Method for hyperaccumulating cadmium in soil by plant roots

Abstract

The invention relates to the field of heavy metal contaminated soil remediation, in particular to a method for hyperaccumulating cadmium in soil by plant roots, plants are sown in the cadmium contaminated soil, and soil remediation is achieved by enriching cadmium in the soil by the plants. The plants are one or more of annual ryegrass, herba houttuyniae and Russian feed vegetables. The method comprises the following steps: investigating the dry matter accumulation amount, the heavy metal enrichment coefficient (BCF) and the transport factor (TF) of different plants, and screening out the plants of which the roots can hyperaccumulate cadmium; by adding a microbial composition and a chelating agent into the screened plants, the cadmium enrichment capacity of the plants is further improved. According to the method, the plants with the roots capable of achieving hyperaccumulation of cadmium are planted in the cadmium-contaminated soil, and remediation of the cadmium-contaminated soil is achieved through hyperaccumulation of the roots of the plants; the enrichment effect of the plants on the cadmium in the soil is enhanced by adding the microbial complex inoculant and the chelating agent to the roots of the plants.

Description

Technical Field

[0001] The invention relates to the field of heavy metal contaminated soil remediation, in particular to a method for super-accumulating cadmium in soil by plant roots. Background Art

[0002] At present, the main anthropogenic sources of cadmium are sewage sludge, cement industry, fuel combustion, power plants, steel protective coatings, polyvinyl chloride stabilizers, phosphate fertilizers, plastics, glass as pigments, electrode materials in nickel-cadmium batteries, mining activities and zinc smelting in various alloys. A large amount of cadmium waste has led to an increasing impact of cadmium pollution on the modern environment. Cadmium pollution in my country's soil is mainly inorganic pollution, and the point-to-point rate of cadmium exceeding the standard is the highest, far exceeding other inorganic pollutants. Cadmium not only damages the growth and development of plants and affects the physiological metabolism of plants, but also accumulates in crops. At the same time, compared with other heavy metals, cadmium has a strong mobility in the soil and is easily absorbed, transferred and accumulated in large quantities by animals, plants and microorganisms, thereby affecting agricultural production activities.

[0003] Physical and chemical remediation methods are restricted and limited. These applied physical and chemical techniques, such as coagulation, electrokinetics, encapsulation, soil washing and soil flushing, are expensive and make the soil unsuitable for plant growth. Among them, in-situ passivation remediation is one of the commonly used chemical remediation techniques. The core of this technology is to add different types of passivators to soil contaminated with low concentrations of cadmium, thereby reducing the effective state and migration capacity of cadmium. However, passivation remediation is currently limited to short-term indoor or field experiments, and there are few studies on long-term monitoring experiments. Passivation only changes the form of cadmium in the soil, and the total amount has not changed. Over time, changes in soil environmental conditions may cause the fixed cadmium to be released again, causing secondary pollution.

[0004] At present, many biological methods have emerged to repair cadmium-contaminated soil. Among them, plant extraction and remediation uses plants as enrichment carriers of cadmium ions, which has the advantages of being green, feasible, and low-cost. However, the disadvantage is that after extraction and remediation, cadmium will be enriched in large quantities inside the plants, and the subsequent treatment of cadmium-enriched biomass is also difficult. At the same time, although plant passivation remediation can stabilize the form of cadmium in the soil, the content of effective cadmium in the soil has not decreased over time.

[0005] For soil cadmium pollution, the use of plant extraction remediation will result in the production of a large amount of cadmium-rich biomass that is difficult to handle, while the use of plant passivation remediation will result in the inability to reduce the content of effective cadmium in the soil. Therefore, there is an urgent need for a more effective method for soil cadmium pollution remediation. Summary of the invention

[0006] In view of the technical problems in the above-mentioned prior art, a method for super-accumulating cadmium in soil at the roots of plants is provided. By examining the dry matter accumulation, heavy metal enrichment factor (BCF) and transport factor (TF) of different plants, plants whose roots can super-accumulate cadmium are screened out; by adding a microbial combination and a chelating agent to the screened plants, the cadmium enrichment capacity of the plants is further improved.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] A method for super-accumulating cadmium in soil by plant roots, wherein plants are sown in cadmium-contaminated soil to achieve soil remediation by enriching cadmium in the soil by plants;

[0009] The plant is one or more of annual ryegrass, houttuynia cordata and Russian fodder grass.

[0010] Annual ryegrass has a strong tolerance to cadmium and can survive in cadmium-contaminated soil and enrich cadmium pollution in the soil through its own metabolism. Houttuynia cordata also has a strong tolerance and enrichment effect on cadmium. Russian fodder grass is a perennial herbaceous perennial plant of the Boraginaceae family. It has a high protein content and various vitamins and is a high-quality forage grass. Compared with other forage grasses, Russian fodder grass has an extremely developed root system and is a preferred plant for studying root enrichment of cadmium.

[0011] Preferably, one or more of a microbial composite agent and a chelating agent are added to the root soil of the plant.

[0012] Soil microorganisms are an important part of the ecosystem and have a complex relationship with plants. Rhizosphere bacterial communities have the potential to directly stimulate root growth, increase plant tolerance to heavy metals, and improve the overall health of plants; compared with adding a single microorganism, microbial combinations can better regulate the symbiotic network of plant rhizosphere microorganisms and promote the improvement of plants' ability to accumulate heavy metals from multiple dimensions; therefore, microbial composite agents can promote plant growth by improving the rhizosphere microenvironment on the one hand, and on the other hand, they can cause the heavy metals around the rhizosphere to undergo morphological changes to improve the efficiency of plant extraction. Chelating agents can improve the efficiency of plants in removing heavy metals.

[0013] Preferably, the microbial composite agent comprises: 15-25% of Bacillus subtilis, 5-15% of Bacillus cereus and 15-25% of Citrobacter rodi.

[0014] Preferably, the microbial composite agent comprises: 20% Bacillus subtilis, 10% Bacillus cereus and 20% Citrobacter rodi.

[0015] The study found that a microbial composite agent of 20% Bacillus subtilis, 10% Bacillus cereus and 20% Citrobacter rodentium can improve the ability of plants to enrich cadmium.

[0016] Preferably, the chelating agent is polyaspartic acid.

[0017] Polyaspartic acid is a highly biodegradable chelating substance. Compared with other chelating agents such as EDTA, polyaspartic acid has high biodegradability. The application of polyaspartic acid can increase the cadmium accumulation in the aboveground parts and roots of plants and promote the enrichment of beneficial bacteria in the roots of plants.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention achieves the restoration of cadmium-contaminated soil by super-accumulating cadmium in the roots of plants planted in cadmium-contaminated soil.

[0020] 2. The present invention enhances the enrichment effect of plants on cadmium in the soil by adding microbial composite agents and chelating agents to the roots of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Diagram of sowing seeds for plants.

[0022] Figure 2 This is a diagram of the plant's growth status during the harvest stage.

[0023] Figure 3 Schematic diagram of the five-point method for inoculating microorganisms.

[0024] Figure 4 This is a graph showing the effects of enhanced treatments on the dry weight of annual ryegrass.

[0025] Figure 5 This is a diagram showing the effect of enhanced treatment on the dry weight of Houttuynia cordata.

[0026] Figure 6 Figure 2 shows the effect of fortification treatment on dry weight of Russian fodder vegetable.

[0027] Figure 7 Graph showing the effect of enhanced treatment on the cadmium content in the aboveground and underground parts of plants (Figures: (a) is the cadmium content in the aboveground part of annual ryegrass; (b) is the cadmium content in the belowground part of annual ryegrass; (c) is the cadmium content in the aboveground part of Houttuynia cordata; (d) is the cadmium content in the belowground part of Houttuynia cordata; (e) is the cadmium content in the aboveground part of Russian fodder vegetable; (f) is the cadmium content in the underground part of Russian fodder vegetable). DETAILED DESCRIPTION

[0028] In order to more clearly describe the invention purpose, technical scheme and technical effect advantages in the specific implementation case of the present invention, the scheme in the specific embodiment will be described in detail in combination with the drawings of the specification of the present invention. The specific technical scheme involved in the following specific embodiments is only for a clear and complete description of the innovative technical scheme of the present invention. It is only a part of the specific implementation scheme that can be adopted by the present invention, not all embodiments, and should not be understood as a limitation on the innovative scheme of the present invention. Any scheme adopting the same inventive concept of the present invention should be included in the protection scope of the present invention.

[0029] Secondly, the relevant descriptions of the drawings in the specific embodiments of the present invention are only for the convenience of technical personnel to understand the scheme of the present invention. Some details in the drawings are displayed for the convenience of clearly presenting the technical scheme. It should not be considered that all technical features in the drawings must be included in the specific implementation cases, and the detailed features in the drawings cannot be regarded as additional limitations on the innovative technical scheme of the present invention.

[0030] For those skilled in the art, when understanding the solutions described in the specific embodiments of the present invention, they can refer to the conventional technical manuals in the field. At the same time, for the places where the above-mentioned terms appear, they can make appropriate understandings or adjustments for reference, and deduce the implementation of the same or similar technical solutions without paying any creative work.

[0031] Example 1

[0032] 1 Materials and Methods

[0033] 1.1 Test plants and soil

[0034] The 9 materials used for the test were annual ryegrass (Loliummultiflorum Lam.), houttuynia cordata Thunb., Russian fodder lettuce (Syrnprtytumperegrinum Ledeb), astragalus sinicus L., bluegrass (Poa annuaL.), amorpha (Amorphafruticosa Linn.), orchard grass (Dactylisglomerata L.), Silphiumperfoliatum L, and Cynodon dactylon L. from Jiangsu Xintiandi Seed Co., Ltd.

[0035] The soil used in the experiment was taken from Mianyang City, Sichuan Province. The background content of cadmium (Cd) in the soil was 0.18 mg kg -1 , pH value is 7.29. Its moisture content is 12.8%, and the organic matter content reaches 22.30 mg kg-1 , alkaline nitrogen is 53.74 mg kg -1 The available phosphorus content and available potassium content were 7.31 mg kg -1 and 21.36 mg kg -1 .

[0036] 1.2 Preparation of Cd-contaminated soil

[0037] Select Cd(NO3)2 and set the Cd concentration to 10 mg kg -1 , with no Cd added as the control group. Cd solutions of different concentrations were prepared according to the Cd gradient setting, and the Cd solutions were evenly sprayed into the soil and then placed in a dark place for 30 days to ensure that the soil reached a balanced state, thus completing the preparation of Cd-contaminated soil.

[0038] 1.3 Potted plant experiment

[0039] Set 10mg·kg -1 and blank control (CK, 10 mg kg -1 The potted plant test container was a round flower pot with a hole and a tray at the bottom, and each pot was filled with 1 kg of soil according to the water content.

[0040] like Figure 1 As shown, seeds with full grains were selected and soaked in deionized water overnight, then placed in a flowerpot, covered with a thin layer of soil and controlled with appropriate temperature and humidity (water holding capacity was maintained at 65% to 75%) during this period. The plants were harvested when they just headed, and relevant indicators were measured.

[0041] 1.4 Analysis methods and data processing

[0042] (1) Biomass determination

[0043] After the samples were harvested, they were repeatedly rinsed with distilled water until no soil remained. The plants were harvested separately according to the aboveground and underground parts, then placed in an oven at 105°C for 2 hours, and then dried at 85°C to constant weight for subsequent dry weight determination.

[0044] (2) Determination of plant Cd content and calculation of enrichment index

[0045] The dried plant samples were ground into powder and then digested using the HNO3-HClO4 method. The Cd content in different parts was determined using an atomic absorption spectrophotometer (Perkin-Elmer, AA700, USA). The Cd content in the aboveground part was recorded as UC, and the Cd content in the underground part was recorded as DC. The enrichment index formula is as follows:

[0046] Plant Cd content (HCC, mg·kg-1 ) = Cd accumulation of the whole plant / whole plant dry weight (2-1)

[0047] BCF = plant Cd content / soil Cd content (2-2)

[0048] Transport coefficient (TF) = aboveground Cd accumulation / underground Cd accumulation (2-3)

[0049] Unit accumulation (BCQ, mg kg -1 ) = whole plant dry weight × plant Cd content / single pot soil weight (2-4)

[0050] 2 Results and analysis

[0051] 2.1 Effects of Cd stress on plant height and biomass

[0052] Figure 2 The results show that the nine plants in non-polluted soil and 10 mg kg -1 Comparison of growth under cadmium pollution conditions. Figure 2 It can be seen that except for annual ryegrass, the other eight plants were significantly stressed by cadmium. Table 1 shows that at 10 mg kg -1 Responses of plant height and biomass of different varieties to cadmium stress under cadmium pollution treatment. As shown in Table 1, at 10 mg kg -1 Under cadmium pollution, the plant heights of the other eight plants except annual ryegrass were significantly inhibited to varying degrees (P<0.05). However, the plant height of annual ryegrass increased under cadmium pollution, indicating that it can be used as a candidate plant for cadmium tolerance. In the absence of Cd stress, the aboveground dry weights of Russian fodder and pine grass were larger, significantly higher than those of the other seven plants (P<0.05), among which the underground dry weight of Russian fodder was the largest, reaching 33.8g. 10mg·kg -1 After cadmium pollution treatment, the aboveground and underground dry weights of the nine plants showed a decreasing trend compared with the CK treatment. Among them, the aboveground and underground dry weights of Herba Lysimachiae, Houttuynia cordata and Russian fodder were more significantly affected by cadmium pollution, which was consistent with the decreasing trend of plant height, indicating that their growth was more sensitive to Cd pollution. However, the Cd10 treatment had little effect on the biomass of annual ryegrass, but had a certain growth-promoting effect on the underground biomass, which increased by 51% compared with CK, indicating that annual ryegrass has a strong tolerance to high cadmium pollution and shows strong Cd tolerance.

[0053] Table 110 mg kg -1 Growth indicators of nine plant species under cadmium pollution

[0054]

[0055] 2.2 Analysis of plant absorption and transport capacity of Cd

[0056] The plant samples were ground into powder and then digested, and the digested solution was quantitatively determined. -1 All of them were subjected to different degrees of stress under cadmium pollution as shown in Table 2. -1 The highest cadmium content in the aboveground parts of the plants was in the bermudagrass, reaching 2.63 mg kg -1 The cadmium content in the upper parts of Dactylis turf and annual ryegrass was 2.57 mg·kg -1 and 2.53 mg kg -1 The highest cadmium content in the underground part was in the annual ryegrass, reaching 82.6 mg kg -1 , which was significantly higher than the other eight plants (P<0.05). The lowest cadmium content in the underground part was in Russian fodder cabbage, which was 6.97 mg kg -1 In terms of the cadmium content of the whole plant, annual ryegrass and Russian fodder mustard had the highest cadmium accumulation, at 69.57 and 63.26 mg kg, respectively. -1 , while the enrichment of Amorpha fruticosa was the lowest, only 8.22 mg kg -1 .

[0057] In terms of single pot accumulation, the single pot accumulation of annual ryegrass and Russian fodder lettuce was the largest under cadmium pollution stress, with 2.77 and 2.66 mg kg -1 The transport coefficient was significantly higher than that of the other seven plants (P<0.05). -1 Under cadmium pollution, the transport coefficients of the nine plants were all <1 compared with CK, proving that they have a strong ability to fix cadmium in the roots of plants. The cadmium transport coefficients of annual ryegrass, Houttuynia cordata and Russian fodder vegetable were all low, 0.04, 0.09 and 0.07 respectively, which were significantly lower than those of the other six plants (P<0.05). The results showed that the above three plants were better candidates for root cadmium enrichment plants. In terms of enrichment coefficient, Amorpha fruticosa had the lowest ability to enrich cadmium, with an enrichment coefficient of only 0.82<1. Similar to the law of transport coefficient, the enrichment coefficients of annual ryegrass, Houttuynia cordata and Russian fodder vegetable were relatively large, 6.33, 2.67 and 5.96 respectively. In summary, we can select annual ryegrass, Houttuynia cordata and Russian fodder vegetable as materials for root cadmium enrichment plants for subsequent research on root enrichment enhancement and cadmium transport control, and improve their advantages as plant enrichment materials.

[0058] Table 210 mg kg -1 Enrichment indexes of nine plants under cadmium pollution conditions

[0059]

[0060]

[0061] Note: UC: Cadmium content of above-ground plants (mg·kg -1 ); DC: underground plant cadmium content (mg·kg -1 ); HCC: plant cadmium content (mg·kg -1 );BCQ:Accumulation in single basin (mg·kg -1 );BCF:enrichment factor;TF:transfer factor

[0062] 3 Discussions

[0063] 3.1 Differences in heavy metal accumulation capacity among different types of plants

[0064] The bioavailability concentration of metals is very important for its absorption and accumulation in plants and is determined by biomass and its enrichment, rather than the total metal concentration in the soil. The absorption efficiency of cadmium by the root system is affected by the biomass of the plant roots in addition to the bioavailability of cadmium at the root-soil common interface in the soil. The nine plants screened in this embodiment all have a certain enrichment capacity for Cd, among which the bioaccumulation capacity of Saururaceae, Gramineae and Boraginaceae is relatively strong, and the enrichment effect on cadmium is remarkable. Among them, the representative species in Gramineae is annual ryegrass. In addition, Sedum ulmoides can tolerate various heavy metals such as Cd and Zn, especially for heavy metal Cd, and is one of the model plants among the soil heavy metal remediation technology. Studies have shown that under heavy metal stress conditions, the aboveground part of Sedum ulmoides is generally higher than the underground part for heavy metal absorption and accumulation, and as the concentration of pollution increases, the accumulation amount also gradually increases.

[0065] The growth of stems, leaves and roots of different plant species in heavy metal contaminated soils was significantly different. The ability of heavy metals to accumulate in different organs of plants varies with plant species. The overall Cd accumulation capacity of the nine plants was root > leaf. In our study, we found that Houttuynia cordata and annual ryegrass had a Cd accumulation rate of 10 mg / kg -1 The growth of the roots of the seven plants was better in the cadmium-contaminated soil, and the growth of its roots was promoted to a certain extent. The root growth and stem and leaf growth of the other seven plants were inhibited to varying degrees. Except for Amorpha fruticosa, the BCF of the other eight plants was >1, indicating that they had a strong absorption capacity for heavy metal Cd. Roots are the main place for plants to absorb heavy metals. The high content of heavy metals in the roots is also related to the well-developed root system and high biomass of the plants. Therefore, the retention of Cd in the roots of the above nine plants is far greater than that in the aboveground parts.

[0066] In addition, Russian fodder grass is a perennial herbaceous perennial plant of the Boraginaceae family. Compared with other forage grasses, Russian fodder grass has a very high root biomass. The results showed that at 10 mg / kg -1In cadmium-contaminated soil, the biomass in the underground part is 33.6g, and the cadmium content in the root is 6.97mg / kg -1 There is no research on using it as a plant accumulator to repair heavy metal contaminated soil. Our research found that although the overall effect of Russian fodder grass in enriching cadmium content is not as good as that of ryegrass, it is a major cadmium enrichment plant in the roots.

[0067] 3.2 Differences in heavy metal transport capacity among different types of plants

[0068] The variation characteristics of Cd content in plant stems and leaves are related to the transport and migration capacity of heavy metals in different parts of plants. Therefore, based on the preliminary understanding of the degree of Cd accumulation of each plant variety, the difference in the ability of Cd to be transported from roots to leaves was further examined through the transport coefficient. It can be seen from the transport coefficient in the table that there are significant differences in the ability of nine plant varieties to transfer Cd from roots to the ground (P<0.05). Their stem and leaf transport coefficients for Cd are 0.04-0.27. Among them, the Cd transport coefficients of annual ryegrass, Houttuynia cordata and Russian fodder vegetable are all small, at 0.04, 0.09 and 0.07, respectively. Cadmium enters the plant body from the soil solution, mainly through the root epidermis, and then is transported to the xylem through two pathways, the symplast and the apoplast, and finally reaches the aboveground part. The ability of plants to absorb heavy metals is mainly affected by their own genetic factors and external environmental conditions. For example, studies have found that compared with leaves, the roots of plants are more sensitive to cadmium stress. After cadmium treatment, except for annual ryegrass and Houttuynia cordata, the root biomass of the other seven plants showed a linear decline, and the decline was significantly greater than that of the aboveground parts. -1 Under cadmium pollution, cadmium stress can promote the root growth of annual ryegrass, help it absorb nutrients, and thus increase the biomass of the plant. This shows that annual ryegrass has a certain resistance to cadmium stress.

[0069] Due to the differences in biomass and heavy metal content in plants, the amount of Cd accumulated by plants also showed significant differences. Except for Amorpha fruticosa, the BCF of the other eight plants was >1, indicating that they had a strong ability to absorb heavy metal Cd. Roots are the main place for plants to absorb heavy metals. The high content of heavy metals in roots is also related to the well-developed root system and high biomass of plants. Therefore, the retention of Cd in the roots of the above nine plants is far greater than that in the aboveground parts.

[0070] 4 Summary

[0071] (1) Different plant species had different responses to Cd stress, except for annual ryegrass, which responded differently to Cd stress at 10 mg / kg -1 Both aboveground and underground biomass were stressed to varying degrees under cadmium pollution. -1Cadmium pollution treatment had no significant effect on the biomass of annual ryegrass, and had a certain growth-promoting effect on the underground biomass. Compared with CK, the underground biomass increased by 51%, indicating that annual ryegrass has a strong ability to tolerate high cadmium pollution and shows strong Cd tolerance.

[0072] (2) There were differences in the enrichment indexes of different plant species. The annual ryegrass had the highest underground cadmium content, reaching 82.6 mg kg-1, which was significantly higher than the other eight plants (P<0.05). In terms of the transport coefficient, the transport coefficients of the nine plants were all <1, proving that they have a strong ability to fix cadmium in the roots of the plants. Annual ryegrass, Houttuynia cordata and Russian fodder mustard had higher enrichment coefficients.

[0073] (3) In summary, we can select annual ryegrass, Houttuynia cordata and Russian fodder lettuce as materials for root cadmium enrichment plants for subsequent research on root enrichment enhancement, thereby improving their advantages as plant enrichment materials.

[0074] Example 2

[0075] 1 Materials and methods

[0076] 1.1 Material preparation

[0077] (1) Preparation of Cd-contaminated soil

[0078] Same as Example 1

[0079] (2) Preparation of materials to assist plant extraction and repair of cadmium

[0080] Bacillus subtilis, Bacillus cereus and Citrobacter Werkman and Gillen were cultured in TGY medium and the bacterial OD 600 When the pH value reaches 0.8, the bacterial solution is mixed and shaken according to the ratio of 20% Bacillus subtilis, 10% Bacillus cereus and 20% Citrobacter. The concentration of polyaspartic acid applied to the soil is 25 mg·L -1 In addition, the microbial combination solution and polyaspartic acid were injected as follows Figure 3 shown.

[0081] 1.2 Construction of plant enhanced extraction and repair system

[0082] According to the screening results in Chapter 2, three dominant forage grasses were obtained: annual ryegrass, houttuynia cordata and Russian fodder grass. The microbial combination, polyaspartic acid and the three dominant forage grasses were combined, and 3 replicates were set for each group.

[0083] 1.3 Method of adding microorganisms and polyaspartic acid

[0084] When the plants grow to the seedling stage, the microbial combination solution is injected into the plant root layer (5 cm underground) with a syringe. The solution is injected using the five-point inoculation method, once every 7 days, 20 mL each time, for a total of 4 injections, and a total of 80 mL of solution is injected into a single pot of forage. Polyaspartic acid (PASP) is also injected into the rhizosphere, with 25 mg·L PASP applied to the roots. -1 , 20 mL per pot, inoculated once every 7 days. The detailed description of each treatment group is shown in Table 3 below.

[0085] Table 3 Experimental design for strengthening Cd accumulation in plant roots

[0086]

[0087] Note: Data analysis is based on different cadmium pollution concentrations.

[0088] 1.4 Plant harvesting and processing

[0089] The plants were harvested when the annual ryegrass grew to 45 days, and the houttuynia cordata and Russian fodder lettuce grew to 60 days, respectively. The plant harvesting and processing methods were the same as those in Example 1.

[0090] 1.5 Analysis and determination methods and data processing

[0091] The analysis and determination methods and data processing are the same as in Example 1.

[0092] 2 Results and analysis

[0093] 2.1 Effects of enhanced treatment on plant dry weight under cadmium pollution

[0094] The effects of polyaspartic acid, microbial combination, and the combined application of polyaspartic acid and microbial combination on the dry weight of three plants under different cadmium pollution conditions were investigated.

[0095] (1) Annual ryegrass

[0096] Effects of polyaspartic acid and microbial assemblages on aboveground and belowground biomass of annual ryegrass Figure 4 As shown in the figure, with the increase of cadmium pollution concentration, the annual ryegrass was more seriously stressed by cadmium, showing the characteristics of reduced biomass and shorter plant growth. Under the three cadmium pollution concentration gradients, the microbial combination and the chelating agent + microbial combination can increase the biomass of the aboveground and underground parts of the annual ryegrass.

[0097] Among them, 3mg·kg -1Under cadmium pollution, the microbial combination treatment made the aboveground dry weight of annual ryegrass reach a maximum of 3.82g, and the aboveground biomass increased by 16.1% compared with the control. However, the difference between microorganisms, microorganisms and polyaspartic acid in increasing the underground biomass was not significant. -1 Under cadmium pollution, the biomass of the aboveground and underground parts of the annual ryegrass grass by the microbial community reached 3.589 g and 2.421 g, respectively, which was significantly increased by 16.1% and 19% compared with the control. -1 Similar to cadmium pollution, the combined application of microorganisms and polyaspartic acid made the underground biomass reach a maximum of 2.721g, an increase of 32.1%.

[0098] Different from the low and medium concentration cadmium pollution, at 10 mg kg -1 Under cadmium pollution, the difference between microorganisms and chelating agents + microorganisms in the aboveground dry weight was not obvious. The combined application of chelating agents + microorganisms made the dry weight of the lower part of the annual ryegrass reach a maximum of 2.89g, an increase of 11.5% compared with the control. It can be seen that the application of microbial combinations can increase the aboveground and underground biomass of annual ryegrass to varying degrees under the three cadmium pollution concentrations.

[0099] (2) Houttuynia cordata

[0100] Effects of polyaspartic acid and microbial combinations on the aboveground and underground biomass of Houttuynia cordata Figure 5 As shown in Figure 2, with the increase of cadmium pollution concentration, the change of biomass of Houttuynia cordata under cadmium stress showed a different trend than that of annual ryegrass. -1 In the cadmium-contaminated soil, only polyaspartic acid had the worst effect on Houttuynia cordata, which was comparable to 3 mg kg -1 Compared with no treatment, the aboveground and underground biomass decreased by 33.3% and 50% respectively. The combined application of microorganisms and microorganisms and polyaspartic acid can increase the aboveground and underground dry weight of Houttuynia cordata to varying degrees, among which the combined application of microorganisms and polyaspartic acid significantly increased the aboveground and underground biomass.

[0101] At 5 mg kg -1 In cadmium-contaminated soil, exogenous application of polyaspartic acid and microbial combination had no significant effect on the increase of underground biomass. In terms of increasing aboveground biomass, different from low-concentration cadmium pollution, only microbial combination treatment made the aboveground dry weight reach a maximum value of 2.188g, which was higher than 5mg·kg -1 Compared with no treatment, it increased by 66.7%. In addition, the difference between polyaspartic acid treatment alone and the combined application of polyaspartic acid and microbial combination was not much, indicating that when the concentration of cadmium is medium, the application of microbial combination is more effective in promoting the ability of plants to extract cadmium.

[0102] As the cadmium pollution concentration further increased, the three measures to promote plant extraction of cadmium could effectively increase the biomass of the above and underground parts of Houttuynia cordata. Among them, the microbial treatment made the above and underground dry weights of the annual ryegrass reach the maximum value, which was higher than 10 mg kg -1 Compared with no treatment, the increases were 118.2% and 164.3%, respectively. And the effect of applying the microbial combination alone was better than that of applying polyaspartic acid alone. It can be seen that the application of the microbial combination can increase the biomass of the aboveground and underground parts of Houttuynia cordata to a certain extent under the three cadmium pollution concentrations.

[0103] (3) Russian fodder vegetables

[0104] Effects of polyaspartic acid and microbial combinations on aboveground and underground biomass of Russian fodder vegetables Figure 6 As shown. Unlike annual ryegrass and Houttuynia cordata, the overall biomass of Russian fodder mustard was larger under cadmium stress, with the aboveground biomass ranging from 10.12 to 15.28 g and 36.56 to 48.15 g. The use of polyaspartic acid alone had no significant effect on increasing the aboveground and underground biomass of Russian fodder mustard under three cadmium pollution concentrations.

[0105] At 3 mg kg -1 In cadmium-contaminated soil, only microbial treatment could increase the aboveground biomass of Russian fodder vegetables to a maximum value of 3 mg kg -1 Compared with the control group, the aboveground biomass increased by 46.9% compared with the control group without adding polyaspartic acid. However, the aboveground biomass increased little when only microbial treatment was used and when polyaspartic acid and microbial treatment were used together. On the contrary, the aboveground biomass increased significantly when both treatments were used together. -1 In cadmium-contaminated soil, microbial treatment alone can increase the aboveground and underground dry weight of Russian fodder vegetables more than polyaspartic acid treatment alone. The combined application of the two can make the aboveground dry weight and underground dry weight of the plants reach the maximum value, respectively. The effect of promoting cadmium enrichment is different with the increase of cadmium pollution concentration. At 10 mg kg -1 In cadmium-contaminated soil, polyaspartic acid alone and 10 mg kg -1 The difference was not obvious when compared with no addition. The effect of microorganism alone and the combined application of the two on increasing the aboveground biomass of Russian fodder vegetables was similar, but the latter was most significant in increasing the underground biomass. -1 Compared with the absence of the addition, it increased by 28.6%.

[0106] It can be seen that, similar to annual ryegrass and Houttuynia cordata, the use of polyaspartic acid alone does not significantly improve the cadmium enrichment ability of the plant. With the increase of cadmium pollution concentration, the combined use of microorganisms and microorganisms and polyaspartic acid can increase the biomass of the aboveground and underground parts of Russian fodder vegetables to varying degrees. Among them, the promotion of cadmium enrichment of annual ryegrass by microbial treatment alone is more obvious.

[0107] 2.2 Effects of enhanced treatment on cadmium absorption of three plants

[0108] like Figure 7 As shown, this example investigates the effects of polyaspartic acid, microbial combination, and polyaspartic acid and microbial combination on the cadmium content and enrichment indexes (BCF, TF) of three plants under different cadmium pollution conditions. Exogenous application of polyaspartic acid or microbial combination has different effects on the Cd content of the aboveground and underground parts of annual ryegrass, Houttuynia cordata, and Russian fodder vegetable.

[0109] (1) Annual ryegrass

[0110] With the increase of cadmium pollution concentration, the degree of cadmium stress on annual ryegrass intensified, and the cadmium content in both the aboveground and underground parts showed an increasing trend. -1 When cadmium was polluted, the differences among the treatments were not obvious, which may be because annual ryegrass was not sensitive to cadmium when the concentration was low. -1 Compared with no addition, the three measures to promote cadmium enrichment did not increase the cadmium content in the aboveground parts of plants.

[0111] At 5 mg kg -1 In cadmium-contaminated soil, the effect of using polyaspartic acid alone was the worst. In contrast, using microbial combination alone increased the cadmium content in the aboveground part most significantly, reaching 6.492 mg kg -1 , which increased by 58.7%. However, the root cadmium absorption was slightly different. The root cadmium content reached a maximum of 38 mg kg when the chelating agent and microorganism were combined. -1 , an increase of 8.1% over the control.

[0112] At 10 mg kg -1 When cadmium was contaminated, the use of polyaspartic acid alone or the use of microbial combination alone increased the cadmium content in the aboveground part of annual ryegrass, and the increase in the cadmium content in the underground part was also very small. The application of microbial combination can significantly increase the cadmium content in the aboveground and underground parts to 13.210 mg kg -1 and 108 mg kg -1, which were 4.7% and 35% higher than those of the control, respectively. This shows that under the three cadmium pollution concentrations, the microbial combination plays a very key role in increasing the cadmium content in the upper and underground parts of the annual ryegrass.

[0113] (2) Houttuynia cordata

[0114] With the different cadmium pollution, the exogenous application of chelating agents or microorganisms has different effects on Houttuynia cordata. When the cadmium pollution is low or medium, the combined application of microorganisms and chelating agents can significantly increase the cadmium content in the aboveground and underground parts. However, at 10 mg kg -1 When cadmium was contaminated, the cadmium content in the underground part reached a maximum of 32 mg kg when microorganisms acted alone, which was higher than that when chelating agents and microorganisms were used together. -1 , an increase of 52.4% over the control.

[0115] (3) Russian fodder vegetables

[0116] Compared with annual ryegrass and Houttuynia cordata, the cadmium enrichment ability of Russian fodder vegetable in the underground part was the worst under the three cadmium pollution concentrations, only at 10 mg·kg -1 The maximum cadmium content in the underground part during cadmium pollution is 8.5 mg kg -1 At 3 mg kg -1 When cadmium was contaminated, the effects of the three exogenous measures to promote plant extraction on Russian fodder lettuce were similar to those on annual ryegrass. There was no significant difference between microbial treatment and the combined treatment of polyaspartic acid and microorganisms in enhancing the plant's ability to extract cadmium. -1 When contaminated with cadmium, the combined application of polyaspartic acid and microbial combination can significantly increase the cadmium content of the aboveground and underground parts of Russian fodder vegetables by 75% and 52%, respectively, compared with the control.

[0117] In summary, under the three cadmium pollution concentrations, the use of microbial combinations alone and the combination of chelators and microorganisms can increase the cadmium content in the aboveground and underground parts to varying degrees. However, since the cadmium absorption capacity of annual ryegrass is much greater than that of Houttuynia cordata and Russian fodder, and the microbial combination has the best effect on improving the cadmium enrichment in the roots of annual ryegrass. Therefore, subsequent studies selected annual ryegrass as plant material and used the application of microbial combinations as an enhanced treatment method.

[0118] 2.3 Effects of enhanced treatment on cadmium transport and enrichment coefficients of three plants

[0119] The promotion of plant's ability to extract cadmium is first reflected in the enrichment coefficient and transport coefficient. The BCF and TF of the plant cadmium remediation system affected by the three exogenous measures are shown in Table 4 below.

[0120] (1) Annual ryegrass

[0121] Among the three plants, annual ryegrass had the highest BCF value (2.46-2.89). -1 In cadmium-contaminated soil, the three treatments had little effect on BCF. The combined use of polyaspartic acid and microorganisms made TF reach a maximum value of 0.315, an increase of 25.8% compared with the control.

[0122] At 5 mg kg -1 In cadmium-contaminated soil, microbial treatment alone and the combined application of polyaspartic acid and microorganisms can significantly increase the BCF value of annual ryegrass (P<0.05), among which the microbial treatment alone has the most significant effect, increasing by 8.7% compared with the control. The change of TF is similar. The microbial treatment alone makes the annual ryegrass reach the maximum TF value of 0.452, which is 41.2% higher than the control.

[0123] At 10 mg kg -1 In cadmium-contaminated soil, the three exogenous measures can increase the BCF and TF values ​​of annual ryegrass to varying degrees. Among them, the microbial treatment alone and the combined application of the two made the BCF reach the maximum value of 2.75 and 2.89, respectively, which increased by 11.7% and 17.5% compared with the control. The maximum value of TF reached by the combined application of the two was 32.4% higher than the control. In summary, among the three methods to improve the cadmium extraction capacity of annual ryegrass, the microbial combination has a significant promoting effect.

[0124] (2) Houttuynia cordata and Russian fodder vegetable

[0125] Compared with annual ryegrass, Houttuynia cordata and Russian fodder grass have lower BCF and TF values. -1 In cadmium-contaminated soil, Houttuynia cordata treated with microorganisms alone reached the maximum BCF and TF of 0.029 and 0.087, respectively, which were 20.8% and 21% higher than the control. Russian fodder vegetable treated with polyaspartic acid and microorganisms reached the maximum BCF and TF of 0.032 and 0.341, respectively. -1 and 10 mg kg -1 In cadmium-contaminated soil, the combined application of both plants can significantly increase the BCF and TF values.

[0126] It can be seen that the application of microbial combination to annual ryegrass can best improve the cadmium enrichment capacity of plant roots. However, for Houttuynia cordata and Russian fodder mustard, two plants with weaker enrichment capacity, the combined application of polyaspartic acid and microbial combination can improve the plant's cadmium enrichment and cadmium transport capacity.

[0127] Table 4 Effects of enhanced treatment on enrichment and translocation indexes of different plants

[0128]

[0129]

[0130] 3 Discussions

[0131] 3.1 The auxiliary role of microorganisms in the extraction and remediation of heavy metals by plants

[0132] Plant-microbe association is a favorable dimension of phytoremediation, which is a low-input, high-yield and sustainable technology. Microorganisms have shown effective effects on metal toxicity through their detoxification and resistance mechanisms and growth-promoting properties. Microbial-assisted phytoremediation is mainly carried out directly or indirectly. They synthesize various nutrients and plant hormone substances, namely growth regulators, siderophores, enzymes, and conversion of mineral nutrients (phosphate, potassium, nitrogen, etc.). In addition, they also regulate the detoxification, accumulation and chelation ability of plants to metals by secreting extracellular components, organic acids, biosurfactants, chelators, etc. Previous studies in this laboratory found that the combination of Bacillus subtilis, Citrobacter and Bacillus showed stronger Cd tolerance and stronger IAA production ability under cadmium pollution conditions, and the growth-promoting effect on plants was also the most obvious. It is inferred that the ability to produce high IAA may be due to the large proportion of Citrobacter in the combination, which is consistent with the research of AbdulWahabAjmal et al.

[0133] Heavy metal resistant bacteria are very important as they play a vital role in bioremediation. To date, several environmentally safe Bacillus cereus strains have been isolated and described, including Bacillus cereus WSE01, MEN8, YL6, SA1, ALT1, ERBP, GGBSTD1, AK1, AR156, C1L and T4S. These strains have been studied under growth chamber, greenhouse and field conditions and show many important properties, such as production of indole-3-acetic acid and siderophores, nitrogen fixation and phosphate solubilization. Bacillus cereus BL4 isolated around pyrite showed high tolerance to Cd and also promoted plant growth, activating the bioavailability of Cd in soil; and after inoculation with strain BL4, the fresh weight, height and Cd accumulation of Miscanthus seedlings increased by 19.08-42.37%, respectively; through its effects on plant physiology and biochemistry and soil microecology, the plant's ability to extract Cd was further improved. Li et al. found that Bacillus subtilis had a strong influence on the fate and mobility of Pb, Sb, Ni, Zn, Cu and Cr, tending to fix heavy metals in rhizosphere soil. It also affected the distribution of heavy metals in rhizosphere soil, corn growth and microbial community structure.

[0134] 3.2 The effect of chelating agents on the enrichment of heavy metals in plant-microorganism systems

[0135] Traditional metal chelators, such as ethylenediaminetetraacetic acid (EDTA), have been gradually replaced due to their poor biodegradability in soil and high risk of heavy metal leaching into groundwater, which poses a high environmental risk to human and animal health. Polyaspartic acid (PASP) is widely used as a fertilizer enhancer in agricultural production. PASP can significantly improve plant growth, Cd accumulation, and total Cd and Pb content in plants. In addition, PASP significantly increased the biomass yield and Cd concentration of Bidens pilosa. Mei-meiHU et al. found that PASP increased the distribution of Cu and Cd in the root system under Cu and Cd combined stress. In terms of basic elements, PASP mainly promoted the absorption and transport of potassium (K), calcium (Ca) and magnesium (Mg), thus greatly exerting physiological effects; PASP seems to mainly promote these favorable factors that play a physiological regulatory function. Under normal soil conditions, PASP mainly acts as a biostimulant or signal molecule to improve nutrient efficiency. .

[0136] Plants and microorganisms can adapt well to heavy metal-containing environments and have been proven to be the best assistants for phytoremediation. The application of green chelators and microorganisms has become a promising alternative method to enhance phytoremediation. The application of EDDS enhances the ability of plant-microorganism joint repair of soil heavy metals. Studies have shown that the application of EDDS creates a good rhizosphere ecological environment for king grass, improves the bioavailability of Cd and increases the biomass of plants. Similar to the results of this chapter, at 10 mg kg -1 The application of polyaspartic acid can increase the biomass and aboveground and underground cadmium concentrations of annual ryegrass, Houttuynia cordata and Russian fodder lettuce when cadmium is contaminated.

[0137] 4 Summary

[0138] (1) The application of three composite bacteria, Bacillus subtilis, Citrobacter rodi and Bacillus cereus, can increase the aboveground (3.21-3.82 g) and underground (2.79-3.56 g) biomass of annual ryegrass to varying degrees. The combined application of polyaspartic acid and microbial combination can increase the biomass of Houttuynia cordata and Russian fodder grass to the greatest extent.

[0139] (2) Combined application of polyaspartic acid and microbial combination at 10 mg kg -1 Cadmium pollution was the best way to increase the cadmium content of the aboveground and underground parts of Houttuynia cordata and Russian fodder vegetables, which were 3.525 mg kg -1 , 25.001mg·kg -1 and 7.233 mg kg -1 and 12.618 mg kg-1 In addition, the cadmium absorption capacity of annual ryegrass was much greater than that of Houttuynia cordata and Russian fodder mustard, and the application of microbial combination played a key role in improving the ability of plants to extract heavy metals under three cadmium pollution concentrations. Especially at 10 mg kg -1 Under cadmium pollution conditions, the application of microbial combinations can significantly increase the cadmium content in the aboveground and underground parts of annual ryegrass to 13.210 mg kg -1 and 98mg·kg -1 .

[0140] (3) Among the three plants, annual ryegrass had the highest BCF value (2.46-2.89), of which the microbial treatment alone and the combined application of the two treatments both resulted in the highest BCF values ​​of 2.75 and 2.89, respectively. The combined application of the two treatments resulted in the maximum TF value, which was 32.4% higher than that of the control. For Houttuynia cordata and Russian fodder mustard, two plants with weaker enrichment abilities, the combined application of polyaspartic acid and microbial combination could increase the ability of the plants to improve cadmium enrichment and cadmium transport.

[0141] The above embodiments describe only the basic principles, main features and / or advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the invention content of the specification only describe the principles or specific cases of the present invention. Without departing from the essence of the innovative idea of ​​the present invention, the innovative scheme of the present invention may be subject to various changes and improvements, and these changes and improvements all fall within the scope of protection required by the present invention.

Claims

1. A method for super-accumulating cadmium in soil by plant roots, characterized in that: Planting plants in cadmium-contaminated soil to achieve soil remediation through plant enrichment of cadmium in the soil; The plant is one or more of annual ryegrass, houttuynia cordata and Russian fodder grass.

2. The method for super-accumulating cadmium in soil by plant roots according to claim 1, characterized in that: One or more of a microbial compound agent and a chelating agent are added to the root soil of the plant.

3. The method for super-accumulating cadmium in soil by plant roots according to claim 2, characterized in that: The microbial composite bacterial agent comprises: 15-25% of Bacillus subtilis, 5-15% of Bacillus cereus and 15-25% of Citrobacter acidophilus.

4. The method for super-accumulating cadmium in soil by plant roots according to claim 3, characterized in that: The microbial composite agent comprises: 20% of Bacillus subtilis, 10% of Bacillus cereus and 20% of Citrobacter acidophilus.

5. The method for super-accumulating cadmium in soil by plant roots according to claim 2, characterized in that: The chelating agent is polyaspartic acid.

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