Microbial composite growth-promoting microbial agent and application thereof

Abstract

The invention relates to a microbial composite growth-promoting bacterial agent and application thereof, the microbial composite bacterial agent is compounded by penicillium (Penicillium) ZJ-1 and Aspergillus flavus ZJ-6, the preservation number of the penicillium (Penicillium) ZJ-1 is CGMCC NO.40559, the preservation number of the Aspergillus flavus ZJ-6 is CGMCC NO.40559, and the preservation number of the Aspergillus flavus ZJ-6 is CGMCC NO.40559. The preservation number of the Aspergillus flavus ZJ-6 is CGMCC (China General Microbiological Culture Collection Center) NO.40560. The Aspergillus flavus ZJ-6 has the advantages that the Aspergillus flavus ZJ-6 is The composite microbial inoculant is suitable for promoting plant growth. The microbial composite growth-promoting microbial agent has the characteristics of dissolving phosphorus, producing IAA and synthesizing iron carriers and ACC deaminase; an eyebright potting experiment proves that the complex microbial inoculant can effectively promote growth of eyebright under uranium stress and enrichment of uranium by eyebright.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental engineering microorganisms and soil remediation, and in particular to a microbial composite growth-promoting agent and application thereof. Background Art

[0002] With the development of social industrialization, the large-scale use of nuclear energy, the increasing mining and smelting of uranium mines, has led to increasingly serious environmental pollution around uranium mines. Affected by natural environments such as rainfall, snowfall, leakage, and surface runoff, uranium-containing wastes have gradually migrated and spread to the surrounding environment, especially the pollution of the soil environment by radioactive nuclides. However, there are still a large number of farmers living around the mining area, engaged in various agricultural activities. Heavy metal uranium can not only cause biological pathology through radioactive decay, but also cause damage to the human body through breathing or food chains, seriously affecting human health. Heavy metal uranium contaminated soil not only restricts the development of the ecological environment around mines, but also poses a threat to human health. Therefore, it is very necessary to treat the contaminated soil around uranium mines.

[0003] Bioremediation technology has attracted widespread attention because of its economical applicability, no secondary pollution during the restoration process, and permanent treatment effects. It can also effectively protect the soil microecological environment. There are many types of soil microorganisms, including bacteria, fungi, actinomycetes and other microbial groups. They are widely and deeply involved in almost all biological activities and biochemical reactions in the soil. They play a key role in direct or indirect ways. Due to their high sensitivity to changes in the soil environment, soil microorganisms are regarded as the most sensitive indicator for assessing soil environmental quality standards. Soil microorganisms can have a significant promoting effect on plant growth through a variety of action mechanisms. These mechanisms cover several key aspects, including but not limited to: secreting plant hormones and enzymes such as indoleacetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which not only promote plant growth but also play an important role in regulating plant physiological processes; through phosphorus solubilization, converting phosphorus in the soil that is difficult for plants to directly absorb and utilize into an absorbable form, thereby increasing the plant's phosphorus uptake; producing iron carriers, which have the ability to specifically bind trivalent iron ions and can transport the bound iron ions into microbial cells to meet the microbial demand for iron; and performing nitrogen fixation, converting free nitrogen in the air into a nitrogen source that can be used by plants. In addition, soil microorganisms can also effectively alleviate the environmental stress faced by plants by increasing the absorption rate of various nutrients in the soil by host plants, such as the adverse effects of adverse conditions such as drought, salinity, and heavy metal pollution on plant growth and development.

[0004] The patent with publication number CN118703343A discloses a Penicillium Q77 which has a growth-promoting effect on Salvia miltiorrhiza seedlings, as shown by the improvement of various growth indicators of Salvia miltiorrhiza (leaf length, leaf width, plant height, stem thickness), and the improvement effect is significant. At the same time, the strain can improve the disease resistance of Salvia miltiorrhiza seedlings.

[0005] The patent with publication number CN111657304A discloses a method for using Aspergillus flavus TL-F3 to enhance ryegrass to repair heavy metal contaminated soil. Aspergillus flavus TL-F3 still has a growth-promoting effect on ryegrass under the stress of heavy metals, which not only improves the enrichment efficiency of ryegrass for Pb(II), Zn(II), Cu(II) and Cd(II), but also reduces the concentration of heavy metals in the soil.

[0006] The above experimental results show that under certain conditions, Penicillium and Aspergillus flavus show good growth-promoting ability. However, most of the strains currently studied are single strains, which have characteristics such as weak competitiveness and susceptibility to inhibition. In addition, in the existing technology, there is a relative lack of research on the combined growth-promoting bacterial agent-plant combined remediation of uranium-contaminated soil, and the bacterial resources that can be actually used in production are still scarce. Therefore, it is urgent to carry out further research and develop more bacterial agents with growth-promoting functions, so as to provide more solid and powerful support for the sustainable development of soil environmental governance. Summary of the invention

[0007] The purpose of the present invention is to provide a microbial composite growth-promoting agent and an application thereof. The microbial composite growth-promoting agent can dissolve phosphorus, synthesize iron carriers, and produce IAA and ACC deaminase. Compounding the microbial composite growth-promoting agent can promote the absorption of nutrients in the soil by eyebright grass, enhance the tolerance of eyebright grass to adverse environments, and thus promote the growth of eyebright grass. In addition, the microbial composite growth-promoting agent can also enhance the repair of uranium-contaminated soil by eyebright grass.

[0008] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a microbial composite growth-promoting agent, which is compounded by Penicillium ZJ-1 and Aspergillus flavus ZJ-6. The Penicillium ZJ-1 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on April 7, 2023, with a deposit number of CGMCC NO.40559; Aspergillus flavus ZJ-6 was deposited in the General Microbiology Center of China National Microbiological Culture Collection on April 7, 2023, with a deposit number of CGMCC NO.40560.

[0010] Furthermore, the preparation method of the microbial composite growth-promoting agent is as follows: Penicillium ZJ-1 and Aspergillus flavus ZJ-6 are inoculated on potato sucrose agar medium respectively, and cultured at a constant temperature of 28°C for 7-10 days until spores are produced, spores on the surface of mycelium are gently scraped and placed in sterile water containing 10% glycerol, the mycelium is filtered to remove the mycelium, and the spore liquid is obtained, and the OD600 value is measured to be 1.0±0.5, that is, the spore concentration is 1×10 8 The method comprises the following steps: inoculating the spore liquid in a potato glucose broth liquid culture medium at a 1% inoculation rate, culturing at 28°C and 120 r / min or 150 r / min for 7 days to obtain a Penicillium ZJ-1 fermentation liquid and an Aspergillus flavus ZJ-6 fermentation liquid, and mixing the Penicillium ZJ-1 fermentation liquid and the Aspergillus flavus ZJ-6 fermentation liquid at different volume ratios to obtain composite bacterial agents of different proportions.

[0011] Preferably, the volume ratio between the fermentation broth of Penicillium ZJ-1 and the fermentation broth of Aspergillus flavus ZJ-6 is 4:1.

[0012] The second aspect of the present invention provides the use of a microbial composite agent in promoting plant growth in soil contaminated with high concentrations of uranium.

[0013] Furthermore, the microbial composite growth-promoting agent improves the plant's absorption of nutrients in the soil by dissolving phosphorus, synthesizing iron carriers, and producing IAA and ACC deaminase, thereby promoting plant growth.

[0014] Preferably, the plant is eyebright; further, the microbial composite agent can enhance the ability of eyebright to repair heavy metal uranium contaminated soil.

[0015] Furthermore, a microbial composite growth-promoting agent was applied to uranium-contaminated soil to promote the growth of eyebright grass and enhance its ability to enrich heavy metal uranium.

[0016] In specific application, after the millet grass seeds are sown and grown for 20 days, the microbial composite growth-promoting agent is added every 5 days. Each time, 400 ml of the microbial composite growth-promoting agent is diluted to 800 ml with deionized water and added. Stop after 6 additions. When adding, the diluted microbial composite growth-promoting agent is directly poured into the soil matrix near the roots of the millet grass seedlings.

[0017] The present invention has the following beneficial effects:

[0018] Penicillium ZJ-1 and Aspergillus flavus ZJ-6 in the present invention are indigenous strains of uranium mines selected by the applicant. After testing, the composite bacterial agent has a good growth-promoting effect, specifically, it has the characteristics of solubilizing phosphate, producing IAA, synthesizing siderophore and ACC deaminase. Potted experiments have proved that the composite bacterial agent can not only promote the growth of eyebright grass, but also enhance its ability to repair uranium-contaminated soil, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the growth status of Penicillium ZJ-1 and Aspergillus flavus ZJ-6 in PDA medium and PDB medium respectively; (a) PDA medium; (b) PDB medium;

[0020] Figure 2 Schematic diagram of the qualitative determination results of the siderophore production capacity of Penicillium ZJ-1 and Aspergillus flavus ZJ-6; (a) Penicillium ZJ-1; (b) Aspergillus flavus ZJ-6;

[0021] Figure 3 The color changes of the microbial composite growth-promoting agent on the 5th and 9th days; (a) 5th day; (b) 9th day;

[0022] Figure 4 It is the phosphate solubilization zone of the strain on NBRIP medium;

[0023] Figure 5 It is a schematic diagram of the determination results of the specific activity of ACC deaminase produced by ZJ-1 and ZJ-6 bacterial liquid alone and in combination;

[0024] Figure 6This is a comparison of the biomass results of different parts of eyebright in the control group and the experimental group;

[0025] Figure 7 This is a comparison chart of the percentage of uranium in different forms in the rhizosphere soil of each treatment group. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with specific embodiments.

[0027] Example 1

[0028] A method for preparing a microbial composite growth-promoting agent comprises the following steps:

[0029] 1) Culture medium preparation: Prepare potato sucrose agar medium (PDA) and potato dextrose broth medium (PDB), sterilize at high temperature for 20-30 min, and set aside;

[0030] 2) Penicillium ZJ-1 with the deposit number of CGMCC NO.40559 was inoculated onto a potato sucrose agar (PDA) medium (eg Figure 1 The spores were cultured at 28 °C for 10 days until spores were produced. The spores on the surface of the mycelium were gently scraped and placed in sterile water containing 10% glycerol. The mycelium was filtered to remove the mycelium to obtain the spore solution. The OD600 value was 1.0 ± 0.5, that is, the spore concentration was 1 × 10 8 The spore solution was inoculated into a potato dextrose broth (PDB) liquid medium at a 1% inoculum amount and cultured at 28°C and 120 r / min for 7 days to obtain ZJ-1 fermentation liquid (such as Figure 1 b), refrigerate for later use;

[0031] 3) Inoculate Aspergillus flavus ZJ-6 with the deposit number of CGMCC NO.40560 onto a potato sucrose agar (PDA) medium (eg Figure 1 a), cultured at 30°C for 7 days until spores were produced, spores on the surface of mycelium were gently scraped and placed in sterile water containing 10% glycerol, mycelium was filtered to remove the mycelium, and spore liquid was obtained. The OD600 value was 1.0±0.5, that is, the spore concentration was 1×10 8 The spore solution was inoculated with 1% inoculum into a potato dextrose broth (PDB) liquid medium, and cultured at 28°C and 150 r / min for 7 days to obtain ZJ-6 fermentation liquid (such as Figure 1 b), refrigerate for later use;

[0032] 4) The ZJ-1 fermentation broth and the ZJ-6 fermentation broth were mixed at volume ratios of 1:4, 3:2, 2:3, 1:1 and 4:1 for later use.

[0033] Example 2: Determination of indoleacetic acid (IAA) production by Penicillium ZJ-1, Aspergillus flavus ZJ-6, and microbial composite growth-promoting agents

[0034] Penicillium ZJ-1, Aspergillus flavus ZJ-6, and the microbial composite growth-promoting agents prepared in Example 1 with different ratios were added with tryptophan filtered by a sterile filter membrane, respectively, so that the tryptophan concentration in the final composite fermentation broth was 200 mg·L -1 , set up a blank control;

[0035] Take the fermentation broth of 48h, 72h, 84h, 96h, 120h and 144h in a centrifuge tube, centrifuge at 8000r / min for 10min, take the supernatant and add Salkowskis colorimetric solution in a ratio of 1:1, place in a dark place to avoid light for 30min, and measure OD at the same time. 530 The absorbance and concentration values ​​at the sites showed that the average IAA content of ZJ-6 bacterial solution was 10.24 mg / L±1.2 mg / L, and the average IAA content of ZJ-1 bacterial solution was 3.19±0.75 mg / L. The content of IAA produced by the mixture of ZJ-1 and ZJ-6 reached the highest on the 5th day, among which the highest content of 4:1 was 10.33 mg / L±1.8 mg / L.

[0036] Example 3: Determination of siderophore production by Penicillium ZJ-1, Aspergillus flavus ZJ-6, and microbial composite growth-promoting agents

[0037] 1) Preparation of improved siderophore detection liquid medium: Weigh 121 g of improved siderophore detection medium, dilute to 900 mL with ionized water, sterilize at 115°C for 20 min, cool to 60°C, slowly add 100 mL of 10× buffer solution and 100 mL of 10× CAS detection solution preheated at 60°C to the medium, mix well and set aside;

[0038] 2) Qualitative screening of the ability of bacterial agents to produce siderophores: Use a sterile inoculation loop to dip the bacterial solution into the improved siderophore detection solid culture medium and culture it at 30°C for 9 days. If the strain does not produce siderophores, the culture medium will not change. If the strain produces siderophores, the culture medium will change, i.e., an obvious yellow hydrolysis zone will appear;

[0039] 3) Quantitative screening of the ability of bacterial agents to produce siderophores: The spore suspension of the above strains was inoculated into a modified siderophore detection liquid medium at a 1% inoculum, and cultured at 30°C and 160 r / min for 9 days. The culture medium was centrifuged for 10 minutes, and 1 mL of the supernatant (reference value (Ar)) and 2×CAS detection solution (10×CAS detection solution mixed with water in a ratio of 1:4) were mixed in equal volumes, and allowed to stand in the dark for 30 minutes for standby use; double distilled water was used as a control to adjust the zero, and the OD value (As) at a wavelength of 630 nm was measured by an enzyme marker. If the siderophore was not produced, the culture medium did not change. If the strain produced siderophores, the culture medium changed to orange-yellow. The As / Ar ratio represented the relative content of siderophores in the sample. The smaller the value, the stronger the ability of the strain to produce siderophores. The ratio of formula (1) represented the activity unit of siderophores in the sample. The higher the activity unit, the stronger the ability to produce siderophores. The results showed that the siderophore production of ZJ-1 reached 52.46%, and that of ZJ-6 reached 75.39%. The As / Ar ratio of the two strains was < 0.5, indicating that both strains were high-producing iron carriers. The 4:1 composite of Penicillium and Aspergillus flavus produced the highest iron carrier content, reaching 91.46% on the ninth day (e.g. Figure 2 and Figure 3 shown).

[0040]

[0041] Example 4: Determination of phosphorus solubilization by Penicillium ZJ-1, Aspergillus flavus ZJ-6, and microbial composite growth-promoting agent

[0042] 1) Preparation of NBRIP medium: 10 g / L glucose, 5 g / L Ca3(PO4)2, 3 g / L NaCl, 0.1 g / L (NH4)2SO4, 6 g / LMgCl2·6H2O, 2.5 g / L KCl, pH 6.8-7.0, autoclave at 108°C for 15 min, set aside;

[0043] 2) The strains were inoculated onto solid plates containing NBRIP, and their phosphate solubilization ability was qualitatively determined by the growth of the strains. The results showed that after 7 days of cultivation, Penicillium ZJ-1, Aspergillus flavus ZJ-6 and the 4:1 composite group all had obvious phosphate solubilization circles (such as Figure 4 );

[0044] 3) After the composite bacterial agents of different proportions were further transferred to NBRIP liquid culture medium at an inoculation rate of 1% and cultured for 10 days, the soluble phosphorus content in the supernatant was 87.37 mg / L-157.64 mg / L, among which the 4:1 composite group of ZJ-1 and ZJ-6 was the highest on the 8th day, reaching 157.64 mg / L.

[0045] Example 5: Determination of ACC deaminase production by Penicillium ZJ-1, Aspergillus flavus ZJ-6, and microbial composite growth-promoting agents

[0046] 1) Preparation of TSB liquid medium: 17.0 g trypticase, 3.0 g soybean papain digest, 5.0 g sodium chloride, 2.5 g potassium dihydrogen phosphate, 2.5 g glucose, pH 7.3 ± 0.2 (25°C), 1000 ml water;

[0047] DF culture medium formula: potassium dihydrogen phosphate (KH2PO4) 0.5g / L, dipotassium hydrogen phosphate (K2HPO4) 0.5g / L, glucose 1.0g / L, gluconic acid 0.5g / L, citric acid 0.5g / L, magnesium sulfate (MgSO4·7H2O) 0.2g / L, calcium chloride (CaCl2) 0.02g / L, manganese sulfate (MnSO4·H2O) 0.01g / L, copper sulfate (CuSO4·5H2O) 0.001g / L, zinc sulfate (ZnSO4·7H2O) 0.001g / L, sodium chloride (NaCl) 0.1g / L.

[0048] Preparation of 0.2% 2,4-dinitrophenylhydrazine solution: Weigh 0.2 g of 2,4-dinitrophenyl, fully dissolve it in an appropriate amount of 2 mol / L HCl, transfer it to a 100 mL volumetric flask, and make up to volume with 2 mol / L HCl.

[0049] Preparation of Coomassie Brilliant Blue staining solution: Weigh 0.1 g of Coomassie Brilliant Blue and dissolve it in 50 mL of 95% ethanol. Then add 100 mL of 85% H3PO4 and mix thoroughly. Transfer to a 1000 mL volumetric flask and make up to volume.

[0050] 2) Determination of ACC deaminase activity: The bacterial agent prepared in Example 1 was inoculated into 7.5 mL of TSB liquid culture medium at a ratio of 1%, and cultured at 28°C at a speed of 160 rpm for 24 hours. Subsequently, the culture was placed in a 4°C environment and centrifuged at a speed of 10000 rpm for 10 minutes to separate the bacteria. The bacteria were resuspended in 7.5 mL of DF culture medium without (NH4)2SO4, and 45 μL of 0.5 mol / L ACC solution was added. After continuing to culture for 48 hours, the centrifugation was repeated, and the precipitate was washed with 5 mL of Tris-HCl buffer (0.1 mol / L, pH7.6), and the precipitate was collected by centrifugation after vortexing. Next, add 1 mL of Tris-HCl buffer (0.1 mol / L, pH 7.6) to the precipitate, shake thoroughly and centrifuge for 10 minutes, resuspend the precipitate in 600 μL Tris-HCl buffer (0.1 mol / L, pH 8.5), add 30 μL of toluene, and vortex rapidly for 30 seconds to prepare a cell disruption solution. Take 200 μL of the cell disruption solution and store it at 4°C for later use.

[0051] 3) Determination of protein concentration: Take 200 μL of the above-preserved cell lysis solution, add 20 μL of 0.5 mol / L ACC solution, mix thoroughly, and keep warm in a 30°C water bath for 15 min. Then, add 1 mL of 0.56 mol / L HCl solution, mix well, and centrifuge at room temperature for 10 min. Take 1 mL of supernatant, add 800 μL of 0.56 mol / L HCl solution, shake well, add 300 μL of 2,4-dinitrophenylhydrazine solution, and keep warm in a 30°C water bath for 30 minutes. Subsequently, add 2 mL of 2 mol / L NaOH solution (color development time 5 minutes), and measure the absorbance at a wavelength of 540 nm to determine the concentration of α-ketobutyric acid generated by the reaction.

[0052] Determination of sample protein concentration: Take 200 μL of cell disruption solution, add 1 mL of Coomassie Brilliant Blue staining solution for reaction, mix thoroughly and let stand (color development time 5 min), and measure the absorbance at 595 nm. Compare the absorbance with the protein standard curve to determine the sample protein concentration.

[0053] 4) The activity of ACC deaminase is determined by measuring the amount of α-ketobutyrate produced when ACC deaminase cleaves ACC, expressed as specific activity, that is, the amount of α-ketobutyrate produced per milligram of protein per hour, in units of μmol α-ketobutyrate / mg·protein / h. The specific activity calculation formula is the ratio of enzyme activity (μmol α-ketobutyrate / L / h) to protein concentration (μg / mL). The ACC deaminase specific activity of ZJ-1 is about 0.37, and that of ZJ-6 is 0.46. The activity of the 4:1 group of ZJ-1 and ZJ-6 composite flora was significantly higher than that of the other composite ratios alone, at 2.84, such as Figure 5 shown.

[0054] Example 6: Microbial composite growth-promoting agent (the volume ratio between the fermentation broth of Penicillium ZJ-1 and the fermentation broth of Aspergillus flavus ZJ-6 is 4:1) promotes the growth of eyebright grass and the enrichment of uranium

[0055] 1) Use potted experiments to grow eyebright grass. The specific method is as follows:

[0056] (1) Uranium concentration in soil is 199.38 g kg -1 The experiment was divided into 2 treatment groups, each with 4 pots in parallel. The control group: no exogenous bacteria were added; the experimental group: the microbial composite growth-promoting agent prepared in Example 1;

[0057] (2) The wheatgrass planting cycle is 55 days. After the wheatgrass seeds are sown and grown for 20 days, the compound bacterial agent is added every 5 days. Each time, 400 ml of the microbial compound growth-promoting agent is diluted to 800 ml with deionized water and then added. When adding, the diluted microbial compound growth-promoting agent is directly poured into the soil matrix near the roots of the wheatgrass seedlings; stop adding after 6 times, and harvest the wheatgrass after the growth cycle ends.

[0058] 2) Biomass determination of eyebright grass:

[0059] (1) Method for detecting aboveground biomass: Cut off all aboveground parts of the plant, wash and dry them to a constant weight, and weigh them on a balance to obtain the aboveground dry biomass.

[0060] (2) Method for detecting underground biomass: Usually the excavation method is used to carefully dig out the plant roots and the surrounding soil. The soil is then rinsed with a sieve to separate the roots. The roots are then washed, dried, and weighed to obtain the underground biomass.

[0061] (3) Biomass directly reflects the growth of plants in uranium-containing soils, and also directly reflects the impact of uranium-containing soils on plants. The biomass of the underground and aboveground parts of the experimental group was higher than that of the control group, indicating that the addition of composite bacterial agents can reduce the toxicity of uranium to plants and effectively promote the growth of eyebright grass. Figure 6 .

[0062] 3) Determination of uranium enrichment and transport by composite bacterial agent and eyebright grass

[0063] (1) The dry ash method was used to determine the uranium content of plant / soil samples. The aboveground and underground parts of the dried millet grass were placed in crucibles, three in parallel, marked, and placed in a muffle furnace for carbonization at 400°C for 3 hours, then heated to 600°C and ashed for another 5 hours. When the temperature in the furnace dropped to room temperature, the crucibles were taken out and 0.1000g of ash samples were accurately weighed in a tetrafluoroethylene crucible; 0.1000g of the dried constant-weight rhizosphere soil was passed through a 100-mesh sieve and weighed in a tetrafluoroethylene crucible. Nitric acid: perchloric acid: hydrofluoric acid (5:3:2) was added to the tetrafluoroethylene crucible in proportion, and the crucible was placed on a graphite hot plate for heating and digestion. When there were no obvious particles in the pyrolysis solution, hydrogen peroxide was added to remove the acid. After the liquid was clarified and cooled, it was filtered through a 0.45um water filter head, acid was added to make the volume constant, and then the uranium concentration was determined by ICP-OES.

[0064] (2) The bioconcentration factor (BCF) reflects the ability of millet grass to absorb uranium in the soil. It is the ratio of the uranium content in the aboveground part and the underground part of the millet grass to the uranium content in the corresponding soil. The larger the enrichment coefficient, the stronger the ability of millet grass to accumulate uranium.

[0065] BCF=C in eyebright grass (mg / kg) / C 土壤部分 (mg / kg)) Formula (2)

[0066] The transfer coefficient (TF) is the distribution of uranium adsorbed by the roots of millet grass in the plant body. It is the ratio of the uranium content in the aboveground part of millet grass to the uranium content in the roots. The larger the transfer coefficient, the stronger the ability of the millet grass roots to absorb uranium and transport it to the stems and leaves.

[0067] TF=C 小米草地上部分 (mg / kg)) / C 小米草地下部分 (mg / kg)) Formula (3)

[0068] (3) In the control group, the uranium content in the aboveground part of millet was 14.56 mg / kg, and the uranium content in the underground part was 243.12 mg / kg, while in the experimental group, the uranium concentration in the aboveground part of millet was 22.39 mg / kg, and the uranium content in the underground part was 398.46 mg / kg. The absorption of uranium by millet in the experimental group was much higher than that in the control group, indicating that the addition of the composite bacterial agent promoted the absorption of active uranium by millet, and also proved that the composite bacterial agent had a synergistic effect on millet, making the effect of millet grass absorbing uranium better.

[0069] According to the data shown in Table 1, the enrichment coefficients of the aboveground and underground parts of the control group were 0.08 and 1.21, respectively, while the enrichment coefficients of the aboveground and underground parts of the experimental group were 0.21 and 2.43, respectively. The enrichment coefficient of the underground part was significantly higher than 1, indicating that with the assistance of the composite microbial agent, the eyebright grass had a significant enrichment ability in the underground part. However, the enrichment effect of the aboveground part was significantly lower than that of the underground part, which indicated that the roots of the eyebright grass had a better ability to store uranium than the stems and leaves. The transport coefficient of the control group was 0.06, and the transport coefficient of the experimental group was 0.08. This result showed that the application of the composite microbial agent increased the content of active uranium in the eyebright grass, enhanced its ability to adsorb uranium and its tolerance to uranium. In addition, the addition of the composite microbial agent also confirmed that it had a positive promoting effect on the growth of eyebright grass and uranium absorption.

[0070] Table 1 BCF and TF of eyebright in each treatment group

[0071]

[0072] 4) Effect of composite microbial agent and eyebright grass on soil uranium speciation Method: Stepwise chemical extraction based on Tessier five-step extraction method, as follows:

[0073] (1) Exchangeable uranium: Take 3.000 g of soil sample and place it in a 150 ml conical flask. Add 20 ml of 1 mol / L MgCl2 solution with a pH of 7, shake for 2 hours, transfer to a 50 ml centrifuge tube to make up the volume, centrifuge at 6000 rpm for 10 min, take the supernatant and transfer it to a 100 ml volumetric flask, rinse the conical flask, transfer the rinse solution to the above volumetric flask after centrifugation in the same way, add 1 ml of nitric acid, and make up the volume with ultrapure water;

[0074] (2) Carbonate-bound uranium: Take the soil sample from the previous step and place it in a conical flask. Add 30 ml of 1 mol / L sodium acetate solution with a pH value of 5. Oscillate at a constant temperature. Take the supernatant and adjust the volume in the same way as step (1).

[0075] (3) Organic matter binding state: Take the soil sample after the previous experiment and place it in a conical flask. Add 20 ml of 30% H2O2 and react at room temperature for 1 hour. Then heat it in a 90°C water bath until it is evaporated; add 20 ml of H2O2 and continue heating in a water bath until the liquid is evaporated; add 50 ml of CH3COONH4 solution and oscillate at a constant temperature for 2 hours. Obtain the supernatant and the volume adjustment process is the same as step (1).

[0076] (4) Amorphous uranium bound to iron-manganese oxides / hydroxides: Take the residual soil sample after the previous experiment in a conical flask, add 20 ml of Tamm's solution, shake at a constant temperature for 5 h, and obtain the supernatant and adjust the volume in the same way as step (1);

[0077] (5) Crystalline iron-manganese oxide / hydroxide combination state: Take the residual soil sample after the previous experiment in a conical flask, add 40 ml of CDB solution with a pH value of 7.0, and oscillate at a constant temperature for 5 h. The supernatant and the volume adjustment process are the same as step (1);

[0078] (6) Residual uranium: digest the soil sample extracted in step (5) and measure it;

[0079] (7) The proportion of each form of uranium in the experimental soil Figure 7 As shown, in the blank control group, the residual state accounted for 28%, the organic matter-bound state accounted for 1%, the crystalline iron-manganese oxidation state accounted for 26%, the amorphous iron-manganese oxidation state accounted for 11%, the carbonate-bound state accounted for 27%, and the exchangeable state accounted for 8%; among them, the inert uranium accounted for 55%, and the active uranium accounted for 45%. In the experimental group of ZJ-1 and ZJ-6 composite bacterial agents, the residual state accounted for 24%, the organic matter-bound state accounted for 1%, the crystalline iron-manganese oxidation state accounted for 22%, the amorphous iron-manganese oxidation state accounted for 18%, the carbonate-bound state accounted for 31%, and the exchangeable state accounted for 5%; among them, the inert uranium accounted for 47%, and the active uranium accounted for 53%. The active uranium in the experimental group was lower than that in the control group, indicating that the addition of mixed bacterial agents promoted the transformation of inert uranium, and the exchangeable state was lower than that in the control group, indicating that the addition of mixed bacterial agents enhanced the absorption of uranium by plants more significantly.

[0080] In summary, the microbial composite agent of the present invention has a good growth-promoting effect, which is specifically manifested in that it has the characteristics of solubilizing phosphate, producing IAA, synthesizing siderophore and ACC deaminase. Potted experiments have proved that the composite agent can not only promote the growth of eyebright grass, but also enhance its ability to repair uranium-contaminated soil, and has a very broad application prospect.

Claims

1. A microbial composite growth-promoting agent, characterized in that: The microbial composite growth-promoting agent is compounded by Penicillium ZJ-1 and Aspergillus flavus ZJ-6. The Penicillium ZJ-1 was deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration on April 7, 2023, with a deposit number of CGMCC NO.40559; Aspergillus flavus ZJ-6 was deposited in the General Microbiology Center of the China National Microbiological Culture Collection Administration on April 7, 2023, with a deposit number of CGMCC NO.40560.

2. A microbial composite growth-promoting agent according to claim 1, characterized in that: The preparation method of the microbial composite growth-promoting agent is as follows: Penicillium ZJ-1 and Aspergillus flavus ZJ-6 were inoculated on potato sucrose agar medium, respectively, and cultured at 28°C for 7-10 days until spores were produced. Spores on the surface of mycelium were gently scraped and placed in sterile water containing 10% glycerol. Mycelium was filtered to remove the mycelium to obtain spore liquid. The OD600 value was determined to be 1.0±0.5, that is, the spore concentration was 1×10 8 The spore liquid is inoculated into a potato glucose broth liquid culture medium at a 1% inoculation rate, and cultured at 28°C, 120r / min and 150r / min for 7 days to obtain Penicillium ZJ-1 fermentation liquid and Aspergillus flavus ZJ-6 fermentation liquid, and the Penicillium ZJ-1 fermentation liquid and the Aspergillus flavus ZJ-6 fermentation liquid are mixed at different volume ratios to obtain composite bacterial agents with different proportions.

3. A microbial composite growth-promoting agent according to claim 2, characterized in that: The volume ratio between the fermentation broth of Penicillium ZJ-1 and the fermentation broth of Aspergillus flavus ZJ-6 was 4:

1.

4. Use of the microbial composite agent according to claim 1 in promoting plant growth in soil contaminated with high concentrations of uranium.

5. The use according to claim 4, characterized in that: The microbial composite growth-promoting agent improves the absorption of nutrients in the soil by plants through dissolving phosphorus, synthesizing iron carriers, and producing IAA and ACC deaminase, thereby promoting plant growth.

6. The use according to claim 4, characterized in that: The plant is eyebright, and the microbial composite agent can enhance the effect of eyebright in repairing soil contaminated by heavy metal uranium.

7. The use according to claim 4, characterized in that: Applying a microbial composite growth-promoting agent to uranium-contaminated soil can promote the growth of eyebright grass and enhance its ability to enrich heavy metal uranium.

Images