aj: acinetobacter junii; a: acinetobacter; ac: acinetobacter calcoaceticus; pm: proteus mirabilis.
And (3) analyzing an experimental result:
the effective phosphorus content in the three culture media inoculated with the bacillus subtilis is about 200mg/L, which shows that the difference of the bacillus subtilis with different sources in the aspect of phosphorus dissolving is not obvious; the experiments of the invention also prove that the Klebsiella and the Bacillus megaterium have very strong capability in the aspect of phosphate solubilizing, the Bacillus megaterium is the strain which is reported to have the phosphate solubilizing capability at the earliest time, the experiments of the invention also prove that the Bacillus licheniformis has very high phosphate solubilizing capability, but the strain with high-efficiency phosphate solubilizing capability is separated, the content of phosphorus in a culture medium exceeds 300mg/L, and the Bacillus calcoaceticus, the Bacillus megaterium also has high phosphate solubilizing capability.
In summary, the following steps: the method for screening bacteria with the phosphate solubilizing function can screen bacteria with different phosphate solubilizing functions from microbial bacteria, proves that bacillus and klebsiella are stronger in phosphate solubilizing than acinetobacter and proteus mirabilis and have species and individual difference, and provides favorable guarantee for the subsequent research of the microbial bacteria.
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 (4)
1. A method for screening bacteria with a phosphate solubilizing function is characterized by comprising the following steps:
s1: preparing an inorganic phosphorus bacteria culture medium, comprising: (N)H4)2SO4：0.5g/L；FeSO4·7H2O：0.03g/L；MnCl2：0.03g/L；KCl：0.3g/L；MgSO4·7H20: 0.3 g/L; glucose: 10 g/L; NaCl: 0.3 g/L; agar: 15 g/L; ca3(PO4)2：5g/L。
S2: preparing a phosphorus standard stock solution, wherein rho P is 100 mu g/mL; weighing 0.439g of monopotassium phosphate, drying in an oven, dissolving in 200mL of water, adding 5mL of concentrated sulfuric acid, transferring into a 1000mL volumetric flask, and diluting to a marked line;
s3: preparing a phosphorus standard vertebral working solution, wherein rho N is 5 mu g/mL, sucking 5.00mL of phosphorus standard stock solution into a 100mL volumetric flask, and diluting to a scale;
s4: respectively adding 0.00, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20, 1.40 and 1.60mL of phosphorus standard working solution into 6 10mL colorimetric tubes, adding 1mL of molybdenum-antimony color-resisting agent, adding water to the marked line, and shaking up; standing at 25 deg.C for 30min, measuring absorbance with 10mm cuvette and water as reference at wavelength of 700 nm;
s5: inoculating the strain: sucking 1-1.5 mL of bacteria liquid into a sterile centrifugal tube, centrifuging for 5min after 5000-5 mL, discarding the supernatant, resuspending with sterile water, repeating for three times, washing off the culture medium with thallus residue, inoculating into a phosphorus liquid culture medium according to the inoculum size of 10%, simultaneously establishing a group of control groups, and culturing for 6 days at 34.5 ℃ after 180 revolutions;
s6: detecting the phosphorus content of the liquid inorganic phosphorus culture medium by adopting a molybdenum-antimony anti-spectrophotometry method: sucking 5 mu L of bacterial liquid, dropping the bacterial liquid on an inorganic phosphorus bacteria culture medium for 3 times, culturing for 6 days, observing the growth condition of the bacteria and whether a phosphorus-dissolving ring exists in 6 days, and selecting the bacterial strain with the solubility index larger than 2 according to the solubility index EDI (solubility index EDI) which is the diameter of the phosphorus-dissolving ring/the diameter of a bacterial colony.
2. The method for screening bacteria with phosphate solubilizing function according to claim 1, wherein agar is not added to the liquid inorganic phosphate medium in S1.
3. The method for screening bacteria with phosphate solubilizing function according to claim 1, wherein the concentration of phosphorus in the standard series after shaking up in S4 is 0.00,0.10,0.20,0.30,0.40,0.50,0.60,0.70,0.80mg/L in sequence.
4. The method for screening bacteria with phosphate solubilizing function according to claim 1, wherein a standard curve is drawn by taking reagent blank corrected absorbance as ordinate and corresponding phosphorus concentration mg/L as abscissa after absorbance measurement in S4.
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