CN114410509B - Microbial inoculum, composition and application of gas-producing enterobacter combined Klebsiella oxytoca - Google Patents
Microbial inoculum, composition and application of gas-producing enterobacter combined Klebsiella oxytoca Download PDFInfo
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Abstract
The invention provides a bacterial agent of gas-producing enterobacter combined gas-producing klebsiella, which is used for treating lead-cadmium polluted soil and comprises gas-producing enterobacter and gas-producing klebsiella; the enterobacter aerogenes is enterobacter aerogenes (Enterobacter aerogenes) W6, latin classification is Enterobacter aerogenes, and is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2021, 7 and 13, and the preservation number is CGMCC No.22888; the Klebsiella oxytoca is Klebsiella oxytoca (Klebsiella aerogenes) Wn, latin classification is Klebsiella aerogenes, and is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2021, 7 months and 13 days, and the preservation number is CGMCC No.22889.
Description
Technical Field
The invention relates to a microorganism and application thereof, in particular to a microbial agent, a composition and application of a gas-producing enterobacter combined Klebsiella aerogenes.
Background
Through industrial development for decades, a large number of mining and smelting causes the excessive heavy metal content of soil, and threatens the survival of human beings. The world average annual emissions of lead (Pb) is statistically about 500 ten thousand tons and cadmium (Cd) about 3 ten thousand tons. The lead-cadmium pollution in the soil has migration capability, and can cause the lead-cadmium content of surface water and underground water to exceed the standard after being subjected to the effects of rain leaching, vertical infiltration, surface runoff and the like. After drinking the underground water polluted by heavy metal lead and cadmium or eating the food chain product polluted by lead and cadmium, the health diseases of cancerogenesis, disease and mutation can be caused. Therefore, the technology of heavy metal pollution which has low cost, no pollution, high efficiency and long-term soil restoration becomes the current primary consideration.
At present, the soil heavy metal restoration technology mainly comprises a physical restoration method, a chemical restoration method, a biological restoration method and the like. Compared with other restoration technologies, the biological-chemical composite restoration technology is a method for treating soil heavy metals by using a heavy metal in-situ restoration technology with wide application range, low cost and short restoration period. The essence of the bio-chemical composite remediation technology is that biological and chemical substrates are put into heavy metal contaminated soil, and chemical reagents and heavy metals react in a series of complex ways under the specific action of the organisms, so that the aim of solidifying and stabilizing the heavy metals in the soil is fulfilled, for example: the method adopts phosphate-dissolving microorganism to match with insoluble phosphorus source to treat heavy metal polluted soil, thus not only solving the problem that the chemical restoration method is easy to cause secondary pollution, but also solving the problem that the restoration period of the biological restoration method is too long.
Although the existing bio-chemical composite remediation technology can be used for treating lead-cadmium polluted soil to a certain extent, microorganisms and treatment methods adopted in the prior art are not particularly outstanding in practical applicability and are easy to cause a reverse dissolution phenomenon. The effectiveness and long-term stability of soil pollution remediation are still difficult technical problems to solve up to now.
In view of the above, it is necessary to provide a bacterial agent, composition and application of a combination of enterobacter aerogenes and klebsiella aerogenes, so as to solve or at least alleviate the above-mentioned technical defects of poor effectiveness and long-term stability of soil remediation.
Disclosure of Invention
The invention mainly aims to provide a microbial inoculum, a composition and application of gas-producing enterobacter combined Klebsiella oxytoca, and aims to solve the technical problems of poor effectiveness and long-term stability of soil treatment in the prior art.
In order to achieve the aim, the invention provides a microbial inoculum of enterobacter aerogenes combined with klebsiella aerogenes, which is used for treating lead-cadmium polluted soil and comprises enterobacter aerogenes and klebsiella aerogenes;
wherein the enterobacter aerogenes is enterobacter aerogenes (Enterobacter aerogenes) W6, latin classification is Enterobacter aerogenes, and is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2021, 7 months and 13 days, and the preservation number is CGMCC No.22888;
the Klebsiella oxytoca is Klebsiella oxytoca (Klebsiella aerogenes) Wn, latin classification is Klebsiella aerogenes, and is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2021, 7 months and 13 days, and the preservation number is CGMCC No.22889.
The invention also provides application of the microbial agent in treating lead-cadmium polluted soil.
The invention also provides a composition for treating lead-cadmium polluted soil, which is characterized by comprising a component A, a component B, a component C and a component D;
wherein the component a comprises a phosphorus source;
the component B comprises the microbial agent as described in any one of the above;
the component C comprises an aqueous solution containing glucose, magnesium chloride, magnesium sulfate, ammonium sulfate and potassium chloride;
the component D comprises an aqueous solution containing tryptone, yeast extract and sodium chloride.
Further, the phosphorus source is tricalcium phosphate.
The invention also provides a treatment method of the lead-cadmium polluted soil, which adopts the composition as any one of the above to treat the lead-cadmium polluted soil.
Further, the method comprises the steps of: applying the component A and a first mixed solution containing the component B and the component C to the soil to be treated in a first time period;
applying a second mixed solution containing the component B and the component D to the soil to be treated for a second period of time;
wherein the second period of time is later than the first period of time.
Further, the method further comprises the following steps: and applying the first mixed liquid to the soil to be remediated between the first time period and the second time period.
Further, between the first time period and the second time period, applying the first mixed liquor to the soil to be treated for a plurality of times, wherein the single application amount is consistent with the total application amount of the first mixed liquor in the first time period;
and applying the second mixed solution to the soil to be treated for a plurality of times in the second time period, wherein the single application amount is consistent with the total application amount of the first mixed solution in the first time period.
Further, the first time period and the second time period have an interval duration of 14-16 days.
The Latin classification of the enterobacter aerogenes (Enterobacter aerogenes) W6 used in the invention is Enterobacter aerogenes, which is stored in China general microbiological culture Collection center (CGMCC), address: no. 3 of North Chen West Lu No. 1 in the Chaoyang area of Beijing, china, the preservation date is 2021, 7, 13 days, and the preservation number is CGMCC No.22888.
The enterobacter aerogenes (Enterobacter aerogenes) W6 is gram-negative bacteria separated from bottom mud of a domestic sewage sedimentation fermentation tank of Hunan leading foreign lake reclaimed water Limited company in Hunan province, and forms round, convex and off-white colonies during culture, the proper growth temperature of the enterobacter aerogenes (Enterobacter aerogenes) W6 is 30-35 ℃, the proper growth PH is 6.9-7.2, and the method is aerobic or facultative anaerobic.
The Latin classification of the Klebsiella oxytoca (Klebsiella aerogenes) Wn used in the invention is Klebsiella aerogenes, which is stored in China general microbiological culture Collection center (CGMCC), address: no. 3 of North Chen Xili No. 1, the preservation date is 2021, 7, 13 days, and the preservation number is CGMCC No.22889.
The Klebsiella aerogenes (Klebsiella aerogenes) Wn is gram-negative bacteria separated from bottom sludge of a domestic sewage sedimentation fermentation tank of a Hunan lead ocean lake reclaimed water Limited company in Hunan province; the colony is in a round shape with obvious bulges, a larger white mucus colony is formed on the agar medium, and yellow mucus is less in the middle zone. The proper growth temperature of Klebsiella aerogenes (Klebsiella aerogenes) Wn is 30-35 ℃, and the proper growth PH=6.9-7.2, aerobic or facultative anaerobic.
Compared with the prior art, the invention has the following advantages:
the invention provides a microbial agent for treating lead-cadmium polluted soil, which is beneficial to the phosphorus containing characteristics of enterobacter aerogenes (Enterobacter aerogenes) W6 and klebsiella aerogenes (Klebsiella aerogenes) Wn, and can be used together by using two strains with the same original sources, so that the strains can produce synergistic effect, and the diversity of phosphate-solubilizing bacteria in the soil is improved; in addition, the invention also utilizes the characteristics of acid and alkali production of microorganisms in different culture mediums, not only can promote the dissolution of phosphorus by the microorganisms, but also can avoid the reverse dissolution of passivation products.
Specifically, the enterobacter aerogenes (Enterobacter aerogenes) W6 combined with tricalcium phosphate can passivate lead and cadmium by 99.7 percent within 15 days; in the soil test, on the 7 th day, the highest removal rate of the effective state contents of Pb and Cd in the soil is 45.5% and 40.9% respectively by the treatment of the enterobacter aerogenes (Enterobacter aerogenes) W6 composite tricalcium phosphate; the highest removal rate of the effective state content is respectively reduced to 30.1 percent and 23.5 percent at 15 days, and the removal rates of the effective state content of Pb and Cd are respectively reduced to 45 percent and 42 percent again at 30 days, so that the phenomenon of reverse dissolution is effectively inhibited.
The Klebsiella aerogenes (Klebsiella aerogenes) Wn combined with tricalcium phosphate can passivate lead and cadmium by 100 percent within 1 day; in a soil test, on the 7 th day, the highest removal rate of the effective state contents of Pb and Cd in the soil is 49% and 49.3% respectively by treating the Klebsiella aerogenes (Klebsiella aerogenes) Wn composite tricalcium phosphate; the highest removal rate of the effective state content is respectively reduced to 45.1% and 46.4% at 15 days, and the removal rates of the effective state content of Pb and Cd are respectively increased to 51% and 51.3% again at 30 days, so that the phenomenon of dissolution is effectively inhibited.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a plot of the phosphate solubilizing circle characteristics of E.aerogenes (Enterobacter aerogenes) W6 and Klebsiella aerogenes (Klebsiella aerogenes) Wn of example 1 grown on solid medium 2 for 7 days;
FIG. 2 is a graph showing the effect of phosphorus content on Enterobacter aerogenes (Enterobacter aerogenes) W6 in example 1;
FIG. 3 is a graph showing the effect of phosphorus content on Klebsiella aerogenes (Klebsiella aerogenes) Wn in example 1;
FIG. 4 is a construction diagram of a phylogenetic tree of E.aerogenes (Enterobacter aerogenes) W6 in example 1;
FIG. 5 is a diagram showing the construction of a phylogenetic tree of Klebsiella aerogenes (Klebsiella aerogenes) Wn in example 1;
FIG. 6 is an XRD pattern of the phosphorus-solubilizing and passivating lead-cadmium reaction product of E.aerogenes (Enterobacter aerogenes) W6 and Klebsiella aerogenes (Klebsiella aerogenes) Wn in example 2;
FIG. 7 shows the phosphorus-solubilizing effect and pH dependence of E.aerogenes (Enterobacter aerogenes) W6 in example 3 in different media;
FIG. 8 shows the phosphorus dissolution effect and pH dependence of Klebsiella aerogenes (Klebsiella aerogenes) Wn in different media in example 3.
The achievement of the object, functional features and advantages of the present invention will be further described with reference to the drawings in connection with the embodiments.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
It should be noted that all directional indicators (such as upper and lower … …) in the embodiments of the present invention are merely used to explain the relative positional relationship, movement conditions, etc. between the components in a specific posture (as shown in the drawings), and if the specific posture is changed, the directional indicator is changed accordingly.
Furthermore, descriptions such as those referred to as "first," "second," and the like, are provided for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying an order of magnitude of the indicated technical features in the present disclosure. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature.
Moreover, the technical solutions of the embodiments of the present invention may be combined with each other, but it is necessary to be based on the fact that those skilled in the art can implement the embodiments, and when the technical solutions are contradictory or cannot be implemented, it should be considered that the combination of the technical solutions does not exist, and is not within the scope of protection claimed by the present invention.
In order to complete the treatment of the lead-cadmium contaminated soil, the invention provides a bacterial agent of enterobacter aerogenes combined with klebsiella aerogenes, which is used for treating the lead-cadmium contaminated soil and comprises enterobacter aerogenes and klebsiella aerogenes;
wherein the enterobacter aerogenes is enterobacter aerogenes (Enterobacter aerogenes) W6, latin classification is Enterobacter aerogenes, and is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2021, 7 months and 13 days, and the preservation number is CGMCC No.22888;
the Klebsiella oxytoca is Klebsiella oxytoca (Klebsiella aerogenes) Wn, latin classification is Klebsiella aerogenes, and is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2021, 7 months and 13 days, and the preservation number is CGMCC No.22889.
The enterobacter aerogenes (Enterobacter aerogenes) W6 and the klebsiella aerogenes (Klebsiella aerogenes) Wn both have better phosphate solubilizing effect, and belong to the category of phosphate solubilizing bacteria. Wherein, within 7 days, the phosphorus dissolving amount of the enterobacter aerogenes (Enterobacter aerogenes) W6 can reach 622.2mg/L, and the phosphorus dissolving amount of the klebsiella aerogenes (Klebsiella aerogenes) Wn can reach 683.1mg/L. Both strains can convert indissolvable compound phosphorus in soil into soluble phosphorus, so that the method is used for passivating lead and cadmium, and the problems of water eutrophication, soil acidification, plant nutrition imbalance and the like caused by directly adding a soluble phosphate compound are avoided. Therefore, the lead-cadmium contaminated soil can be effectively treated by using the enterobacter aerogenes (Enterobacter aerogenes) W6 and the klebsiella aerogenes (Klebsiella aerogenes) Wn.
Specific passivation processes may include: the microorganism and the insoluble phosphorus source such as tricalcium phosphate in the microbial agent enter the contaminated soil, can fix heavy metals (such as Pb, cd, cu and Zn) and form highly insoluble metal phosphate precipitate, and remarkably reduces the mobility and usability of Pb and Cd, wherein the passivation product of cadmium is Ca 7.7 Cd 0.8 (PO 4 ) 8 (H 2 O) 2.4 The method comprises the steps of carrying out a first treatment on the surface of the The passivation product of lead is Pb 5 (PO 4 ) 3 Cl。
It should be understood that the invention also makes comparative researches on some strains with phosphate-dissolving capability in the existing phosphate-dissolving bacteria. For example: in the existing research, according to the disclosure in screening of a high-efficiency phosphate solubilizing bacterium and verification of phosphate solubilizing effect, tang Min (2020) and the like screen a high-efficiency phosphate solubilizing bacterium from farmlands: the bacillus belicus is identified by 16S r DNA analysis, the phosphorus dissolving amount reaches 495.4mg/L, and the phosphorus dissolving amount reaches 582.4mg/L after condition optimization. However, the phosphorus dissolution effect is still lower than those of W6 and Wn listed in the present embodiment of the invention.
On the basis, the enterobacter aerogenes (Enterobacter aerogenes) W6 and the klebsiella aerogenes (Klebsiella aerogenes) Wn are simultaneously used as the microbial inoculum for treating lead-separated polluted soil, so that the diversity of phosphate-dissolving bacteria can be improved; moreover, since the original sources of the two species of the present invention are the same and the efficacy is similar, a synergistic effect can be produced, for example: both can produce acid, and under the combined action, the PH dropping speed in the environment can be increased, so that the phosphorus dissolving efficiency can be further improved; in addition, the enterobacter aerogenes (Enterobacter aerogenes) W6 and the klebsiella aerogenes (Klebsiella aerogenes) Wn can produce organic acids and carbon dioxide for promoting the stabilization of heavy metal minerals.
In view of the above, the invention also provides an application of the microbial inoculum in the treatment of lead-cadmium polluted soil.
In order to facilitate the development of treatment work and further improve the treatment efficiency, the invention also provides a composition for treating lead-cadmium polluted soil, which comprises a component A, a component B, a component C and a component D;
wherein the component A comprises a phosphorus source, which refers to a poorly soluble phosphorus source, preferably tricalcium phosphate.
The component B comprises the microbial inoculum according to any embodiment.
The component C comprises an aqueous solution containing glucose, magnesium chloride, magnesium sulfate, ammonium sulfate and potassium chloride.
The component C is in a liquid state, and the preparation process of the component C can be as follows: weighing 48.7% of glucose, 24.3% of magnesium chloride, 1.2% of magnesium sulfate, 0.97% of ammonium sulfate, 0.49% of potassium chloride and 24.34% of tricalcium phosphate by mass percent (tricalcium phosphate can be sprayed into soil, so that tricalcium phosphate can not be added), and then adding water into a container for holding the weighed components to fix the volume; wherein the first preset mass is the sum of the masses of the glucose, the magnesium chloride, the magnesium sulfate, the ammonium sulfate, the potassium chloride and the tricalcium phosphate, and the mass-volume ratio of the first preset mass to the component C can be 2.055g:100ml.
For example: in preparing 100ml of component C, 1g of glucose, 0.5g of magnesium chloride, 0.025g of magnesium sulfate, 0.02g of ammonium sulfate, 0.01g of potassium chloride and 0.5g of tricalcium phosphate are added to 100ml of water as calculated; however, since tricalcium phosphate is previously applied to the soil in the actual treatment process, tricalcium phosphate may not be added to the component C, and 1g of glucose, 0.5g of magnesium chloride, 0.025g of magnesium sulfate, 0.02g of ammonium sulfate, and 0.01g of potassium chloride may be added to 100ml of water; of course, the solid may be weighed and then the volume may be fixed to 100ml.
The component D comprises an aqueous solution containing tryptone, yeast extract and sodium chloride.
The component D is in a liquid state, and the preparation process of the component D can be as follows: weighing 40% of tryptone, 20% of yeast powder and 40% of sodium chloride which account for the second preset mass according to the mass percentage, and then adding water into a container for holding the components to fix the volume; wherein the second preset mass is the total mass of tryptone, yeast extract and sodium chloride expected to be applied into the soil, and the mass-to-volume ratio of the second preset mass to the component D may be 2.5g:100ml.
For example: when preparing 100ml of component D, 1g of tryptone, 0.5g of yeast extract and 1g of sodium chloride can be added to 100ml of water; of course, the solid may be weighed and then the volume may be fixed to 100ml.
It is noted that although lead-cadmium and the like can be passivated by the dual action of microorganisms and a poorly soluble phosphorus source, although the phenomenon of dissolution occurs with the increase of the reaction time, for example: highly insoluble metal phosphate precipitates produce CdCO 3 、CdPO 4 And the like.
It is known that, through experimental study, the microorganisms in the microbial inoculum can generate acid production and phosphorus dissolution when being cultured in the component C; while in said component D, the alkali production phenomenon occurs and the occurrence of the reverse dissolution phenomenon is suppressed.
Therefore, the technology utilizes the characteristics of microorganisms cultivated in different components from the perspective of reaction products, regulates and controls the pH intensity from acid (cultivated in component C) to alkali (cultivated in component D), naturally fixes and stabilizes lead and cadmium, avoids occurrence of reverse dissolution, and creatively finds a long-acting mechanism for producing phosphorus and being applied to heavy metal pollution treatment through pH regulation and control of microorganisms.
Based on the composition, the invention also provides a treatment method of the lead-cadmium polluted soil, which adopts the composition according to any embodiment to treat the lead-cadmium polluted soil.
Specifically, the method for treating the lead-cadmium polluted soil comprises the following steps: applying the component A and the first mixed solution containing the component B and the component C to the soil to be treated in a first time period.
It should be noted that the first mixed solution may be similar to a culture solution after the microorganisms in the microbial inoculum are propagated in the component C, and the two microorganisms in the microbial inoculum may be separately propagated in the component C and applied to the soil, or may be co-cultured and applied to the soil. In the specific test process, liquid is usually directly taken from the culture solution of the microorganism, and part of tricalcium phosphate is contained in the culture solution during the early-stage strain culture, so that the first mixed solution can also contain tricalcium phosphate, and the actual effect is not influenced. In particular applications, the person skilled in the art can optionally decide whether to add part of the tricalcium phosphate to the component C, depending on the circumstances.
Applying a second mixed solution containing the component B and the component D to the soil to be treated for a second period of time; the second mixed solution can be analogized to a culture solution after the microorganisms are propagated in the component D, and two microorganisms in the microbial inoculum can be separately applied to soil after the culture of the component D or can be applied to the soil after the co-culture.
Wherein the second period of time is later than the first period of time.
In addition, the first mixed solution may be applied to the soil to be treated between the first period and the second period to maintain the soil moisture content and the activity of bacteria.
As an explanation of the above embodiment: the first time period is the starting time, namely, the component A (comprising a indissolvable phosphorus source) and the first mixed solution (namely, the component C with microorganisms in the microbial inoculum) are applied to the soil to be treated at the beginning, so that the passivation phenomenon of lead and cadmium in the soil occurs.
The interval duration between the first time period and the second time period may be 14-16 days, specifically, the interval between the second time period and the first time period may be 15 days, that is, after 15 days of the first time period, the second mixed solution may be applied to the soil to be treated, so as to inhibit occurrence of the dissolution phenomenon, thereby enhancing the passivation effect.
In addition, the first mixed solution is applied between the first time period and the second time period to enhance the phosphorus dissolving efficiency, and ensure the earlier generation of the phosphorus dissolving effect while promoting the growth of microorganisms so as to ensure the passivation of lead and cadmium in the earlier stage.
Further, the first mixed liquid is applied to the soil to be treated for a plurality of times between the first time period and the second time period, and the single application amount is consistent with the total application amount of the first mixed liquid in the first time period. And applying the second mixed solution to the soil to be treated for a plurality of times in the second time period, wherein the single application amount is consistent with the total application amount of the first mixed solution in the first time period.
For a further understanding of the invention, an illustration is now given:
example 1
Screening and identification of Enterobacter aerogenes (Enterobacter aerogenes) W6
1. Culture conditions: aerobic, temperature 30 ℃, initial ph=7 (i.e. initial PH of the culture medium is 7); wherein 150rpm/min shaking culture is adopted in the culture under the liquid condition.
2. Preparing a culture medium: liquid medium 1, liquid medium 2, solid medium 2.
The preparation process of the liquid culture medium 1 comprises the following steps: calculating the mass of the solid component according to the capacity and the solid-liquid concentration (25 g/L) which are prepared according to the requirement, weighing 40% tryptone, 20% yeast powder and 40% sodium chloride which account for the mass percent of the solid component, and fixing the volume after weighing;
the preparation process of the liquid culture medium 2 comprises the following steps: calculating the mass of solid components according to the capacity and the solid-liquid concentration (20.55 g/L) prepared according to the requirement, weighing 48.7% glucose, 24.3% magnesium chloride, 1.2% magnesium sulfate, 0.97% ammonium sulfate, 0.49% potassium chloride and 24.34% tricalcium phosphate according to the mass percentage of the solid components, and fixing the volume after weighing;
the solid medium 2 was added with 1.5% agar (mass/volume ratio, i.e., 1.5g agar was added to 100ml of the liquid medium 2) in comparison with the liquid medium 2.
3.1 g of sediment obtained from a domestic sewage sedimentation fermentation tank of Hunan precursor ocean lake reclaimed water Limited company in Hunan province, long-time sandy city is weighed and put into a sterilization centrifuge tube, 9ml of 0.9% physiological saline is added to prepare 10 -1 Placing the fungus suspension in a shaking table at 150rpm/min for culturingShaking for 20min in a incubator, taking out and standing for 2h.
4. Standing the suspension, and inoculating 2% of supernatant to the liquid culture medium 2 for specific culture; placing the mixture on a shaking table, keeping the temperature at 30 ℃ and 150rpm/min, and shake culturing for 2-3 d. Culturing for a period of time, and gradient diluting with 4.5ml of ultrapure water with dilution ratio of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Is a sample bacterial suspension of 10 -2 、10 -4 、10 -6 And respectively taking 200 mu l of bacterial suspension with multiple concentration gradients, sequentially and uniformly coating the bacterial suspension on a solid culture medium 2, inversely placing the coated culture dish into a constant temperature incubator, culturing at a constant temperature of 30 ℃ for 24 hours, observing and recording the sizes of bacterial colonies and phosphate solubilizing rings, marking the bacterial strains, and screening out 5 strains of phosphate solubilizing bacteria.
5. Respectively carrying out plate streak purification on the screened 5 phosphate solubilizing bacteria on a solid culture medium 2 (respectively selecting phosphate solubilizing bacteria standard plates with obvious phosphate solubilizing rings from the 5 phosphate solubilizing bacteria in a sterile operation table to be streaked on different solid culture mediums 2, placing the solid culture mediums in a 30 ℃ incubator for culturing for one week), and then further selecting two phosphate solubilizing bacteria with obvious phosphate solubilizing effects, and marking the two phosphate solubilizing bacteria as W6 and Wn; wherein, the characteristic of the phosphorus dissolving ring of W6 and Wn is shown in figure 1.
6. After phosphate solubilizing bacteria dissolve phosphate, qualitatively analyzing the effective phosphorus content in the fermentation liquor: molybdenum-antimony colorimetric method.
The single strains of W6 and Wn are respectively placed in different liquid culture mediums 2 for 7 days to be detected, and the specific detection process is as follows: taking 1% of bacterial suspension in a sterilized centrifuge tube, centrifuging at 10000rpm, taking 100 mu L of supernatant, adding a 50ml colorimetric tube, adding two thirds of ultrapure water to a scale line, adding 1ml of the prepared solution A and 2ml of the prepared solution B, uniformly mixing, fixing the volume to the scale line, standing for 15min, measuring an absorbance value on an ultraviolet-visible spectrophotometer, setting the wavelength to 700nm, and calculating the corresponding effective phosphorus content according to a standard curve. In addition, this example also performed a sterile blank under equivalent conditions.
7. After the measurement, it was concluded that: referring to FIGS. 2 and 3, the available phosphorus concentration in the blank test was far lower than that in the W6 and Wn culture solutions, and the phosphorus-dissolving effect was excellent when the W6 and Wn culture medium 2 was used as the nutrient source. Within 7 days, the phosphorus dissolving amount of W6 can reach 622.2mg/L, and the phosphorus dissolving amount of Wn can reach 683.1mg/L.
8. Identification of phosphate-solubilizing bacteria W6 and Wn: the strains were amplified and forward sequenced using primers and by comparing the sequences to those in the NCBI database.
(1) W6 has 99% similarity with Enterobacter aerogenes, and as shown in FIG. 4, the development tree is constructed and identified as Enterobacter aerogenes.
The 16s DNA gene sequence splicing measurement result of the enterobacter aerogenes W6 strain is as follows:
Tgcaagtcgagcggtagcacagagagcttgctctcgggtgacgagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaagtgggggaccttcgggcctcatgccatcagatgtgcccagatgggattagctagtaggtggggtaatggctcacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtatgaagaaggccttcgggttgtaaagtactttcagcgaggaggaaggcgttaaggttaataaccttagcgattgacgttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggctagagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctactcttgacatccagagaacttagcagagatgctttggtgccttcgggaactctgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcgattcggtcgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaagcgacctcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagatcagaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggagggcgcttacc
(2) Wn and Klebsiella aerogenes are 99% similar, and as shown in FIG. 5, the development tree is constructed and identified as Klebsiella aerogenes.
The 16s DNA gene sequence splicing measurement result of the Klebsiella aerogenes Wn strain is as follows:
Gccctcccgaaggttaagctacctacttcttttgcaacccactcccatggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgtagcattctgatctacgattactagcgattccgacttcatggagtcgagttgcagactccaatccggactacgacatactttatgaggtccgcttgctctcgcgaggtcgcttctctttgtatatgccattgtagcacgtgtgtagccctactcgtaagggccatgatgacttgacgtcatccccaccttcctccagtttatcactggcagtctcctttgagttcccgaccgaatcgctggcaacaaaggataagggttgcgctcgttgcgggacttaacccaacatttcacaacacgagctgacgacagccatgcagcacctgtctcagagttcccgaaggcaccaaagcatctctgctaagttctctggatgtcaagagtaggtaaggttcttcgcgttgcatcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcatttgagttttaaccttgcggccgtactccccaggcggtcgacttaacgcgttagctccggaagccacgcctcaagggcacaacctccaagtcgacatcgtttacggcgtggactaccagggtatctaatcctgtttgctccccacgctttcgcacctgagcgtcagtctttgtccagggggccgccttcgccaccggtattcctccagatctctacgcatttcaccgctacacctggaattctacccccctctacaagactctagcctgccagtttcgaatgcagttcccaggttgagcccggggatttcacatccgacttgacagaccgcctgcgtgcgctttacgcccagtaattccgattaacgcttgcaccctccgtattaccgcggctgctggcacggagttagccggtgcttcttctgcgagtaacgtcaatcgctaaggttattaaccttaacgccttcctcctcgctgaaagtactttacaacccgaaggccttcttcatacacgcggcatggctgcatcaggcttgcgcccattgtgcaatattccccactgctgcctcccgtaggagtctggaccgtgtctcagttccagtgtggctggtcatcctctcagaccagctagggatcgtcgcctaggtgagccattaccccacctactagctaatcccatctgggcacatctgatggcatgaggcccgaaggtcccccactttggtcttgcgacattatgcggtattagctaccgtttccagtagttatccccctccatcaggcagtttcccagacattactcacccgtccgccgctcgtcacccgagagcaagctctctgtgttaccgctcgacttgca
example 2
Preparing lead and cadmium mother solution: 1.5985g of lead nitrate and 20.317g of cadmium chloride are weighed and respectively dissolved in a beaker containing 500ml of ultrapure water, and simultaneously are continuously stirred by a glass rod until dissolved, and then are respectively transferred into a 1L volumetric flask to fix volume to scale marks, and are labeled for standby.
1. Action of enterobacter aerogenes (Enterobacter aerogenes) W6 in passivation of lead and cadmium
1. Single colonies were picked from plates of W6 strain and cultured overnight in 20mL of liquid medium 1 (same as in example 1), and then transferred to 100mL of liquid medium 1 for expansion culture.
2. The growth density of the W6 strain was observed, and its growth curve was measured at a wavelength of 600nm with an ultraviolet spectrophotometer to determine the logarithmic growth phase and stationary phase OD of the W6 strain 600 Time instant=1 was used for subsequent experiments; at 5.5h of incubation, W6 reached OD 600 Stationary phase=1.
4. Lead-cadmium solution experiments were performed by adding 1ml of lead mother liquor and 1ml of cadmium mother liquor (1 ml of lead mother liquor and 1ml of cadmium mother liquor were added to 100ml of liquid medium 2) through sterile filter heads, respectively, and simultaneously performing expansion culture to OD 600 Adding about 1% bacterial solution according to bacterial inoculation amount, performing water bath shake culture at 30deg.C under the condition of initial pH=7 and 150rpm/min, and determining Pb in the solution by ICP at regular time 2+ 、Cd 2+ Is a concentration change of (c).
In addition, this example also performed a sterile blank under equivalent conditions.
The results show that: under the conditions of aerobic, normal pressure, 30 ℃ and constant temperature shaking culture with initial PH=7 and 150rpm/min, the experimental group can not detect Pb basically at 15 days 2+ 、Cd 2+ Is present. At this time, the passivation rate of lead and cadmium was 99.7%.
2. Application of Klebsiella oxytoca Wn (Klebsiella aerogenes) in passivation of lead and cadmium
1. Single colonies were picked from plates of Wn strain and cultured overnight in 20mL of liquid medium 1 (same as in example 1), and then transferred to 100mL of liquid medium 1 for expansion culture.
2. Observing the growth density of Wn strain, measuring its growth curve at 600nm wavelength with ultraviolet spectrophotometer to determine logarithmic growth phase and stationary phase OD of Wn strain 600 Time instant=1 was used for subsequent experiments; at 5h of cultivation, wn reached OD 600 Stationary phase=1.
4. Lead-cadmium solution experiments were performed by adding 1ml of lead mother liquor and 1ml of cadmium mother liquor (1 ml of lead mother liquor and 1ml of cadmium mother liquor were added to 100ml of liquid medium 2) through sterile filter heads, respectively, and simultaneously performing expansion culture to OD 600 Adding about 1% bacterial solution according to bacterial inoculation amount, performing water bath shake culture at 30deg.C under the condition of initial pH=7 and 150rpm/min, and determining Pb in the solution by ICP at regular time 2+ 、Cd 2+ Is a concentration change of (c).
In addition, this example also performed a sterile blank under equivalent conditions.
The results show that: ICP was unable to measure Pb on day 1 under conditions of aerobic, normal pressure, 30℃and initial pH=7, 150rpm/min constant temperature shaking culture 2+ 、Cd 2+ Is present, the passivation lead-cadmium is 100%.
3. Identification of passivation products
The passivation reaction products after the action of W6 and Wn are respectively centrifuged (centrifugation condition: 10000rpm for 10 min), reaction precipitate is left (washed by 3 times of ultrapure water and centrifuged), and the reaction product is put into a 30-DEG oven for drying for one week, and the reaction product can be completely dried and ground into powder for XRD test.
The results show that: as understood with reference to FIG. 6, in both sets of experiments with W6 and Wn, the reaction product of cadmium included Ca 7.7 Cd 0.8 (PO 4 ) 8 (H 2 O) 2.4 The method comprises the steps of carrying out a first treatment on the surface of the The lead reaction products all include Pb 5 (PO 4 ) 3 Cl。
Example 3
The liquid medium 1 used in this example was the same as that in example 1, and glucose and tricalcium phosphate were additionally added in the same amount as that of the liquid medium 2; the liquid medium 2 used in this example was the same as that used in example 1; the initial pH of the medium used in this example was 7.
1. Effect of different media on Enterobacter aerogenes (Enterobacter aerogenes) W6
Culturing bacterial liquid of 1% W6 in culture medium 1 and culture medium 2 as different nutrient sources, culturing at constant temperature of 30deg.C on a shaking table at 150rpm/min, shake culturing for one week, taking 3ml sample on a sterile operation table at the same time point every day in a 5ml centrifuge tube, centrifuging at 8000rpm for 5min to obtain supernatant, measuring content change of available phosphorus by molybdenum-antimony colorimetric method, and recording corresponding pH value.
The results after measurement show that: as is understood from FIG. 7, when the nutrient source is medium 2, the phosphate solubilizing effect is remarkable, and the pH value in the medium solution system is drastically lowered from neutral to about 4.
As a result, it was found that the phosphate solubilizing efficiency of W6 was correlated with the pH value to some extent, since the pH value of the medium 1 used as a control test was not substantially increased to about 9.
In fig. 7, the solid line corresponds to the amount of dissolved phosphorus, and the broken line corresponds to PH.
2. Effect of different media on Klebsiella aerogenes (Klebsiella aerogenes) Wn
Culturing bacterial liquid of which the Wn is 1% is respectively absorbed and put into a culture medium 1 and a culture medium 2 to be used as different nutrition sources to be cultured, placing the culture liquid on a shaking table to keep the temperature constant at 30 ℃ and 150rpm/min, shake-culturing for one week, taking 3ml of sample on a sterile operation table at the same time point every day, centrifuging the sample in a 5ml centrifuge tube at 8000rpm for 5 minutes to obtain supernatant, measuring the content change of available phosphorus by a molybdenum-antimony colorimetric method, and recording the corresponding PH value.
The results after measurement show that: as is understood from FIG. 8, when the nutrient source is medium 2, the phosphate solubilizing effect is remarkable, and the pH value in the medium solution system is drastically lowered from neutral to about 4.
As a result, it was found that the pH of the medium 1 used as a control test was steadily increased to about 9 without phosphate dissolution, and that the phosphate dissolution efficiency of Wn was correlated with the pH.
In fig. 8, the solid line corresponds to the amount of dissolved phosphorus, and the broken line corresponds to PH.
To sum up: the culture medium 1 and the culture medium 2 have the same effect on W6 and Wn, so that both strains can produce acid in the culture medium 2 and produce alkali in the culture medium 1.
Example 4
The liquid medium 1 used in this example was identical to the liquid medium 1 in example 1; the liquid medium 2 contained in the bacterial liquid sprayed for the first time in this example was the same as that in example 1; in the microorganism-containing medium 2 sprayed at subsequent intervals, no tricalcium phosphate was added to the liquid medium 2 to avoid the effect of the continued addition of tricalcium phosphate on the soil, and the amounts of the remaining components of the liquid medium 2 were kept consistent with those in example 1.
1. Application of enterobacter aerogenes (Enterobacter aerogenes) W6 in lead-cadmium polluted soil
1. Weighing 10g (40 meshes) of air-dried soil, placing the air-dried soil into a 50ml centrifuge tube, adding a phosphorus source (insoluble phosphorus source: tricalcium phosphate) in a direct throwing manner, and stirring by using a clean plastic rod to uniformly distribute the tricalcium phosphate in a soil sample;
2. spraying bacterial liquid of enterobacter aerogenes W6 prepared in advance (bacterial agent grows to OD in the culture medium 2) 600 Stationary phase or logarithmic phase of=1) and stirred uniformly, eventually making the soil moisture content (bacterial liquid) 30%;
3. medium 2 containing W6 (od=1) was added every 3 days for the first 15 days to maintain the water content of the soil at 30%, and medium 1 containing W6 (od=1) was added every 3 days for the second 15 days to maintain the water content of the soil at 30%. The added phosphate radical content and the content of Pb and Cd heavy metal elements in the soil keep a certain proportion relation, specifically, the added amount of tricalcium phosphate is related to the molar mass ratio of Pb (P: HMs =1:1) and Cd (P: HMs =5:1) in the soil, and the added amount is finally determined to be 4mg/g.
In addition, this example also performed a sterile blank under equivalent conditions.
The experimental results show that: compared with a blank control test, the treatment process has the advantages that the highest removal rate of the effective state contents of Pb and Cd in the soil is 45.5% and 40.9% respectively after the treatment of the microbial W6 microbial agent composite tricalcium phosphate in 7 days; at 15 days, the highest removal rates of the active state contents were reduced to 30.1% and 23.5%, respectively, indicating that the dissolution occurred only by the action of phosphate dissolution. At 30 days, the removal rates of the effective state contents of Pb and Cd reach 45% and 42% again, respectively, which shows that the use of the culture medium can inhibit the phenomenon of reverse dissolution.
2. Application of Klebsiella aerogenes (Klebsiella aerogenes) Wn in lead-cadmium contaminated soil
1. Weighing 10g (40 meshes) of air-dried soil, placing the air-dried soil into a 50ml centrifuge tube, adding a phosphorus source (insoluble phosphorus source: tricalcium phosphate) in a direct throwing manner, and stirring by using a clean plastic rod to uniformly distribute the tricalcium phosphate in a soil sample;
2. spraying the bacterial solution of Klebsiella aerogenes Wn prepared in advance (the bacterial agent grows to OD in the culture medium 2) 600 Stationary phase or logarithmic phase of=1) and stirred uniformly, eventually making the soil moisture content (bacterial liquid) 30%;
3. medium 2 containing Wn (od=1) was added every 3 days for the first 15 days to maintain the water content of the soil at 30%, and medium 1 containing Wn (od=1) was added every 3 days for the second 15 days to maintain the water content of the soil at 30%. The added phosphate radical content and the content of Pb and Cd heavy metal elements in the soil keep a certain proportion relation, specifically, the added amount of tricalcium phosphate is related to the molar mass ratio of Pb (P: HMs =1:1) and Cd (P: HMs =5:1) in the soil, and the added amount is finally determined to be 4mg/g.
In addition, this example also performed a sterile blank under equivalent conditions.
The experimental results show that: compared with a blank control test, the treatment process has the advantages that the highest removal rate of the effective state contents of Pb and Cd in the soil is 49% and 49.3% respectively after the treatment of the microbial Wn microbial agent and the tricalcium phosphate is carried out in 7 days; at 15 days, the highest removal rates of the active state content were reduced to 45.1% and 46.4%, respectively, indicating that the dissolution occurred only by phosphate dissolution. At 30 days, the removal rates of the effective state contents of Pb and Cd reach 51% and 51.3% again, respectively, which shows that the use of the culture medium can inhibit the phenomenon of reverse dissolution.
In the above technical solution of the present invention, the above is only a preferred embodiment of the present invention, and therefore, the patent scope of the present invention is not limited thereto, and all the equivalent structural changes made by the description of the present invention and the content of the accompanying drawings or the direct/indirect application in other related technical fields are included in the patent protection scope of the present invention.
Sequence listing
<110> university of south-middle school
<120> bacterial agent, composition and application of enterobacter aerogenes combined with klebsiella aerogenes
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1410
<212> DNA
<213> Enterobacter aerogenes (Enterobacter aerogenes)
<400> 1
tgcaagtcga gcggtagcac agagagcttg ctctcgggtg acgagcggcg gacgggtgag 60
taatgtctgg gaaactgcct gatggagggg gataactact ggaaacggta gctaataccg 120
cataacgtcg caagaccaaa gtgggggacc ttcgggcctc atgccatcag atgtgcccag 180
atgggattag ctagtaggtg gggtaatggc tcacctaggc gacgatccct agctggtctg 240
agaggatgac cagccacact ggaactgaga cacggtccag actcctacgg gaggcagcag 300
tggggaatat tgcacaatgg gcgcaagcct gatgcagcca tgccgcgtgt atgaagaagg 360
ccttcgggtt gtaaagtact ttcagcgagg aggaaggcgt taaggttaat aaccttagcg 420
attgacgtta ctcgcagaag aagcaccggc taactccgtg ccagcagccg cggtaatacg 480
gagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg cacgcaggcg gtctgtcaag 540
tcggatgtga aatccccggg ctcaacctgg gaactgcatt cgaaactggc aggctagagt 600
cttgtagagg ggggtagaat tccaggtgta gcggtgaaat gcgtagagat ctggaggaat 660
accggtggcg aaggcggccc cctggacaaa gactgacgct caggtgcgaa agcgtgggga 720
gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgtcgact tggaggttgt 780
gcccttgagg cgtggcttcc ggagctaacg cgttaagtcg accgcctggg gagtacggcc 840
gcaaggttaa aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt 900
aattcgatgc aacgcgaaga accttaccta ctcttgacat ccagagaact tagcagagat 960
gctttggtgc cttcgggaac tctgagacag gtgctgcatg gctgtcgtca gctcgtgttg 1020
tgaaatgttg ggttaagtcc cgcaacgagc gcaaccctta tcctttgttg ccagcgattc 1080
ggtcgggaac tcaaaggaga ctgccagtga taaactggag gaaggtgggg atgacgtcaa 1140
gtcatcatgg cccttacgag tagggctaca cacgtgctac aatggcatat acaaagagaa 1200
gcgacctcgc gagagcaagc ggacctcata aagtatgtcg tagtccggat tggagtctgc 1260
aactcgactc catgaagtcg gaatcgctag taatcgtaga tcagaatgct acggtgaata 1320
cgttcccggg ccttgtacac accgcccgtc acaccatggg agtgggttgc aaaagaagta 1380
ggtagcttaa ccttcgggag ggcgcttacc 1410
<210> 2
<211> 1403
<212> DNA
<213> Klebsiella aerogenes (Klebsiella aerogenes)
<400> 2
gccctcccga aggttaagct acctacttct tttgcaaccc actcccatgg tgtgacgggc 60
ggtgtgtaca aggcccggga acgtattcac cgtagcattc tgatctacga ttactagcga 120
ttccgacttc atggagtcga gttgcagact ccaatccgga ctacgacata ctttatgagg 180
tccgcttgct ctcgcgaggt cgcttctctt tgtatatgcc attgtagcac gtgtgtagcc 240
ctactcgtaa gggccatgat gacttgacgt catccccacc ttcctccagt ttatcactgg 300
cagtctcctt tgagttcccg accgaatcgc tggcaacaaa ggataagggt tgcgctcgtt 360
gcgggactta acccaacatt tcacaacacg agctgacgac agccatgcag cacctgtctc 420
agagttcccg aaggcaccaa agcatctctg ctaagttctc tggatgtcaa gagtaggtaa 480
ggttcttcgc gttgcatcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca 540
attcatttga gttttaacct tgcggccgta ctccccaggc ggtcgactta acgcgttagc 600
tccggaagcc acgcctcaag ggcacaacct ccaagtcgac atcgtttacg gcgtggacta 660
ccagggtatc taatcctgtt tgctccccac gctttcgcac ctgagcgtca gtctttgtcc 720
agggggccgc cttcgccacc ggtattcctc cagatctcta cgcatttcac cgctacacct 780
ggaattctac ccccctctac aagactctag cctgccagtt tcgaatgcag ttcccaggtt 840
gagcccgggg atttcacatc cgacttgaca gaccgcctgc gtgcgcttta cgcccagtaa 900
ttccgattaa cgcttgcacc ctccgtatta ccgcggctgc tggcacggag ttagccggtg 960
cttcttctgc gagtaacgtc aatcgctaag gttattaacc ttaacgcctt cctcctcgct 1020
gaaagtactt tacaacccga aggccttctt catacacgcg gcatggctgc atcaggcttg 1080
cgcccattgt gcaatattcc ccactgctgc ctcccgtagg agtctggacc gtgtctcagt 1140
tccagtgtgg ctggtcatcc tctcagacca gctagggatc gtcgcctagg tgagccatta 1200
ccccacctac tagctaatcc catctgggca catctgatgg catgaggccc gaaggtcccc 1260
cactttggtc ttgcgacatt atgcggtatt agctaccgtt tccagtagtt atccccctcc 1320
atcaggcagt ttcccagaca ttactcaccc gtccgccgct cgtcacccga gagcaagctc 1380
tctgtgttac cgctcgactt gca 1403
Claims (9)
1. The microbial agent for combining enterobacter aerogenes with klebsiella aerogenes is characterized by being used for treating lead-cadmium polluted soil, and comprises enterobacter aerogenes and klebsiella aerogenes;
wherein the enterobacter aerogenes is enterobacter aerogenes @Enterobacter aerogenes) W6 Latin classification designationEnterobacter aerogenesThe strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2021, 7 and 13, and the preservation number is CGMCC No.22888;
the Klebsiella aerogenes is Klebsiella aerogenes @Klebsiella aerogenes) Wn, latin classification designationKlebsiella aerogenesThe strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2021, 7 and 13 days, and the preservation number is CGMCC No.22889.
2. Use of the microbial inoculum of claim 1 in the treatment of lead-cadmium contaminated soil.
3. The composition for treating the lead-cadmium polluted soil is characterized by comprising a component A, a component B, a component C and a component D;
wherein the component a comprises a phosphorus source;
the component B comprises the microbial agent of claim 1;
the component C comprises an aqueous solution containing glucose, magnesium chloride, magnesium sulfate, ammonium sulfate and potassium chloride;
the component D comprises an aqueous solution containing tryptone, yeast extract and sodium chloride.
4. A composition according to claim 3, wherein the phosphorus source is tricalcium phosphate.
5. A method for treating lead-cadmium contaminated soil, characterized in that the composition according to claim 3 or 4 is used for treating the lead-cadmium contaminated soil.
6. The method for treating lead-cadmium contaminated soil according to claim 5, comprising: applying the component A and a first mixed solution containing the component B and the component C to the soil to be treated in a first time period;
applying a second mixed solution containing the component B and the component D to the soil to be treated for a second period of time;
wherein the second period of time is later than the first period of time.
7. The method for treating lead-cadmium contaminated soil according to claim 6, further comprising: and applying the first mixed liquid to the soil to be remediated between the first time period and the second time period.
8. The method for treating lead-cadmium polluted soil according to claim 7, wherein,
applying the first mixed liquid to the soil to be treated for a plurality of times between the first time period and the second time period, wherein the single application amount is consistent with the total application amount of the first mixed liquid in the first time period;
and applying the second mixed solution to the soil to be treated for a plurality of times in the second time period, wherein the single application amount is consistent with the total application amount of the first mixed solution in the first time period.
9. The method for remediation of lead-cadmium contaminated soil according to any one of claims 6 to 8, wherein the first time period and the second time period are separated by a period of 14 to 16 days.
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