A method for planting plants in cooperation with microorganisms in heavy metal contaminated soil

Abstract

The present application belongs to the field of agricultural planting, and particularly relates to a microbial synergistic plant planting method in heavy metal contaminated soil. The planting method comprises the following steps: planting plants mainly in the form of crops in the heavy metal contaminated soil to be repaired; applying 2-keto-L-gulonic acid mother liquor, plant growth promoting bacteria and plant biological stimulants to the soil around the roots and / or the leaves of the plants in the vegetative growth stage of the plants; and applying the gulonic acid mother liquor and endophytic bacillus in the reproductive growth stage of the plants. In the vegetative growth stage of the crops, the 2-keto-L-gulonic acid mother liquor, the plant growth promoting bacteria and the plant biological stimulants are applied, and in the reproductive growth stage, the 2-keto-L-gulonic acid mother liquor and the endophytic bacillus are applied, which not only greatly improves the heavy metal enrichment efficiency of the roots, stems and leaves of the crops, but also greatly reduces the heavy metal concentration of the seeds of the crops and meets the national feed or food standards.

Description

Technical Field

[0001] This invention belongs to the field of agricultural planting, specifically relating to a method for plant cultivation using microbial synergy in heavy metal contaminated soil. Background Technology

[0002] Phytoremediation, as an emerging green and environmentally friendly remediation technology, possesses many advantages that traditional remediation techniques cannot match. Phytoremediation offers advantages such as low cost, no damage to site structure, no secondary pollution, environmental friendliness, and easy acceptance. However, phytoremediation technology still has some shortcomings, hindering its widespread application. These shortcomings include: 1) Long remediation cycle and low remediation efficiency. The effectiveness and speed of phytoremediation depend on the biomass of plants per unit area and the concentration of heavy metals in the aboveground parts. While hyperaccumulating plants have strong accumulation capabilities, they generally have small biomass and slow growth; while high-biomass plants, although having large aboveground biomass, do not have high concentrations of heavy metals within their bodies and have weak accumulation capabilities, resulting in low remediation efficiency. Currently, most hyperaccumulating plants have drawbacks such as slow growth, small biomass, limited root extension depth, metal selectivity, and low heavy metal transfer rate from roots to aboveground parts, resulting in very low actual remediation efficiency and long remediation time; 2) The remediation capacity of a single plant is limited, usually only able to accumulate one or two heavy metals; 3) Phytoremediation technology is easily affected by environmental factors, leading to unstable remediation effects; 4) Phytoremediation has low economic benefits; 5) In northern regions with short frost-free periods, the suitable period for plant growth is very short.

[0003] In addition, planting plants, especially crops, in soil contaminated with heavy metals often results in the accumulation of heavy metals in the seeds or fruits, making it difficult for the plants to enter the food chain or be transformed into high-value products, thus hindering economic benefits.

[0004] These problems have severely hampered the large-scale application of phytoremediation technology for contaminated soil, and research on new technologies in this field has almost stagnated for many years, with no substantial breakthroughs. Summary of the Invention

[0005] This invention proposes a novel plant cultivation method with microbial synergy in heavy metal contaminated soil. This method can improve the heavy metal accumulation efficiency of plant roots, stems and leaves, shorten the remediation cycle, and at the same time reduce the heavy metal content of crop fruits / seeds (edible parts) to meet feed and / or food standards, thereby generating economic benefits and reducing remediation costs.

[0006] The technical solution adopted by this invention to solve the technical problem is:

[0007] A method for plant cultivation with microbial synergy in heavy metal contaminated soil, the method comprising the following steps:

[0008] Step 1: Planting plants in the heavy metal contaminated soil to be remediated, preferably, the plants are mainly crops;

[0009] Step 2: During the vegetative growth stage of the plant, apply 2-keto-L-gulonic acid stock solution (hereinafter referred to as gulonic acid stock solution), plant growth-promoting bacteria and plant biostimulants to the soil around the roots and / or the leaves.

[0010] Step 3: Apply gulonic acid stock solution and plant endophytic Bacillus to the soil around the roots and / or leaves of the plant during its reproductive growth stage.

[0011] In the above-mentioned method for plant cultivation with microbial synergy in heavy metal contaminated soil, the 2-keto-L-gulonic acid mother liquor is the residue after evaporation (RAE) in the second step of vitamin C industrial fermentation, after ultrafiltration, evaporation, concentration, crystallization and extraction of 2-keto-L-gulonic acid from the 2-keto-L-gulonic acid fermentation broth.

[0012] In the aforementioned method for synergistic microbial plant cultivation in heavy metal contaminated soil, the main components of the 2-keto-L-gulonic acid mother liquor include: 2-keto-L-gulonic acid, small amounts of oxalic acid, formic acid, acetic acid, and trace amounts of amino acids, polypeptides, oligosaccharides, and nucleotides. The molecular weights of all these components are below 500 Daltons, and most are below 200 Daltons. Therefore, this gulonic acid mother liquor is also known as small-molecule organic carbon or low-molecular-weight organic acid.

[0013] In the aforementioned method for synergistic microbial cultivation in heavy metal contaminated soil, the pH of the 2-keto-L-gulonic acid stock solution is adjusted to 2.0-8.0. The pH value of the stock solution needs to be determined based on the pH of the soil to be used and the suitable pH for the plant, and adjusted to this value using an alkaline substance. The gulonic acid stock solution needs to be diluted 50-1000 times with water and applied via drip irrigation / watering / sprinkler irrigation at a rate of 45-1500 kg / ha / application, preferably 150-300 kg / ha / application, for 3-6 applications.

[0014] In the above-mentioned method for plant cultivation with synergistic microbial activity in heavy metal contaminated soil, the plant growth-promoting bacteria are single or compound liquid or solid inoculants of Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens.

[0015] In the above-mentioned method for plant cultivation using microbial synergy in heavy metal contaminated soil, the strain of endophytic Bacillus is named CBS-5 (Bacillus endophyticus CBS-5), which is deposited at the China Industrial Microbial Culture Collection Center with accession number CICC 10622.

[0016] In the above-mentioned method for planting plants with microbial synergy in heavy metal contaminated soil, the plant is corn, the vegetative growth stage is from seedling emergence to the large trumpet stage, and the reproductive growth stage is from the tasseling stage to maturity.

[0017] In the aforementioned method for synergistic microbial cultivation in heavy metal-contaminated soil, the plant biostimulants include, but are not limited to, one or more of the following: humic acid, fulvic acid, polyglutamic acid, itaconic acid, sorbitol, citric acid, and γ-aminobutyric acid. The method of using plant biostimulants must be determined according to the product instructions, plant species and growth stage, soil and climate conditions, etc.

[0018] In the above-mentioned plant cultivation method with microbial synergy in heavy metal contaminated soil, the method increases the content of heavy metals such as cadmium, chromium, lead, mercury, and arsenic in corn roots, stems, and leaves by 2 to 4 times; the method strengthens the blocking of the transmission of heavy metals from crop roots, stems, and leaves to fruits / grains (edible parts) and reduces the content of heavy metals in crop grains, which can reduce the content of heavy metals such as cadmium, chromium, lead, mercury, and arsenic in corn grains to 1 / 8 to 3 / 4 of the control group.

[0019] In the above-mentioned method for synergistic microbial cultivation in heavy metal contaminated soil, the plants mentioned are wheat, rice, broad beans, soybeans, millet, rapeseed, sesame, watermelon, apples, or melons, but are not limited to the above-mentioned plants.

[0020] By employing the above technical solution, the present invention has at least the following advantages:

[0021] 1. Significantly Improves Heavy Metal Accumulation Efficiency in Crop Roots, Stems, and Leaves: Firstly, although corn has a large biomass, deep root system, and is easy to cultivate, showing potential for soil remediation, its heavy metal accumulation efficiency is relatively low compared to super-accumulating plants (mostly herbaceous plants). Therefore, using corn as a soil remediation plant results in a relatively long remediation cycle, making practical application difficult. This invention significantly improves the heavy metal accumulation efficiency in corn roots, stems, and leaves by applying gulonic acid stock solution (small molecule organic carbon / low molecular weight organic acid), plant growth-promoting bacteria, and plant biostimulants during the corn growing season, especially the vegetative growth stage, thereby achieving efficient removal of heavy metals from the soil. The principle behind this improved efficiency of heavy metal accumulation in plants is as follows: First, the main components of gulonic acid stock solution, such as 2-keto-L-gulonic acid, oxalic acid, formic acid, and acetic acid, are all low-molecular-weight organic acids. Plant growth-promoting bacteria applied to the soil also secrete low-molecular-weight organic acids. All of these factors significantly increase the concentration of low-molecular-weight organic acids in the soil, making it easier for bound and exchangeable heavy metals fixed or bound by soil aggregates to be released, thus facilitating absorption by plant roots. Second, gulonic acid stock solution, plant growth-promoting bacteria, and plant biostimulants (such as fulvic acid, itaconic acid, and γ-aminobutyric acid) all improve the plant's tolerance / resistance to heavy metals, thereby increasing the concentration of heavy metals in plant roots, stems, and leaves. Third, plant growth-promoting bacteria and plant biostimulants can accelerate the transport of heavy metals from the soil to the roots and from the roots to the stems and leaves. Fourth, gulonic acid mother liquor (small molecule organic carbon), plant growth-promoting bacteria, biostimulants, and endophytic Bacillus can all significantly increase plant biomass, thereby increasing the total accumulation of heavy metals in plants.

[0022] This invention, verified through pot experiments and field demonstrations, shows that the application of small-molecule organic carbon, plant growth-promoting bacteria, and plant biostimulants increased the content of heavy metals (such as cadmium, lead, arsenic, and zinc) in the roots, stems, and leaves of corn by 2 to 4 times. The heavy metal concentration in the corn roots, stems, and leaves can reach 1.5 to 200 times the concentration of heavy metals in the soil. Therefore, this technology can shorten the phytoremediation cycle to 1 / 4 to 1 / 2 of the cycle of traditional methods.

[0023] 2. Effectively reducing heavy metal content in corn kernels and realizing their economic value: As an economic crop, corn has been explored for remediation of soil contaminated with heavy metals. However, the heavy metal content in its kernels (edible parts) often exceeds the standard, making it unsuitable for use as feed or food, thus hindering its economic value. This invention applies gulonic acid mother liquor (small molecule organic carbon) and endophytic Bacillus endophyticus CBS-5 during the reproductive growth stage of corn, namely the tasseling, fruit setting, and fruit ripening stages. This further strengthens the blockage of heavy metal transport from plant roots, stems, and leaves to kernels, thereby significantly reducing the heavy metal content in corn kernels. We hypothesize that the enhanced transport blockade is due to the following mechanisms: First, small-molecule organic carbon and endophytic Bacillus promote the unloading of heavy metals by heavy metal transport proteins before they enter the seeds, thus strengthening the transport blockade. Second, the large number of endophytic Bacillus bacteria entering the plant can complex / chelate, electrostatically adsorb, and encapsulate heavy metals by secreting extracellular polysaccharides, resulting in greater retention of heavy metals in the roots, stems, and leaves, thereby increasing the concentration of heavy metals in the roots, stems, and leaves and reducing the amount of heavy metals transported to the seeds. Third, the application of 2-keto-L-gulonic acid stock solution can significantly increase the vitamin C level in plants. Vitamin C can form stable complexes with heavy metal ions in plants, thereby reducing the activity and bioavailability of heavy metal ions and decreasing the direct toxicity of heavy metal ions to plant cells. Vitamin C can also scavenge excess reactive oxygen species (ROS) produced in plants under heavy metal stress, reducing oxidative damage and protecting the structure and function of plant cells. Vitamin C may also participate in regulating the hormonal balance in plants, thus indirectly affecting the plant's tolerance to heavy metals. In conclusion, we hypothesize that gulonic acid mother liquor increases vitamin C content, thereby enabling plant roots, stems, and leaves to carry more heavy metals, thus reducing the transfer of heavy metals from roots, stems, and leaves to grains.

[0024] This invention, verified through pot experiments and field demonstrations, shows that the application of gulonic acid mother liquor (small molecule organic carbon / low molecular weight organic acid) and plant endophytic Bacillus reduces the content of heavy metals (such as cadmium, arsenic, lead, and copper) in corn kernels to 1 / 8 to 3 / 4 of the untreated group. The heavy metal concentration in corn kernels is only 1 / 400 to 1 / 5 of the heavy metal concentration in roots, stems, and leaves, making the heavy metal content of corn kernels meet national feed or food standards, thus demonstrating its economic value.

[0025] 3. Outstanding comprehensive advantages: Compared with existing heavy metal contaminated soil remediation technologies such as chemical leaching, electrokinetic remediation, and traditional phytoremediation, the technology of this invention has significant advantages such as high remediation efficiency, low cost, minimal impact on the soil ecological environment, simple operation, applicability to multiple types of pollution and a wide range of soil conditions.

[0026] In summary, this invention utilizes the application of gulonic acid mother liquor (small molecule organic carbon), plant biostimulants, and plant growth-promoting bacteria during the crop's growing season, especially the vegetative growth stage, and the application of endophytic Bacillus and gulonic acid mother liquor during the crop's reproductive growth stage. This significantly improves the heavy metal accumulation efficiency in crop roots, stems, and leaves, while simultaneously reducing the heavy metal concentration in crop seeds to meet national feed or food standards. This technology shortens the remediation cycle of heavy metal-contaminated soil, ensures crop seeds meet feed or food standards, generates economic benefits, and reduces remediation costs. This new technology can efficiently remediate heavy metal pollution in soil while ensuring agricultural production benefits, achieving a win-win situation for ecological restoration and economic development. It provides a highly promising and innovative solution for the remediation of heavy metal-contaminated sites, and is of great significance for ensuring national food security and promoting the construction of beautiful villages.

[0027] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0028] Figure 1 This is a flowchart of a method for synergistic microbial plant cultivation in heavy metal contaminated soil, provided by an embodiment of the present invention. Detailed Implementation

[0029] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with preferred embodiments and accompanying drawings, is provided below.

[0030] The method for plant cultivation in heavy metal contaminated soil using microbial synergy provided by this invention includes the following steps:

[0031] Step 1: Plant plants in the heavy metal contaminated soil to be remediated. To improve economic benefits, the plants are mainly agricultural crops.

[0032] Step 2: During the vegetative growth stage of the plant, apply 2-keto-L-gulonic acid stock solution, plant growth-promoting bacteria, and plant biostimulants to the soil around the roots and / or the leaves.

[0033] Step 3: Apply gulonic acid stock solution and endophytic Bacillus to the soil around the roots and / or leaves of the plant during its reproductive growth stage.

[0034] The plant cultivation method of the present invention, which uses microorganisms in synergistic microbial cultivation in heavy metal contaminated soil, can improve the efficiency of heavy metal enrichment in plant roots, stems and leaves, while reducing the heavy metal content in plant seeds.

[0035] The 2-keto-L-gulonic acid mother liquor is the residual liquid at the bottom of the reactor after the second step of the 2-keto-L-gulonic acid fermentation broth in the vitamin C industrial fermentation process has undergone ultrafiltration, evaporation, concentration, crystallization and extraction of 2-keto-L-gulonic acid.

[0036] The main components of the 2-keto-L-gulonic acid mother liquor include: 2-keto-L-gulonic acid (content 22~30%), oxalic acid (2~4%), formic acid (4~9%), acetic acid (1~3%), and trace amounts of amino acids, polypeptides, oligosaccharides, and nucleotides, with the remainder being water (content 50~55%).

[0037] The pH value of the 2-keto-L-gulonic acid mother liquor should be adjusted to 2.0-8.0. The pH value needs to be determined based on the pH of the soil to be used and the suitable pH for the plant, and adjusted to this value using an alkaline substance. The gulonic acid mother liquor needs to be diluted 50-1000 times with water and applied via drip irrigation / watering / sprinkler irrigation. In field trials, the application rate is 45-1500 kg / ha / application, with an optimal application rate of 150-300 kg / ha / application, applied 3-6 times.

[0038] The plant growth-promoting bacteria mentioned are single or compound liquid or solid inoculants of Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens, but are not limited to these plant growth-promoting bacteria. The effective viable count of the above inoculants is greater than 0.5 × 10⁻⁶. 9 The concentration of CFU / ml or g needs to be diluted with water 50 to 500 times (preferably, the dilution ratio can also be 150 to 300 times) and then applied by drip irrigation / watering / sprinkler irrigation. When applied in the field, the application rate is 10 to 300 kg / ha / time, preferably 45 to 75 kg / ha / time, and 3 to 6 times.

[0039] The plant endophytic Bacillus strain described is named CBS-5 (Bacillus endophyticus CBS-5), and is deposited at the China Industrial Microbiological Culture Collection Center (CICC) with accession number CICC 10622. It was deposited on February 4, 2013, at Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing. The effective viable count of this plant endophytic Bacillus preparation is greater than 2.0 × 10⁻⁶. 9 The concentration of CFU / ml or g needs to be diluted with water 50 to 500 times (preferably, the dilution ratio can also be 150 to 300 times) and then applied by drip irrigation / watering / sprinkler irrigation. When applied in the field, the application rate is 15 to 450 kg / ha / time, preferably 65 to 150 kg / ha / time, and 2 to 5 times.

[0040] In practical application, 2-keto-L-gulonic acid stock solution can be applied alone or in combination with plant growth-promoting bacteria or endophytic Bacillus. When adjusting the pH of the 2-keto-L-gulonic acid stock solution, either adjust the pH first, or adjust the pH after combining the gulonic acid stock solution with the growth-promoting bacteria / endophytic Bacillus. Both methods are acceptable. After combining the two, the pH will be lower (pH 2-5), which is more conducive to the dormancy of the Bacillus and maintaining its higher survival rate.

[0041] In this embodiment, the plant is corn, the vegetative growth stage is from seedling emergence to the large trumpet stage, and the reproductive growth stage is from tasseling to maturity.

[0042] In the field of heavy metal contaminated soil remediation, corn, as a widely cultivated crop, has shown considerable potential. The main advantages of corn as a soil remediation plant are: 1) Large biomass and well-developed root system: Corn, with its large biomass and extensive root system, has an advantage in absorbing and accumulating heavy metals in the soil. 2) Convenient cultivation and management: Corn cultivation techniques are mature, facilitating mechanized operations and large-scale management. 3) Corn can complete a growth cycle even in short frost-free areas in northern regions. 4) Potential economic value: During soil remediation, if the harvested corn kernels (edible parts) meet feed or food safety and hygiene standards, they can enter the market as feed or industrial raw materials, potentially achieving both soil remediation and economic benefits.

[0043] The plant cultivation method provided by this invention can improve the heavy metal accumulation efficiency of plant roots, stems and leaves, shorten the remediation cycle, and at the same time reduce the heavy metal content of seeds, thus forming an economic benefit and / or reducing the remediation cost of heavy metal contaminated soil.

[0044] In the above-mentioned method of plant cultivation with microbial synergy in heavy metal contaminated soil, the plant biostimulants include one or more of humic acid, fulvic acid, polyglutamic acid, itaconic acid, sorbitol, citric acid, and γ-aminobutyric acid, but are not limited to the above-mentioned plant biostimulants. Their usage should be determined according to the product instructions, plant species and growth stage, soil and climate conditions, etc.

[0045] The method increases the content of heavy metals such as cadmium, chromium, lead, mercury, and arsenic in corn roots, stems, and leaves by 2 to 4 times. The method strengthens the blocking of the transport of heavy metals from crop roots, stems, and leaves to fruits / grains and reduces the content of heavy metals in crop grains, which can reduce the content of heavy metals such as cadmium, chromium, lead, mercury, and arsenic in corn grains to 1 / 8 to 3 / 4 of the control group.

[0046] In the above-mentioned method for synergistic microbial cultivation in heavy metal contaminated soil, the plants are corn, wheat, rice, broad beans, soybeans, millet, rapeseed, sesame, watermelon, apples or melons, but are not limited to the above-mentioned plants.

[0047] Example 1

[0048] This embodiment uses a corn pot experiment to further illustrate the implementation method and remediation effect of the microbial synergistic plant cultivation method in heavy metal contaminated soil according to the present invention.

[0049] The soil sample used in this embodiment was collected from a heavy metal-contaminated soil in Huludao City, Liaoning Province. The soil type is brown soil, and its main relevant indicators are as follows: soil pH 6.12, organic matter 18.90 g / kg, total nitrogen 1.40 g / kg, total phosphorus 0.91 g / kg, and available potassium 105.76 mg / kg. The heavy metal content in the soil was 1.63 mg / kg for Cd, 75.8 mg / kg for Pb, 331 mg / kg for Zn, 10.5 mg / kg for As, 2.2 mg / kg for Hg, and 47.2 mg / kg for Cr. The Cd and Zn contents exceeded the screening risk values ​​for agricultural land soil pollution according to the National Soil Environmental Quality Standard (GB15618-2018) (this soil is from the vicinity of a zinc plant, hence its high zinc content).

[0050] The experimental setup for potted maize cultivation in this embodiment is as follows: Cylindrical plastic pots with a base diameter of 40 cm and a height of 50 cm were used, each containing approximately 6 kg of air-dried soil. A maize variety with a moderate growth cycle, strong adaptability, and good tolerance to pollutants, namely Dongdan 1331, was selected. Five maize seeds were sown in each pot. When the seedlings reached the 3-leaf stage, thinning was performed, leaving one seedling with uniform growth. Macronutrient fertilizers included urea, superphosphate, and potassium sulfate. Routine management included regular watering to keep the soil moist and fertilization according to the maize's growth stage. Sample collection and index determination: At harvest, samples were collected and measured from various parts of the maize root, stem, leaves, and kernels, as well as the heavy metal content in the soil, and the maize plant height and biomass.

[0051] In this embodiment, the dilution factor of the gulonic acid mother liquor (RAE) is 150 times (by volume). Alternatively, the dilution factor of the RAE can also be any value between 50 and 1000 times.

[0052] The maize pot experiment was conducted in two batches. The first batch of maize pot experiments included four treatment groups: 1) control group (CK), 2) gulonic acid stock solution application group (RAE), 3) endophytic Bacillus application group (B), and 4) RAE, endophytic Bacillus, and humic acid combined application group (RB). Each group had four replicates. The second batch of pot experiments included two treatment groups: 1) control group (CK) and 2) RAE and endophytic Bacillus combined application group (RB). Each group had three replicates.

[0053] The soil used in the second batch of maize pot experiments was the soil retained from the first batch of experiments, and the soil used was from the corresponding treatment group of the first batch of pot experiments. Each treatment received the same amount of basal fertilizer, and then received three top-dressing applications of macro- and micronutrient fertilizers via drip irrigation at the jointing and grain-filling stages. The amounts of nitrogen, phosphorus, and potassium in the drip irrigation fertilizers were kept consistent across all treatment groups. The application rate of RAE was 0.45 g / pot / time, the application rate of plant growth-promoting bacteria (a compound inoculant of Bacillus subtilis and Bacillus licheniformis) was 0.15 g / pot / time, and the application rate of plant biostimulant fulvic acid was 0.05 g / pot / time, applied 5 times during the vegetative growth stage of maize; the application rate of plant endophytic Bacillus was 0.3 g / pot / time, applied 4 times during the reproductive growth stage of maize. In the second batch of maize pot trials, all treatment groups were the same as in the first batch of pot trials, except that the RB group was given additional application of γ-aminobutyric acid (GABA, a plant biostimulant). The GABA was diluted 800 times and applied as a foliar spray twice during the seedling and jointing stages.

[0054] Plant and soil samples were collected from each treatment group at harvest time, and the contents of maize roots, stems, leaves and grains, as well as the heavy metals Cd, Pb, Zn and As in the soil, were determined.

[0055] The present invention further illustrates the dosage and usage of the repair materials or reagents used in the embodiments of the present invention through a table (Table 1).

[0056] Table 1. Dosage and application methods of materials and reagents in pot experiments of soil remediation technology

[0057]

[0058] Remark:

[0059] 1. The above-mentioned gulonic acid mother liquor, plant growth-promoting bacteria (Bacillus subtilis and Bacillus licheniformis), and plant endophytic Bacillus were all diluted 200 times with water before being applied by irrigation.

[0060] 2. In the second batch of potted plant trials, γ-aminobutyric acid (GABA) was added to the RB group, diluted 800 times, and sprayed on the leaves twice during the seedling and jointing stages.

[0061] 3. Bacillus subtilis and Bacillus licheniformis inoculants were purchased from Hubei Qiming Bioengineering Co., Ltd.; endophytic Bacillus inoculant was purchased from Beijing Naphthalene Biochemical Technology Co., Ltd.

[0062] After collecting the first batch of potted maize samples, the results of the determination of the main heavy metal content in the roots, stems, leaves, and kernels of maize are shown in Table 2. The results show that the distribution pattern of most heavy metals in different maize organs is: leaves > roots > stems > kernels or roots > leaves > stems > kernels. We speculate that this is because plants have certain protective mechanisms for fruits / kernels (genetic organs or reproductive sites), resulting in much lower heavy metal concentrations in fruits / kernels compared to those in roots, stems, and leaves. This is a relatively common phenomenon in plants such as maize, wheat, rice, and apples. However, in another study, the observation results of heavy metal content in different parts of peanuts showed that the heavy metal transport blocking phenomenon from roots, stems, and leaves to kernels differed from previous results on maize—peanuts had a significantly weaker ability to block cadmium transport than maize.

[0063] The results of heavy metal analysis in the roots, stems, and leaves of maize from the first batch of potted maize experiments (Table 2) show that the application of RAE significantly increased the heavy metal content in roots, stems, and leaves compared to the control group. Specifically, Cd content increased by 60.9%, 29.3%, and 101.6% in roots, stems, and leaves, respectively; Pb content increased by 52.9%, 73.1%, and 125.8%; and Zn content increased by 46.6%, 45.6%, and 31.4%. The RAE + growth-promoting bacteria + endophytic bacteria treatment group (RB group) showed an even greater increase in heavy metal content compared to the control group. Specifically, Cd content increased by 67.9%, 111.0%, and 170.4% in roots, stems, and leaves, respectively; Pb content increased by 118.0%, 151.1%, and 219.5%; and Zn content increased by 62.7%, 64.5%, and 59.5%, respectively. Although the heavy metal content in roots, stems and leaves of the group treated with growth-promoting bacteria + endophytic Bacillus (Group B) was higher than that of the CK group, the increase was not as significant as that of the RAE group.

[0064] On the other hand, Table 2 shows the results of heavy metal determination in maize kernels from the first batch of pot experiments. The application of RAE significantly reduced the content of heavy metals Cd, Pb, Zn, and As in the kernels compared to the control group. Specifically, the contents of Cd, Pb, Zn, and As in maize kernels decreased by 31.3%, 37.6%, 27.6%, and 16.7%, respectively. The application of RAE + growth-promoting bacteria + endophytic bacteria + biostimulants (RB group) reduced the heavy metal content in maize kernels even more significantly than the control group. Specifically, the contents of Cd, Pb, Zn, and As in maize kernels decreased by 43.7%, 39.7%, 31.5%, and 66.7%, respectively.

[0065] Table 2. Results of heavy metal content determination in maize during pot experiments (first batch) of soil phyto-microbial remediation technology.

[0066]

[0067] Note: Different lowercase letters after the values ​​in the table indicate significant differences between treatments (P < 0.05).

[0068] The results of heavy metal content detection in various parts of maize in the second batch of pot experiments (Table 3) showed the same trend as the first batch. The treatment group (RB group) treated with RAE + growth-promoting bacteria + endophytic bacteria + biostimulants showed a greater increase in heavy metal content than the control group. Specifically, Cd content in roots, stems, and leaves increased by 74.5%, 156.1%, and 146.8%, respectively; Pb content increased by 188.6%, 241.6%, and 295.5%, respectively; and Zn content increased by 96.0%, 117.3%, and 99.6%, respectively. The Cd, Pb, Zn, and As contents in maize kernels decreased by 42.8%, 40.5%, 38.4%, and 68.2%, respectively, in the second batch of pot experiments. Because the second batch of pot experiments used the same soil as the first batch, the low molecular weight organic carbon and microbial agents applied in the first batch had a certain delayed effect, meaning they also exerted a certain fertilizing effect in the second batch. Therefore, the increase in root, stem, and leaf content in the second batch was greater than that in the first batch. Furthermore, the increase in Hg enrichment efficiency in maize roots, stems, and leaves was less than that for Cd, Pb, Zn, and As; simultaneously, the decrease in Hg content in maize kernels was less than that for Cd, Pb, Zn, and As. Overall, the RB treatment had the greatest impact on As in plants, followed by Cd and Pb, and the least impact on Hg.

[0069] The results of heavy metal detection in the corn kernels showed that in the first batch of pot experiments, the contents of Cd, Pb, Zn, and As in the corn kernels of group RB were 0.09, 2.07, 18.31, and 0.008 mg / kg, respectively; in the second batch of pot experiments, the contents of Cd, Pb, Zn, and As in the corn kernels of group RB were 0.08, 1.88, 14.26, and 0.007 mg / kg, respectively. The heavy metal content in the corn kernels of both batches of pot experiments met the national standard "Feed Hygiene Standard GB13078-2017". However, the Pb content in the corn kernels exceeded the limit standard of "National Food Safety Standard Limits for Contaminants in Food 2762-2022", which requires further research and improvement.

[0070] The above test results all indicate that the combined application of RAE, growth-promoting bacteria, endophytic bacteria, and biostimulants not only effectively promoted the accumulation of heavy metals in the roots, stems, and leaves of corn, but also strengthened the blockage of heavy metal transport from stems and leaves to kernels, thereby significantly reducing the heavy metal content in corn kernels.

[0071] Table 3. Results of heavy metal content determination in maize from pot experiments using soil phyto-microbial remediation technology (batch 2)

[0072]

[0073] Note: Different lowercase letters after the values ​​in the table indicate significant differences between treatments (P < 0.05).

[0074] Furthermore, in the soil heavy metal content detection results after two consecutive batches of pot experiments, we observed that the contents of Cd, Pb, Zn, and AS in the soil of the RB treatment group were significantly lower than those in the CK group, decreasing by 10.90%, 13.07%, 11.59%, and 9.07%, respectively (Table 4). This is because the maize in the RB group absorbed more heavy metals from the soil and accumulated them in the roots, stems, and leaves.

[0075] Meanwhile, the maize plant height and biomass of the RB treatment group were significantly improved, increasing by 9.73% and 19.65% (first batch) and 9.50% and 21.24% (second batch) respectively (Table 5). This is because the RB group provided the plant with more small molecule organic carbon and beneficial microorganisms, which improved the plant's utilization rate of macronutrients, thereby increasing maize biomass.

[0076] In summary, the heavy metal detection results of the plants and soil in the two consecutive batches of pot experiments show that the remediation effect of the microbial synergistic plant cultivation method in heavy metal contaminated soil of the present invention is ideal and has achieved the expected goal.

[0077] In summary, this invention utilizes a plant-microbe co-remediation technology for heavy metal pollution using corn as a carrier. The main remediation process includes: during the crop's vegetative growth stage, applying gulonic acid stock solution, plant growth promoters, and plant biostimulants significantly enhances the absorption, accumulation, and translocation efficiency of heavy metals in the roots, stems, and leaves of the corn planting system in heavy metal-contaminated soil; during the crop's reproductive growth stage, applying small-molecule organic carbon fertilizer and endophytic Bacillus preparations strengthens the blocking of heavy metal transport from stems and leaves to grains and other fruit organs. This approach not only improves the efficiency of phytoremediation of heavy metal-contaminated soil but also ensures that the heavy metal content in the corn grains, the remediation carrier, is lower than national feed and food standards, thus generating economic benefits and achieving the goals of shortening the remediation cycle and reducing remediation costs.

[0078] Table 4. Results of soil heavy metal content detection in pot experiments using plant-microbe combined remediation technology

[0079]

[0080] Note: Different lowercase letters after the values ​​in the table indicate significant differences between treatments (P < 0.05).

[0081] Table 5. Results of maize plant height and biomass in pot experiments using plant-microbe co-remediation technology

[0082]

[0083] Note: Different lowercase letters after the values ​​in the table indicate significant differences between treatments (P < 0.05).

[0084] Example 2

[0085] In the specific implementation of the microbial synergistic plant cultivation method in heavy metal contaminated soil of the present invention, a field demonstration was conducted to further investigate and verify the effects of the combined application of gulonic acid mother liquor (RAE) + growth-promoting bacteria + endophytic bacteria + biostimulants on the heavy metal content of various organs of maize planted in heavy metal contaminated farmland soil. The difference between Example 2 and Example 1 is that the demonstration farmland in this example is located in Changtu County, Tieling City, Liaoning Province.

[0086] (1) Overview of the demonstration site

[0087] This experiment was conducted on farmland soil in Changtu County, Tieling City, Liaoning Province. The farmland soil was brown soil with a pH of 6.15, organic matter of 23.58 g / kg, total nitrogen of 1.65 g / kg, total phosphorus of 0.84 g / kg, and available potassium of 96.0 mg / kg. The soil contained heavy metals cadmium (Cd) of 0.46 mg / kg, lead (Pb) of 92.38 mg / kg, arsenic (As) of 12.05 mg / kg, and zinc (Zn) of 59.17 mg / kg. Cd and Pb exceeded the screening values ​​for agricultural land soil pollution risk according to the National Soil Environmental Quality Standard (GB15618-2018).

[0088] (2) Treatment setup and corn planting and fertilization process

[0089] The maize variety used was Xianyu 1483. The experiment consisted of two treatment groups: a control group (CK) and a group receiving RAE + growth-promoting bacteria + endophytic bacteria + biostimulants (RB). Each treatment group had three plots, each with an area of ​​39.84 m². 2 (4.8 m × 8.3 m).

[0090] Corn was sown in spring at a planting density of 4800 plants / mu. At sowing, all treatment groups received basal fertilizer (NPK) via hole application, consisting of 6 kg urea, 15 kg diammonium phosphate, and 8 kg potassium chloride. Topdressing was applied three times: at the seedling stage (4-6 leaf stage, 6 kg urea), the tasseling stage (large trumpet stage, 18 kg urea + 7 kg potassium chloride), and the grain-filling stage (silking to grain-filling stage, 5 kg urea, plus foliar spraying of 0.2%-0.3% potassium dihydrogen phosphate solution), via drip irrigation or foliar spraying. The amounts of NPK fertilizer in the basal and topdressing fertilizers were consistent across all treatment groups.

[0091] The fertilization schedule for RAE, growth-promoting bacteria, endophytic bacteria, and plant biostimulants for Group RB is as follows: ① During the vegetative growth stage: RAE is applied at a rate of 15 kg / mu / time, and the plant growth-promoting bacteria (Bacillus subtilis + Bacillus licheniformis) is applied at a rate of 5 kg / mu / time, for 5 applications during the vegetative growth stage of corn; the plant biostimulant fulvic acid is applied at a rate of 2 kg / mu / time, once during the seedling stage; the plant biostimulant γ-aminobutyric acid is applied, diluted 800 times, and sprayed foliarly twice during the seedling and jointing stages. ② During the reproductive growth stage: RAE is applied at a rate of 15 kg / mu / time, and the endophytic Bacillus bacteria inoculant is applied at a rate of 7 kg / mu / time, for 4 applications during the reproductive growth stage of corn.

[0092] RAE, plant growth promoter, humic acid, and plant endophytic Bacillus are applied by the integrated water and fertilizer device according to the usage amount, along with watering or fertilizer application.

[0093] Plant and soil samples were collected from each treatment group at harvest time to determine maize yield and heavy metal concentrations in maize roots, stems, leaves, grains, and soil (0-20 cm depth).

[0094] (3) Demonstration results

[0095] Table 6 shows the results of the determination of the main related heavy metals in maize samples collected after the autumn harvest in a field experiment, specifically in the roots, stems, leaves, and kernels. The results in Table 6 show that the distribution pattern of most heavy metals in different maize organs remains: leaves > roots > stems > kernels or roots > leaves > stems > kernels.

[0096] Table 6 shows the results of heavy metal content determination in maize roots, stems, and leaves. The RAE + endophytic bacteria group (RB group) significantly increased heavy metal content compared to the control group. Specifically, Cd content increased by 95.1%, 75.6%, and 101.7% in roots, stems, and leaves; Pb content increased by 45.3%, 52.7%, and 74.7%; Zn content increased by 59.3%, 74.4%, and 50.7%; and As content increased by 141.0%, 64.6%, and 118.8%. On the other hand, Table 5 shows the results of heavy metal determination in maize kernels. The RAE + endophytic bacteria group (RB group) significantly reduced heavy metal content in maize kernels compared to the control group. Specifically, Cd, Pb, Zn, and As content decreased by 36.4%, 42.1%, 39.5%, and 76.2% in maize kernels, respectively. This further verifies the good effect of this combination. The results in the table show that the contents of heavy metals Cd, Pb, Zn, and As in corn kernels of group RB were 0.07, 2.45, 3.61, and 0.005 mg / kg, respectively, all of which meet the requirements for heavy metal limits in the national standard "Feed Hygiene Standard GB13078-2017". However, the contents of lead and cadmium do not yet meet the requirements of the national standard "National Food Safety Standard Limits of Contaminants in Food GB2762-2022".

[0097] The results of the field demonstration further validated the pot experiment results, namely, that the application of RAE, especially the combined application of RAE and endophytic bacteria, not only effectively promoted the accumulation of heavy metals in maize roots, stems, and leaves, but also strengthened the blocking of heavy metal migration from stems and leaves to grains, thereby significantly reducing the heavy metal content in maize kernels. In the field demonstration results in Changtu, the soil heavy metal contents of Cd, Pb, Zn, and AS in the RB treatment group were significantly lower than those in the CK group, decreasing by 4.55%, 4.19%, 3.21%, and 2.67%, respectively (Table 6). Simultaneously, the maize plant height and biomass in the RB treatment group were significantly increased, increasing by 5.50% and 17.86%, respectively (Table 7).

[0098] Table 6. Results of heavy metal content determination in maize from a field demonstration project of plant-microbe co-remediation technology (Changtu County)

[0099]

[0100] Note: Different lowercase letters after the values ​​in the table indicate significant differences between treatments (P < 0.05).

[0101] Table 7. Results of heavy metal content and maize biomass in soil from field demonstration of plant-microbe combined remediation technology (Changtu County)

[0102]

[0103] Note: Different lowercase letters after the values ​​in the table indicate significant differences between treatments (P < 0.05).

[0104] Example 3

[0105] In the specific implementation of the microbial synergistic plant cultivation method in heavy metal contaminated soil of the present invention, another field experiment was also carried out. The difference between this experiment and Example 1 is that the field of this specific example is a heavy metal contaminated farmland in Huludao City, Liaoning Province.

[0106] (1) Overview of the test site

[0107] The experimental site was located in a heavy metal-contaminated area in Huludao City, Liaoning Province. The soil in this plot was brown soil, and its main indicators included: pH 6.34, organic matter content 15.13 g / kg, total nitrogen 1.28 g / kg, total phosphorus 0.75 g / kg, and available potassium 121.49 mg / kg. The heavy metal content in the soil was 1.27 mg / kg for Cd, 82.5 mg / kg for Pb, 12.3 mg / kg for As, 306.0 mg / kg for Zn, 2.1 mg / kg for Hg, and 40.2 mg / kg for Cr. The Cd and Zn contents exceeded the screening risk values ​​for agricultural land soil pollution according to the National Soil Environmental Quality Standard (GB15618-2018).

[0108] (2) Treatment setup and corn planting and fertilization process

[0109] The maize variety used was Xianyu 1483. Maize was sown in spring at a planting density of 5200 plants / mu. At sowing, all treatment groups received basal fertilizer (NPK) via hole application, consisting of 7 kg urea, 15 kg diammonium phosphate, and 8 kg potassium chloride. Topdressing was applied three times via drip irrigation at the seedling stage (4-6 leaf stage, 7 kg urea), the tasseling stage (large trumpet stage, 18 kg urea + 7 kg potassium chloride), and the grain-filling stage (silking to grain-filling stage, 5 kg urea). The amounts of NPK fertilizer in the basal and topdressing fertilizers were consistent across all treatment groups.

[0110] The experiment consisted of two treatments: a control group (CK) and a group receiving both RAE and microbial agent combined application (RB). Each treatment had three plots, each with an area of ​​360 m². 2(15m×24m). Each treatment received three top-dressing applications of macro- and micronutrient fertilizers via drip irrigation during the jointing stage (mid-June) and the grain-filling stage (early and late August). The amounts of nitrogen, phosphorus, and potassium in the drip irrigation fertilizers were kept consistent across all treatments. The application rate of RAE was 10 kg / mu / time, the application rate of plant growth promoter (Bacillus subtilis + Bacillus licheniformis) was 5 kg / mu / time, and the application rate of plant biostimulant fulvic acid was 1 kg / mu / time, applied four times during the vegetative growth stage of maize; the application rate of endophytic Bacillus inoculant was 7 kg / mu / time, applied three times during the reproductive growth stage of maize.

[0111] RAE, plant growth promoter, humic acid, and plant endophytic Bacillus are applied by the integrated water and fertilizer device according to the usage amount, along with watering or fertilizer application.

[0112] Samples were taken from each treatment group at harvest time, and the contents of heavy metals Cd, Pb, Zn and As in maize roots, stems, leaves and grains, as well as in the soil (0-20 cm depth), were measured.

[0113] (3) Demonstration results

[0114] Field experiments were conducted after the autumn harvest, collecting maize and soil samples. The results of determining the content of major heavy metals in the roots, stems, leaves, and kernels of maize were shown in Table 8. The results of the heavy metal content determination in plants show that the distribution pattern of most heavy metals in maize organs remains: leaves > roots > stems > kernels or roots > leaves > stems > kernels.

[0115] Table 8. Results of heavy metal content determination in maize from a field demonstration project of plant-microbe combined remediation technology (Huludao City)

[0116]

[0117] Note: Different lowercase letters after the values ​​in the table indicate significant differences between treatments (P < 0.05).

[0118] Table 8 shows the results of heavy metal content determination in maize roots, stems, and leaves. The group treated with RAE + growth-promoting bacteria + biostimulants + endophytic bacteria significantly increased heavy metal content compared to the control group. Specifically, Cd content increased by 69.3%, 110.3%, and 176.7% in roots, stems, and leaves, respectively; Pb content increased by 60.4%, 108.6%, and 273.8%; Zn content increased by 57.4%, 71.2%, and 56.2%; and As content increased by 90.0%, 80.6%, and 135.8%. On the other hand, Table 7 shows the results of heavy metal determination in maize kernels. The group treated with RAE + endophytic bacteria (RB group) reduced heavy metal content in maize kernels more significantly than the control group. Specifically, Cd, Pb, Zn, and As content in maize kernels decreased by 29.4%, 9.7%, 14.0%, and 78.4%, respectively. As shown in Table 7, the contents of heavy metals Cd, Pb, Zn and As in corn kernels of group RB were 0.12, 4.58, 27.18 and 0.008 mg / kg, respectively, all of which met the requirements of the national standard "Feed Hygiene Standard GB13078-2017" regarding the limits of heavy metals.

[0119] The results of the field demonstration further validated the results of the pot experiment. The combined application of RAE + growth-promoting bacteria + biostimulants + endophytic bacteria (RB group) not only effectively promoted the accumulation of heavy metals in maize roots, stems, and leaves, but also significantly reduced the heavy metal content in maize kernels by strengthening the inhibition of heavy metal migration from stems and leaves to kernels. The contents of Cd, Pb, Zn, and As in the maize kernels of the RAE + growth-promoting bacteria + biostimulants + endophytic bacteria combined treatment group were all lower than the corresponding limits in the maize feed standard (GB13078-2017), therefore, these kernels can be used as feed for livestock and poultry farming. The stems, leaves, and roots of each treatment were collected and incinerated to ultimately remove heavy metals from the soil.

[0120] In field demonstration tests conducted in Huludao City, the soil heavy metals Cd, Pb, Zn, and As contents in the RB treatment group were significantly lower than those in the CK group, decreasing by 4.88%, 4.12%, 3.47%, and 4.82%, respectively (Table 9). Simultaneously, the maize plant height and biomass in the RB treatment group were significantly increased, by 8.61% and 18.23% respectively compared to the control group (Table 9).

[0121] Table 9. Results of heavy metal content and maize biomass in soil from field demonstration projects using plant-microbe combined remediation technology (Huludao City)

[0122]

[0123] Note: Different lowercase letters after the values ​​in the table indicate significant differences between treatments (P < 0.05).

[0124] This invention verifies through pot experiments and field demonstrations that the application of small molecule organic carbon / plant biostimulants and plant growth-promoting bacteria increases the content of heavy metals (such as cadmium, lead, arsenic and zinc) in the roots, stems and leaves of corn by 2 to 4 times, making the heavy metal concentration in the roots, stems and leaves of corn reach 1.5 to 200 times the heavy metal concentration in the soil.

[0125] The above embodiments fully demonstrate that the method of the present invention has significant effects on improving the efficiency of heavy metal enrichment in plant roots, stems and leaves and reducing the heavy metal content in seeds, as well as good application prospects.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for growing plants in cooperation with microorganisms in heavy metal contaminated soil, characterized by, It comprises the following steps: Step 1, planting plants in the heavy metal contaminated soil to be repaired; Step 2, applying 2-keto-L-gulonic acid mother liquor, plant growth promoting bacteria and plant biological stimulants to the rhizosphere soil and / or leaf surface of the plants in the vegetative growth stage of the plants, the plant biological stimulants being one or more of humic acid, fulvic acid, polyglutamic acid, itaconic acid, sorbitol, citric acid and gamma-aminobutyric acid; Step 3, applying gulonic acid mother liquor and endophytic Bacillus to the rhizosphere soil and / or leaf of the plants in the reproductive growth stage of the plants, the strain of the endophytic Bacillus being named as CBS-5 (Bacillus endophyticus CBS-5) and preserved in China Industrial Microbial Culture Collection Center with the preservation number of CICC 10622; The plants are corns, the vegetative growth stage is from the emergence stage to the large trumpet stage, and the reproductive growth stage is from the tasseling stage to the mature stage.

2. The method of claim 1, wherein the method is characterized by, The 2-keto-L-gulonic acid mother liquor is the residual liquid at the bottom of the kettle after the second step 2-keto-L-gulonic acid fermentation liquid in the industrial fermentation of vitamin C is subjected to ultrafiltration, evaporation, concentration, crystallization and extraction of 2-keto-L-gulonic acid.

3. The method for microbial cooperation in plant cultivation in heavy metal contaminated soil according to claim 2, characterized in that, The 2-keto-L-gulonic acid mother liquor comprises 22-30% of 2-keto-L-gulonic acid, 2-4% of oxalic acid, 4-9% of formic acid, 1-3% of acetic acid, less than 1% of amino acids, polypeptides, oligosaccharides and nucleotides, and the rest is water.

4. The method of claim 1, wherein the method is characterized by, The pH value of the 2-keto-L-gulonic acid mother liquor is 2.0-8.

0.

5. The method of claim 1, wherein the method is characterized by, The plant growth promoting bacteria are single or complex bacterial agents of Bacillus subtilis, Bacillus licheniformis or Bacillus amyloliquefaciens, which are liquid bacterial agents or solid bacterial agents.

6. The method of claim 1, wherein the method is characterized by, The plant planting method can increase the contents of heavy metals such as cadmium, chromium, lead, mercury and arsenic in the roots, stems and leaves of corns by 2-4 times, and can reduce the contents of heavy metals in corn kernels to 1 / 8-3 / 4 of those in the control group.

7. The method for microbial cooperation in plant cultivation in heavy metal contaminated soil according to claim 1, characterized in that, The plants are wheat, rice, broad bean, soybean, millet, rape, sesame, watermelon, apple or melon.

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