Microbial composite growth promoting agent and application thereof

Abstract

The application relates to a microbial composite growth-promoting agent and an application thereof, wherein the microbial composite agent is compounded by Penicillium ZJ-1 and Aspergillus flavus ZJ-6; the preservation number of the Penicillium ZJ-1 is CGMCC NO.40559; and the preservation number of the Aspergillus flavus ZJ-6 is CGMCC NO.40560. The microbial composite agent is suitable for application in promoting plant growth. The microbial composite growth-promoting agent has the characteristics of dissolving phosphorus, producing IAA, synthesizing iron carrier and ACC deaminase; and through millet grass potting experiments, it is proved that the composite agent can effectively promote the growth of millet grass under uranium stress and the enrichment of uranium by the millet grass.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental engineering microorganisms and soil remediation technology, and particularly relates to a microbial compound growth promoting microbial inoculant and application thereof. BACKGROUND

[0002] With the development of social industrialization, the use of nuclear energy is large, and the mining and smelting of uranium mines are increasing, which leads to the increasingly serious pollution of the environment around the uranium mine. Influenced by natural environment such as rainfall, snowfall, leakage, and surface runoff, the uranium-containing waste also gradually migrates and spreads to the surrounding environment, especially the pollution of the soil environment by radionuclides. However, at present, there are still a large number of farmers living around the mining area and engaging in various agricultural activities. Heavy metal uranium can not only cause biological lesions through radioactive decay, but also cause damage to the human body through breathing or food chain, seriously affecting human health. The heavy metal uranium contaminated soil not only restricts the development of the ecological environment around the mine, but also threatens human health. Therefore, it is necessary to treat the contaminated soil around the uranium mine.

[0003] Bioremediation technology is widely concerned because of its economic applicability, no secondary pollution in the remediation process, permanent treatment effect, and the ability to effectively protect the soil micro-ecological environment. Soil microorganisms are diverse, including bacteria, fungi, actinomycetes, and other microbial populations, which widely and deeply participate in almost all biological activities and biochemical reactions in the soil. They play a key role in a direct or indirect way. Due to its high sensitivity to changes in the soil environment, soil microorganisms are considered as the most sensitive indicators for assessing the soil environmental quality standard. Soil microorganisms can produce significant promoting effects on plant growth by means of diverse mechanisms. These mechanisms cover multiple key aspects, including but not limited to: secreting plant hormones and enzyme substances such as indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which can not only promote plant growth, but also play an important role in regulating plant physiological processes; through phosphorus dissolution, converting phosphorus elements in the soil that are difficult for plants to directly absorb and utilize into absorbable forms, thereby improving plant uptake of phosphorus; producing siderophores, which have the ability to specifically bind to trivalent iron ions and can transport the bound iron ions into microbial cells to meet the demand of microorganisms for iron elements; and performing nitrogen fixation, converting free nitrogen in the air into nitrogen sources available to plants. In addition, soil microorganisms can also effectively alleviate the environmental stress faced by plants, such as drought, salinity, heavy metal pollution, and other adverse conditions that adversely affect plant growth and development, by increasing the absorption rate of host plants to various nutrients in the soil.

[0004] The patent with publication number CN118703343A discloses that a strain of Penicillium Q77 has a growth-promoting effect on Danshen seedlings, which is manifested in that various growth indicators (leaf length, leaf width, plant height, and stem thickness) of Danshen are improved, and the improvement effect is significant. Meanwhile, the strain can improve the disease resistance of Danshen seedlings.

[0005] The patent with publication number CN111657304A discloses a method for strengthening ryegrass to repair heavy metal contaminated soil by Aspergillus flavus TL-F3. Under the stress of heavy metals, Aspergillus flavus TL-F3 still has a growth-promoting effect on ryegrass, which not only improves the enrichment efficiency of ryegrass on 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 exhibit good growth-promoting ability. However, most of the strains studied at present are single strains, which have weak competitive ability and are easily inhibited, and in the existing technology, the research on composite growth-promoting microbial agents-plant combined repair of uranium contaminated soil is relatively scarce, and the microbial resources that can be practically applied in production are still scarce. Therefore, it is urgent to carry out further research to develop more growth-promoting microbial agents, so as to provide more solid support for the sustainable development of soil environment governance. SUMMARY

[0007] The purpose of the present application is to provide a microbial composite growth-promoting agent and its application. The microbial composite growth-promoting agent can dissolve phosphorus, synthesize iron carriers, produce IAA and ACC deaminase. The microbial composite agent can promote the absorption of nutrients in the soil by small millet grass and enhance the tolerance of small millet grass to harsh environments, thereby promoting the growth of small millet grass. In addition, the microbial composite growth-promoting agent can also strengthen the repair of uranium contaminated soil by small millet grass.

[0008] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

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

[0010] Further, the preparation method of the microbial composite growth-promoting agent is as follows: the Penicillium ZJ-1 and the Aspergillus flavus ZJ-6 are inoculated into potato sucrose agar medium respectively, and spores are produced after incubation at 28°C for 7-10 days. The spores on the surface of the mycelium are scraped gently and placed in sterile water containing 10% glycerol. The mycelium is removed by filtration to obtain a spore solution with an OD600 value of 1.0±0.5, i.e. a spore concentration of 1×10 8 The spore solution is inoculated into potato glucose broth liquid medium at an inoculation amount of 1%, and incubated at 28°C, 120r / min or 150r / min for 7 days to obtain Penicillium ZJ-1 fermentation broth and Aspergillus flavus ZJ-6 fermentation broth. The Penicillium ZJ-1 fermentation broth and the Aspergillus flavus ZJ-6 fermentation broth are mixed in different volume ratios to obtain composite microbial agents with different ratios.

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

[0012] In a second aspect, the present application provides the application of the microbial composite microbial agent in promoting the growth of plants in high-concentration uranium-contaminated soil.

[0013] Further, the microbial composite growth-promoting agent promotes plant growth by dissolving phosphorus, synthesizing iron carriers, producing IAA and ACC deaminase to improve the absorption of nutrient elements in the soil by plants.

[0014] Preferably, the plant is small grass. Further, the microbial composite microbial agent can strengthen the repair of heavy metal uranium-contaminated soil by small grass.

[0015] Further, the microbial compound growth promoting agent is applied in the uranium contaminated soil to promote the growth of the little millet grass and improve the uranium enrichment capacity of the little millet grass.

[0016] In a specific application, the microbial compound growth promoting agent is added once every 5 days after the little millet grass seeds are sowed for 20 days, 400ml of the microbial compound growth promoting agent is diluted to 800ml with deionized water, and then added to the soil matrix close to the roots of the little millet grass seedlings, and the addition is stopped after 6 times.

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

[0018] The Penicillium ZJ-1 and the Aspergillus flavus ZJ-6 in the present application are uranium mine indigenous strains screened by the applicant, and the compound microbial agent has good growth promoting effect, and the specific performance is that the compound microbial agent has the characteristics of phosphorus dissolution, IAA production, iron carrier synthesis and ACC deaminase, and the pot experiment proves that the compound microbial agent can not only promote the growth of the little millet grass, but also strengthen the repair of the uranium contaminated soil, and has a very wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The growth state schematic diagrams of the Penicillium ZJ-1 and the Aspergillus flavus ZJ-6 in the PDA culture medium and the PDB culture medium are shown in Fig. 1, wherein (a) is the PDA culture medium, and (b) is the PDB culture medium;

[0020] Figure 2 The qualitative determination result schematic diagrams of the iron carrier production capacity of the Penicillium ZJ-1 and the Aspergillus flavus ZJ-6 are shown in Fig. 2, wherein (a) is the Penicillium ZJ-1, and (b) is the Aspergillus flavus ZJ-6;

[0021] Figure 3 The color development changes of the microbial compound growth promoting agent on the 5th day and the 9th day are shown in Fig. 3, wherein (a) is the 5th day, and (b) is the 9th day;

[0022] Figure 4 The phosphorus dissolution circles of the strains on the NBRIP culture medium are shown in Fig. 4;

[0023] Figure 5 The determination result schematic diagrams of the ACC deaminase specific activity of the ZJ-1 and ZJ-6 microbial liquid alone and in combination are shown in Fig. 5;

[0024] Figure 6The biomass of different parts of Eragrostis poaeoides in the control group and the experimental group is compared in the following graph;

[0025] Figure 7 The percentage of different forms of uranium in the rhizosphere soil of each treatment group is compared in the following graph. DETAILED DESCRIPTION

[0026] The application will be further described in detail below in combination with specific examples.

[0027] Example 1

[0028] A preparation method of a microbial composite growth-promoting microbial agent, comprising the following steps:

[0029] 1) Preparation of culture medium: prepare potato sucrose agar medium (PDA) and potato glucose broth (PDB), high-temperature sterilization for 20-30 min, and stand by;

[0030] 2) inoculate Penicillium ZJ-1 with the preservation number of CGMCC NO.40559 into potato sucrose agar (PDA) medium (as shown in Figure 1 a), and incubate at 28℃ for 10 days to produce spores, gently scrape the spores on the surface of the mycelium into sterile water containing 10% glycerol, filter out the mycelium to obtain a spore solution, and measure the OD600 value to be 1.0±0.5, that is, the spore concentration is 1×10 8 above / mL, inoculate the spore solution into potato glucose broth (PDB) liquid medium at an inoculation amount of 1%, and incubate at 28℃ and 120 r / min for 7 days to obtain ZJ-1 fermentation liquor (as shown in Figure 1 b), and store in a refrigerator for standby;

[0031] 3) inoculate Aspergillus flavus ZJ-6 with the preservation number of CGMCC NO.40560 into potato sucrose agar (PDA) medium (as shown in Figure 1 a), and incubate at 30℃ for 7 days to produce spores, gently scrape the spores on the surface of the mycelium into sterile water containing 10% glycerol, filter out the mycelium to obtain a spore solution, and measure the OD600 value to be 1.0±0.5, that is, the spore concentration is 1×10 8 above / mL; inoculate the spore solution into potato glucose broth (PDB) liquid medium at an inoculation amount of 1%, and incubate at 28℃ and 150 r / min for 7 days to obtain ZJ-6 fermentation liquor (as shown in Figure 1 b), and store in a refrigerator for standby;

[0032] 4) mix ZJ-1 fermentation liquor and ZJ-6 fermentation liquor according to the volume ratio of 1:4, 3:2, 2:3, 1:1 and 4:1, and stand by.

[0033] Example 2: Penicillium ZJ-1, Aspergillus flavus ZJ-6, and determination of indole acetic acid (IAA) produced by microbial complex growth promoting microbial inoculant

[0034] Take Penicillium ZJ-1, Aspergillus flavus ZJ-6, and different proportions of microbial complex growth promoting microbial inoculant prepared in Example 1, respectively, add tryptophan filtered by sterile filter, so that the concentration of tryptophan in the final complex fermentation broth is 200 mg / L -1 , set up blank control;

[0035] Take 48 h, 72 h, 84 h, 96 h, 120 h, and 144 h fermentation broth in centrifuge tubes, centrifuge at 8000 r / min for 10 min, add salkowskis colorimetric solution at 1:1 after taking the supernatant, place in the dark to avoid coloration for 30 min, and measure the absorbance value and concentration value at OD 530 The average content of IAA produced by ZJ-6 strain solution is 10.24 mg / L ± 1.2 mg / L, the average content of IAA produced by ZJ-1 strain solution is 3.19 ± 0.75 mg / L, and the content of IAA produced by the mixture of ZJ-1 and ZJ-6 reaches the highest on the 5th day, among which the content of 4:1 is the highest, which is 10.33 mg / L ± 1.8 mg / L.

[0036] Example 3: Determination of siderophore produced by Penicillium ZJ-1, Aspergillus flavus ZJ-6, and microbial complex growth promoting microbial inoculant

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

[0038] 2) Qualitative screening of siderophore production capacity of microbial inoculant: use a sterile inoculation ring to dip the bacterial solution into the modified siderophore detection solid medium, and incubate at 30°C for 9 days. If the strain does not produce siderophore, the medium will not change. If the strain produces siderophore, the medium will change, i.e. a clear yellow hydrolysis ring will appear;

[0039] 3) Quantitative screening of siderophore production ability of bacterial agent: take the spore suspension of the above-mentioned strains and inoculate in the modified siderophore detection liquid medium at 1% inoculation amount, and culture at 30°C and 160r / min for 9d. Centrifuge the culture liquid for 10min, mix 1ml supernatant (reference value (Ar)) and 2x CAS detection liquid (10x CAS detection liquid mixed with water at 1:4) in equal volume, and stand in dark for 30min for standby; take double distilled water as control to adjust zero, and measure the OD value at 630nm wavelength (As) by enzyme labeling instrument. If no siderophore is produced, the culture medium does not change, and if the strain produces siderophore, the culture medium changes to orange. The relative content of siderophore in the sample is expressed by the ratio of As / Ar, and the smaller the value, the stronger the siderophore production ability of the strain. The active unit of siderophore in the sample is expressed by the ratio of formula (1), and the higher the active unit, the stronger the siderophore production ability. It is found that the siderophore production of ZJ-1 reaches 52.46%, and the siderophore production of ZJ-6 reaches 75.39%. And the As / Ar of the two strains is less than 0.5, indicating that they are both high-yield siderophore-producing bacteria. The content of siderophore produced by the 4:1 complex of Penicillium and Aspergillus flavus is the highest, reaching 91.46% on the ninth day (as shown in Figure 2 and Figure 3 ).

[0040]

[0041] Example 4: Phosphorus-dissolving determination of Penicillium ZJ-1, Aspergillus flavus ZJ-6 and microbial complex microbial inoculant

[0042] 1) Preparation of NBRIP medium: 10g / L glucose, 5g / L Ca3(PO4)2, 3g / L NaCl, 0.1g / L (NH4)2SO4, 6g / L MgCl2·6H2O, 2.5g / L KCl, pH 6.8-7.0, autoclaved at 108°C for 15min for standby;

[0043] 2) Inoculate the strains to NBRIP solid plate, and qualitatively determine the phosphorus-dissolving ability by the growth condition of the strains. The results show that after 7d of culture, Penicillium ZJ-1, Aspergillus flavus ZJ-6 and the 4:1 complex group all have obvious phosphorus-dissolving ring (as shown in Figure 4 );

[0044] 3) Further transfer the complex microbial inoculant with different proportions at 1% inoculation amount to NBRIP liquid medium and culture for 10d. The soluble phosphorus content in the supernatant is 87.37mg / L-157.64mg / L, and the highest is 157.64mg / L for the 4:1 complex group of ZJ-1 and ZJ-6 on the 8th day.

[0045] Example 5: Penicillium ZJ-1, Aspergillus flavus ZJ-6, and microbial complex growth promoting agent ACC deaminase production determination

[0046] 1) TSB liquid medium preparation: Tryptone 17.0 g, soybean papain digest 3.0 g, sodium chloride 5.0 g, potassium phosphate dibasic 2.5 g, glucose 2.5 g, pH 7.3±0.2 (25℃), water 1000 ml;

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

[0048] 0.2% 2,4-dinitrophenylhydrazine solution preparation: weigh 0.2 g of 2,4-dinitrophenyl, dissolve it in an appropriate amount of 2 mol / L HC1, transfer it to a 100 mL volumetric flask, and dilute it to volume with 2 mol / L HC1.

[0049] Coomassie brilliant blue staining solution preparation: weigh 0.1 g of Coomassie brilliant blue, dissolve it in 50 mL of 95% ethanol, then add 100 mL of 85% H3PO4 and mix well, transfer it to a 1000 mL volumetric flask and dilute to volume.

[0050] 2) ACC deaminase activity determination: The bacterial agent prepared in Example 1 was inoculated into 7.5 mL TSB liquid medium at a ratio of 1%, and cultured at 28°C with a rotation speed of 160 rpm for 24 hours. Subsequently, the culture was placed in a 4°C environment and centrifuged at a rotation speed of 10,000 rpm for 10 minutes to separate the bacterial cells. The bacterial cells were resuspended in 7.5 mL DF medium without (NH4)2SO4, and 45 μL of ACC solution with a concentration of 0.5 mol / L was added. After 48 hours of continuous culture and centrifugation, the precipitate was washed with 5 mL of Tris-HCI buffer (0.1 mol / L, pH 7.6), and after vortexing, the precipitate was collected by centrifugation. Next, the precipitate was added to 1 mL of Tris-HCI buffer (0.1 mol / L, pH 7.6), and after centrifugation for 10 minutes, the precipitate was resuspended in 600 μL of Tris-HCI buffer (0.1 mol / L, pH 8.5) and 30 μL of toluene was added. The mixture was quickly vortexed for 30 seconds to prepare a cell lysate. 200 μL of the cell lysate was stored at 4°C for later use.

[0051] 3) Protein concentration determination: 200 μL of the above-mentioned stored cell lysate was taken, 20 μL of ACC solution with a concentration of 0.5 mol / L was added, and the mixture was thoroughly mixed and incubated in a 30°C water bath for 15 minutes. Then, 1 mL of HCl solution with a concentration of 0.56 mol / L was added, and after mixing, the mixture was centrifuged at room temperature for 10 minutes. 1 mL of the supernatant was taken, 800 μL of 0.56 mol / L HCl solution was added, and after thoroughly mixing, 300 μL of 2,4-dinitrophenylhydrazine solution was added. The mixture was incubated in a 30°C water bath for 30 minutes. Subsequently, 2 mL of 2 mol / L NaOH solution was added (color development time 5 minutes), and the absorbance was measured at a wavelength of 540 nm to determine the concentration of α-ketobutyric acid generated in the reaction.

[0052] Sample protein concentration determination: 200 μL of the cell lysate was taken, 1 mL of Coomassie brilliant blue staining solution was added for reaction, and after thoroughly mixing, the mixture was allowed to stand (color development time 5 minutes). The absorbance was measured at 595 nm. The absorbance value was compared with the protein standard curve to determine the protein concentration of the sample.

[0053] 4) The activity of ACC deaminase was determined by measuring the amount of α-ketobutyric acid produced when ACC deaminase cleaved ACC. The activity was expressed as specific activity, i.e., the amount of α-ketobutyric acid produced per milligram of protein per hour, with units of μmol α-ketobutyric acid / mg·protein / h. The specific activity calculation formula was the ratio of enzyme activity (μmol α-ketobutyric acid / L / h) to protein concentration (μg / mL). The specific activity of ACC deaminase of ZJ-1 was about 0.37, and that of ZJ-6 was 0.46. The activity of the ZJ-1 and ZJ-6 complex bacterial population in the 4:1 group was significantly higher than that of the individual and other complex ratios, reaching 2.84, which wasFigure 5 As shown.

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

[0055] 1) Pot experiment planting small millet grass, the specific method is as follows:

[0056] (1) The uranium concentration in the soil is 199.38 g·kg -1 , the experiment is divided into 2 treatment groups, 4 pots in each group, the control group: without adding any exogenous bacteria; the experimental group: the microbial composite growth-promoting inoculant prepared in Example 1;

[0057] (2) The planting period of small millet grass is 55 days, 20 days after the small millet grass seeds are sown, the composite inoculant is added every 5 days, 400 ml of microbial composite growth-promoting inoculant is diluted to 800 ml with deionized water each time, and then added, and the diluted microbial composite growth-promoting inoculant is directly poured into the soil medium near the root of the small millet grass seedling during the addition; after 6 times of addition, stop, and harvest the small millet grass after the growth period ends for standby.

[0058] 2) Biomass determination of small millet grass:

[0059] (1) The above-ground biomass detection method: cut off the above-ground part of the plant, wash and dry to constant weight, and weigh with a balance to obtain the dry biomass of the above-ground part.

[0060] (2) The underground biomass detection method: usually adopt the digging method, carefully dig out the plant roots and surrounding soil together, then wash and dry the roots, and weigh to obtain the underground biomass.

[0061] (3) Biomass is a direct reflection of the growth of plants in uranium-containing soil, and also directly reflects the influence of uranium ore soil on plants. The biomass of the underground part and the above-ground part of the experimental group is higher than that of the control group, which shows that the addition of the composite inoculant can reduce the toxicity of uranium to plants, effectively promote the growth of small millet grass, and see Figure 6 .

[0062] 3) Determination of uranium enrichment and transportation by composite inoculant and small millet grass

[0063] (1) The plant / soil sample uranium content is determined by dry ash method. The dried little millet aboveground and underground parts are respectively put into crucibles, three parallel, marked and placed in the muffle furnace at 400°C for carbonization for 3 hours, and then heated to 600°C for ashing for 5 hours. When the furnace temperature drops to room temperature, the crucibles are taken out, and 0.1000 g of ash sample in each crucible is accurately weighed in a tetrafluoroethylene crucible; the rhizosphere soil sieved through a 100 mesh sieve is dried to constant weight and weighed 0.1000 g in a tetrafluoroethylene crucible. Nitric acid, perchloric acid and hydrofluoric acid (5:3:2) are added in the tetrafluoroethylene crucible in proportion, the crucible is placed on a graphite hot plate for heating and digestion, hydrogen peroxide is added to remove acid when there is no obvious particle in the pyrolysis liquid, the liquid is clarified and cooled, and then filtered through a 0.45 um water system filter head, acid is added to constant volume, and then the uranium concentration is determined by ICP-OES.

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

[0065] BCF = C 土壤部分 (mg / kg) / C

[0066] The transfer coefficient (TF) is the distribution of uranium adsorbed by the roots of little millet in the plant body, which is the ratio of the uranium content in the aboveground part of little millet to the uranium content in the root part. The larger the transfer coefficient, the stronger the ability of little millet to transport uranium from the root to the stem and leaf part.

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

[0068] (3) The uranium content in the aboveground part of little millet in the control group is 14.56 mg / kg, and the uranium content in the underground part is 243.12 mg / kg. The uranium concentration in the aboveground part of little millet in the experimental group is 22.39 mg / kg, and the uranium content in the underground part is 398.46 mg / kg. The absorption of little millet in the experimental group is much higher than that in the control group, which shows that the addition of compound microbial agent promotes the absorption of active uranium by little millet, and also proves that the compound microbial agent has a synergistic effect on little millet, making the absorption of uranium by little millet better.

[0069] According to the data shown in Table 1, the enrichment coefficients of the aboveground parts and underground parts of the control group plants are 0.08 and 1.21 respectively, while the enrichment coefficients of the aboveground parts and underground parts of the experimental group plants are 0.21 and 2.43 respectively. The enrichment coefficient of the underground part is significantly higher than 1, indicating that the small reed has a significant enrichment capacity in the underground part under the auxiliary action of the compound microbial agent. However, the enrichment effect of the aboveground part is obviously lower than that of the underground part, which shows that the root of the small reed has a more outstanding ability to store uranium than the stem and leaf part. The translocation coefficient of the control group is 0.06, and the translocation coefficient of the experimental group is 0.08, which shows that the application of the compound microbial agent increases the content of active uranium in the small reed, enhances its adsorption capacity of uranium and its tolerance to uranium. In addition, the addition of the compound microbial agent also proves to have a positive promoting effect on the growth of the small reed and the absorption of uranium.

[0070] Table 1 BCF and TF of the small reed in each treatment group

[0071]

[0072] 4) Effect of compound microbial agent on uranium speciation in soil in cooperation with small reed Method: Based on the Tessier five-step extraction method, the step-by-step chemical extraction is as follows:

[0073] (1) Exchangeable uranium: take 3.000g of soil sample, put it in a 150ml conical flask, add 20ml of 1mol / L MgCl2 solution with pH value of 7, shake for 2 hours, transfer to a 50ml centrifuge tube, and centrifuge at 6000rpm for 10min, take the supernatant and move it into a 100ml volumetric flask, rinse the conical flask, rinse the liquid in the same way and move it into the above-mentioned volumetric flask, add 1ml of nitric acid, and dilute with ultrapure water;

[0074] (2) Carbonate-bound uranium: take the soil sample after the above step experiment and put it in a conical flask, add 30ml of 1mol / L sodium acetate solution with pH value of 5, constant temperature oscillation, take the supernatant and the constant volume process of step (1);

[0075] (3) Organic matter combination: take the soil sample after the above step experiment and put it in a conical flask, add 20ml of 30% H2O2 respectively, react at room temperature for 1h, then heat in a water bath at 90℃ until dry; add 20ml of H2O2, continue to heat in a water bath until dry; add 50ml of CH3COONH4 solution, constant temperature oscillation for 2h, get the supernatant and the constant volume process of step (1).

[0076] (4) Amorphous iron and manganese oxide / hydroxide-bound uranium: take the residual soil sample after the above step experiment in a conical flask, add 20ml of Tamm's solution, constant temperature oscillation for 5h, get the supernatant and the constant volume process of step (1);

[0077] (5) Crystalline iron-manganese oxide / hydroxide combination: 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 shake 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 uranium accounted for 28%, the organically bound uranium accounted for 1%, the crystalline ferromanganese oxidized uranium accounted for 26%, the amorphous ferromanganese oxidized uranium accounted for 11%, the carbonate-bound uranium accounted for 27%, and the exchangeable uranium accounted for 8%. Of these, the inert uranium accounted for 55%, and the active uranium accounted for 45%. In the experimental groups treated with the ZJ-1 and ZJ-6 composite inoculants, the residual uranium accounted for 24%, the organically bound uranium accounted for 1%, the crystalline ferromanganese oxidized uranium accounted for 22%, the amorphous ferromanganese oxidized uranium accounted for 18%, the carbonate-bound uranium accounted for 31%, and the exchangeable uranium accounted for 5%. Of these, the inert uranium accounted for 47%, and the active uranium accounted for 53%. The active uranium content in the experimental groups was lower than that in the control group, indicating that the addition of the composite inoculant promoted the conversion of inert uranium. Furthermore, the exchangeable uranium content was lower than that in the control group, indicating that the addition of the composite inoculant significantly enhanced plant uranium absorption.

[0080] In summary, the microbial composite agent of the present invention has a good growth-promoting effect, specifically, it has the characteristics of solubilizing phosphate, producing IAA, synthesizing siderophores 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 broad application prospects.

Claims

1. Application of a microbial complex inoculant in the plant growth promotion in high-concentration uranium-contaminated soil; the plant is millet grass; the microbial complex growth-promoting inoculant is compounded by Penicillium sp. ( Penicillium ) ZJ-1 and Aspergillus flavus sp. ( Aspergillus flavus ) ZJ-6, wherein the Penicillium sp. ( Penicillium ) ZJ-1 is preserved in the China General Microbiological Culture Collection Center on April 7, 2023, with a preservation number of CGMCC NO. 40559; and the Aspergillus flavus sp. ( Aspergillus flavus ) ZJ-6 is preserved in the China General Microbiological Culture Collection Center on April 7, 2023, with a preservation number of CGMCC NO. 40560.​​​​ 2. Use according to claim 1, characterized in that, The microbial complex growth-promoting agent can promote plant growth by dissolving phosphorus, synthesizing iron carriers, producing IAA and ACC deaminase to improve the absorption of nutrient elements in the soil by plants.

3. Use according to claim 1, characterized in that, The preparation method of the microbial composite growth-promoting agent is as follows: Penicillium ( Penicillium )ZJ-1 and Aspergillus flavus( Aspergillus flavus )ZJ-6 were inoculated onto potato sucrose agar medium and cultured at 28°C for 7-10 days until spores were produced. Spores were gently scraped from the surface of the mycelium 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 determined to be 1.0±0.5, that is, the spore concentration was 1×10 8 The spores were inoculated into potato glucose broth at a 1% inoculum volume, and cultured at 28°C, 120 rpm and 150 rpm for 7 days to obtain Penicillium ( Penicillium )ZJ-1 fermentation broth and Aspergillus flavus( Aspergillus flavus ) ZJ-6 fermentation broth, the fermentation broth of Penicillium ZJ-1 and the fermentation broth of Aspergillus flavus ZJ-6 were mixed in different volume ratios to obtain composite bacterial agents of different proportions.

4. Use according to claim 3, characterized in that, Penicillium ( Penicillium )ZJ-1 fermentation broth and Aspergillus flavus ( Aspergillus flavus )The volume ratio of ZJ-6 fermentation broth was 4:

1.

5. The use according to claim 1, characterized in that, The microbial complex agent can strengthen the repair of millet grass to the heavy metal uranium contaminated soil.

6. Use according to claim 5, characterized in that, The microbial complex growth-promoting agent is applied in the uranium contaminated soil to promote the growth of the millet grass and improve the ability of the millet grass to enrich heavy metal uranium.

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