CN107841472B - Non-decarboxylation lechlenibacter with phosphate solubilizing capability and application thereof - Google Patents
Non-decarboxylation lechlenibacter with phosphate solubilizing capability and application thereof Download PDFInfo
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- CN107841472B CN107841472B CN201711101750.8A CN201711101750A CN107841472B CN 107841472 B CN107841472 B CN 107841472B CN 201711101750 A CN201711101750 A CN 201711101750A CN 107841472 B CN107841472 B CN 107841472B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
Abstract
The invention relates to the field of microorganisms, and particularly relates to non-decarboxylation lechlenibacter with phosphate solubilizing capability and application thereof. The preservation number of the non-decarboxylation lux strain MRP-1 is CGMCC No. 14561. The strain has a stable activation effect on insoluble phosphorus, can survive and play a role in a high heavy metal pressure environment, and has the characteristics of easy culture and low cost. The strain obtained by screening is environment-friendly, does not produce secondary pollution, and can be used for plant-microorganism combined remediation of soil heavy metal pollution.
Description
Technical Field
The invention relates to the field of microorganisms, and particularly relates to non-decarboxylation lechlenibacter with phosphate solubilizing capability and application thereof.
Background
At present, the problem of heavy metal pollution of soil is increasingly highlighted, a soil bioremediation technology is a process of absorbing, converting, enriching and transferring heavy metals by using specific plants, microorganisms or protozoa so as to restore the normal ecological function of a soil system, wherein the plant-microorganism combined remediation is developed into an important way of the bioremediation technology.
The phosphate solubilizing microorganism can convert the insoluble phosphorus in the soil into the phosphorus which can be absorbed and utilized by plants by depending on the metabolite of the phosphate solubilizing microorganism or by the synergistic effect with other organisms, and has multiple plant growth promoting functions. However, the existing phosphate solubilizing microorganisms cannot play a role in the heavy metal contaminated soil area because the existing phosphate solubilizing microorganisms do not have heavy metal resistance. Therefore, it is necessary to find suitable phosphate solubilizing microorganisms with heavy metal resistance, so that the phosphate solubilizing microorganisms can protect plant roots and promote plant growth in the plant-microorganism combined remediation process of heavy metal contaminated soil, and the survival competitiveness and remediation efficiency of heavy metal remediation plants are enhanced.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a non-decarboxylation lux strain with higher phosphate solubilizing capability.
It is a further object of the present invention to provide the use of the above-mentioned bacteria, under heavy metal pressure, heavy metals including: lead, cadmium and zinc.
The invention screens the non-decarboxylation lux strain MRP-1 with higher phosphate-solubilizing ability from the heavy metal contaminated soil, and the preservation number is CGMCC No. 14561.
The strain obtained by screening has a stable activation effect on insoluble phosphorus, can survive and play a role in a high heavy metal pressure environment, and has the characteristics of easy culture and low cost. The strain obtained by screening is environment-friendly, does not produce secondary pollution, and can be used for plant-microorganism combined remediation of soil heavy metal pollution.
Drawings
FIG. 1 shows the effect of non-decarboxylating lechlehem bacteria on the activation of insoluble tricalcium phosphate;
FIG. 2 is a graph showing the change in the soluble phosphorus content of activated bacteria of the species Lectochilus decarbolyticus during 7 days of culture;
FIG. 3 shows the 7-day growth and pH change of non-decarboxylated lechlenibacter under lead pressure;
FIG. 4 shows the 7-day growth and pH change of non-decarboxylated luxes under cadmium pressure;
FIG. 5 shows the change in the amount of growth of non-decarboxylated lechlenibacter 7 days and pH under zinc pressure.
The non-decarboxylating leclerian strain (Leclercia adecaboylata) MRP-1 was deposited in the general microbiological center of the china committee for culture collection of microorganisms at 22.08.2017 (No. 3 of west way No.1 of north chen facing yang, institute of microbiology, china academy of sciences, 100101) under the following deposition numbers: CGMCC No. 14561.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1 selection of resistant phosphate solubilizing Strain
Using heavy metal contaminated soil as a bacterial sourceThe collected soil samples were stored at-20 ℃. When in experiment, the soil sample is thawed to normal temperature, 10g of soil is weighed into 90mL of sterile water, and the soil is made into a soil suspension after shaking for 30min at 120 rpm. Gradient dilution, pair 10-5～10-30.1mL of bacterial liquid is respectively taken by a pipette for dilution gradient to be put into a tricalcium phosphate solid culture medium, the bacterial liquid is evenly coated and placed in a constant temperature incubator at 28 ℃ for 7d, the growth condition of bacterial colonies and the generation of phosphate solubilizing rings in a flat plate are observed (as shown in figure 1), the strains generating the phosphate solubilizing rings are separated and purified for many times by adopting a flat plate coating and partition marking method, and finally the strains with high-efficiency phosphate solubilizing capability are obtained and inoculated on a beef extract peptone slant culture medium and stored for later use in a refrigerator at 4 ℃.
Wherein, the tricalcium phosphate culture medium comprises the following components: 10.0g of glucose, 0.3g of sodium chloride, 0.3g of magnesium sulfate, 0.03g of manganese sulfate, 0.5g of ammonium sulfate, 0.3g of potassium chloride, 0.03g of ferric sulfate, 5.0g of calcium phosphate and 1000mL of distilled water, wherein the pH value is 7.2-7.4, 2.0% (m/v) of agar powder is required to be added into a solid culture medium, and all the culture media are sterilized for 30min at the temperature of 121 ℃ and the pressure of 110 Kpa.
The beef extract peptone liquid medium comprises the following components: 3.0g of beef extract, 5.0g of sodium chloride, 10g of peptone and 1000mL of distilled water, adjusting the pH to about 7.2 by using sodium hydroxide or hydrochloric acid solution, and sterilizing for 30min at the temperature of 121 ℃ and under the condition of 110 kPa.
Inoculating the screened strains into a tricalcium phosphate culture medium under the conditions that the concentrations of heavy metals of lead, cadmium and zinc are 0, 0.5, 1, 2, 4, 8 and 12mM, culturing for 7 days in a constant temperature incubator at 28 ℃, and then selecting strains which can grow colonies on a plate culture medium.
And (3) storing the screened strain in a slant culture medium, amplifying a 16SrDNA fragment by using primers 27F (5'-AGA GTT TGA TCC TGGCTC AG-3') and 1492R (5'-TAC GGC TAC CTT GTT ACG ACT T-3') and using a PCR (polymerase chain reaction) technology, sequencing an amplification product to obtain a 16S rDNA sequence of the strain, comparing, and identifying the strain as non-decarboxylating lechleri (Leclercia adecaboxylinata).
Example 2 determination of phosphate solubilizing ability of Strain non-decarboxylating Leuconostoc (Leclercia adecaboxylata)
Preparing a seed solution: and (3) filling 50mL of beef extract peptone liquid culture medium in a 150mL triangular conical flask, and sterilizing. A ring of non-decarboxylating lechi (Leclercia adecarboxylata) thallus stored in a slant culture medium is selected and inoculated into a beef extract peptone liquid culture medium, and the beef extract peptone liquid culture medium is cultured at 120rpm and 37 ℃ for 24 hours to obtain a seed liquid.
And taking 1mL to 50mL of activated bacterium liquid tricalcium phosphate liquid culture medium, culturing for 7d under the conditions of 120rpm and 30 ℃, and setting a control.
Drawing a soluble phosphorus standard curve: respectively absorb 10mg/L of K2HPO40.0, 1.0, 2.0, 3.0, 4.0 and 5.0mL of standard solution is put into a 50mL colorimetric tube with a plug, a little water is firstly added, 2-3 drops of dinitrophenol are added, 100g/L of sodium carbonate and 50mL/L of sulfuric acid solution are used for regulating the color to be yellow, 5mL of molybdenum-antimony color-resisting agent is added, the mixture is shaken up and down reversely, water is added for fixing the volume to 50mL, and the solution with the concentration of 0, 0.2, 0.4, 0.6, 0.8 and 1.0mg/L is prepared. Standing for 30min, measuring absorbance at wavelength of 700nm, and drawing standard curve with absorbance as ordinate and phosphorus concentration (mg/L) as abscissa.
Determining the content of soluble phosphorus in the culture solution: taking 5mL of bacterial liquid into a 10mL centrifuge tube, centrifuging at 5000rpm for 10min, taking supernatant to pass through a membrane, then taking 0.1mL to 50mL of colorimetric tube with a plug, adding 2-3 drops of dinitrophenol, adjusting the color to be yellow by using 100g/L sodium carbonate and 50mL/L sulfuric acid solution, adding 5mL of molybdenum-antimony anti-color-developing agent, shaking up and down in an inverted manner, adding water to fix the volume to 50mL, standing for 30min, measuring absorbance at 700nm wavelength by using an ultraviolet-visible spectrophotometer, and calculating the content of soluble phosphorus in the supernatant by contrasting a standard curve of soluble phosphorus.
As shown in FIG. 2, after culturing the non-decarboxylating bacterium Leclercia adeccoria (Leclercia adeccarboxylata) in a tricalcium phosphate liquid culture medium for 24h, the soluble phosphorus content is increased from 5mg/L to 158mg/L, the slightly soluble tricalcium phosphate is continuously dissolved in the subsequent growth process, and the soluble phosphorus content reaches 218mg/L after culturing for 7 days.
Example 3 growth of non-decarboxylated Lexella under heavy Metal lead pressure and pH Change of the culture
Preparing a seed solution: and (3) filling 50mL of beef extract peptone liquid culture medium in a 150mL triangular conical flask, and sterilizing. A ring of non-decarboxylating lechi (Leclercia adecarboxylata) thallus stored in a slant culture medium is selected and inoculated into a beef extract peptone liquid culture medium, and the beef extract peptone liquid culture medium is cultured at 120rpm and 37 ℃ for 24 hours to obtain a seed liquid.
Inoculating the activated strain into a sterilized tricalcium phosphate culture medium containing heavy metal lead, wherein the preparation method comprises the following steps: the lead nitrate stock solution with the concentration of 500mM is used, and 0.1, 0.2mL to 50mL of tricalcium phosphate culture medium is absorbed to prepare the culture conditions with the heavy metal concentration of 1, 2 mM. Culturing at 30 deg.C and 120rpm for 7 d. Measuring OD600And pH.
As shown in FIG. 3, the non-decarboxylated bacterium lux was slightly inhibited in the growth of the first three days, OD600The value decreased, but as the culture time extended, the amount of growth gradually recovered and approached the blank. In addition, in the presence of heavy metal lead, the non-decarboxylated lechlenibacter bacteria still maintain high phosphate solubilizing ability, and after 3 days of culture, the pH of the culture solution is reduced to about 5, and the pH of the blank culture solution is about 4.5.
Example 4 growth of non-decarboxylated Lexella under heavy metal cadmium pressure and pH Change of the culture
Inoculating the activated strain into a sterilized tricalcium phosphate culture medium containing heavy metal cadmium, wherein the preparation method comprises the following steps: and (3) sucking 0.01mL to 50mL of tricalcium phosphate culture medium by using a cadmium chloride stock solution with the concentration of 500mM to prepare a culture condition with the heavy metal concentration of 0.1 mM. Cultured at 30 ℃ for 7d at 120rpm, and then the OD thereof was measured600And the pH.
As shown in FIG. 4, in the presence of 0.1mM cadmium, the growth of the non-decarboxylated bacterium Leuconostoc was slightly inhibited on the first day, but as the culture time was prolonged, the amount of growth gradually recovered and approached the blank. The non-decarboxylated lechlenibacter can keep higher phosphate-solubilizing capability under the condition of cadmium, and after 2 days of culture, the pH value of the culture solution is reduced to about 4.5, which is close to the blank.
Example 5 growth of Nodecarboxylic luxes under pressure of heavy Metal Zinc and pH Change of the culture solution
Inoculating the activated strain into sterilized tricalcium phosphate culture medium containing heavy metal zinc, and its preparation methodComprises the following steps: the zinc nitrate stock solution with the concentration of 500mM is used, and 0.03mL to 50mL of tricalcium phosphate culture medium is absorbed to prepare the culture condition with the heavy metal concentration of 0.3 mM. Cultured at 30 ℃ for 7d at 120rpm, and then the OD thereof was measured600And the pH.
As shown in FIG. 5, in the presence of 0.3mM zinc, the growth of non-decarboxylated bacteria was stable, OD 3 days before culture600Slightly decreased, but as the culture time was prolonged, the amount of growth gradually recovered and approached the blank. The non-decarboxylation lechler bacterium can keep good phosphate-solubilizing capability under the condition of zinc, and the pH value of the culture solution can be reduced to about 4.5 after 1 day of culture, which is basically consistent with the blank.
Claims (3)
1. The decarboxylation-free lux strain MRP-1 (Leclercia adecaboxylinata) is characterized in that the preservation number of the decarboxylation-free lux strain MRP-1 is CGMCC No. 14561.
2. Use of the non-decarboxylating lecr strain MRP-1 according to claim 1 for the degradation of phosphorus.
3. Use of the strain of decarboxylative lux MRP-1 according to claim 1 for the remediation of heavy metals contaminated with metals selected from the group consisting of lead, cadmium and zinc.
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| CN109971781B (en) * | 2019-03-29 | 2020-12-18 | 中国人民解放军军事科学院军事医学研究院 | Novel transposon Tn6505 and application thereof |
| CN110699348B (en) * | 2019-06-28 | 2021-08-17 | 北京林业大学 | Phosphorus-containing microbial capsule and preparation method and application thereof |
| CN110592066B (en) * | 2019-09-06 | 2021-09-14 | 北京林业大学 | Charcoal-loaded nano zero-valent iron coupled phosphate solubilizing bacterium immobilized pellet and preparation method and application thereof |
| CN110624949B (en) * | 2019-09-30 | 2021-10-12 | 武汉工程大学 | Method for repairing excessive phosphorus pollution of phosphorite waste land by combining indigenous microorganisms and plants |
| CN111909708B (en) * | 2020-09-03 | 2022-08-26 | 中南大学 | Mining area soil remediation agent and preparation method and application thereof |
| CN115029287A (en) * | 2020-09-03 | 2022-09-09 | 中南大学 | Phosphate solubilizing bacterium agent and preparation method and application thereof |
| CN113186116B (en) * | 2021-02-08 | 2022-09-23 | 茅台学院 | Non-decarboxylation lechlenibacter NJ22 with lactic acid as carbon source and application thereof |
| CN114891688B (en) * | 2022-06-06 | 2023-07-07 | 中国长江三峡集团有限公司 | Phosphate-dissolving bacteria coupled iron-catabolite reducing bacteria system and application thereof in heavy metal fixation |
| CN115975884B (en) * | 2022-12-21 | 2023-06-30 | 中国科学院沈阳应用生态研究所 | Lekkera CYIJM6 and application thereof |
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| CN102690763B (en) * | 2012-02-23 | 2014-01-01 | 西北大学 | Enterobacter agglomerans with phosphate-solubilizing capacity, and application thereof |
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