Remediation method and system for soil heavy metal pollution based on bamboo nano material

Abstract

The invention discloses a remediation method and system for soil heavy metal pollution based on a bamboo nano material, bamboo is cut in the axial direction of fibers, ultrasonic cleaning and vacuum drying are carried out, after energy gradient grinding is carried out in sequence, high-pressure homogenization and in-situ dispersion nanocrystallization treatment are carried out, and the porous bamboo nano material is obtained; the method comprises the following steps: inserting an anti-heavy metal target gene into a selected microbial expression vector, introducing the expression vector into microorganisms by using an electroporation method, detecting the expression level of the target gene, inoculating the microorganisms over-expressing the target gene into a culture medium, and determining the heavy metal removal rate based on microbial growth and the heavy metal removal rate. The microorganism with high heavy metal tolerance and biotransformation capability is obtained; the method comprises the following steps: uniformly distributing microorganisms under the assistance of a dispersing aid, carrying out low-temperature standing crosslinking by adopting a cross-linking agent, adsorbing the microorganisms on the surface of a bamboo nano material to obtain a microorganism and bamboo nano material composite repairing agent, and applying the microorganism and bamboo nano material composite repairing agent to polluted soil for heavy metal pollution repairing.

Description

Technical Field

[0001] The present invention belongs to the technical field of soil remediation, and particularly relates to a method and a system for remediating soil heavy metal pollution based on bamboo nanomaterials. Background Art

[0002] Soil heavy metal pollution is one of the important challenges in global environmental governance. Traditional remediation methods have problems such as low efficiency, high cost, and high risk of secondary pollution. In recent years, nanomaterials and microbial remediation technologies have received wide attention, but the application effects of single technologies are limited, and there is a lack of intelligent regulation means. Therefore, there is an urgent need for an efficient, intelligent, and sustainable soil heavy metal remediation method.

[0003] Bamboo nanomaterials are a kind of natural, renewable, and biodegradable materials, with high specific surface area, rich functional groups, and good adsorption properties. Compared with traditional synthetic nanomaterials, bamboo nanomaterials are more environmentally friendly, lower in cost, and have a wide range of sources. As a carrier, bamboo nanomaterials can provide a good attachment environment and nutrients for microorganisms, enhancing the activity of microorganisms.

[0004] Rhodococcus erythropolis is a Gram-positive bacterium existing in the environment and has strong heavy metal tolerance.

[0005] How to intelligently combine the high adsorption property of bamboo nanomaterials and the bioremediation ability of Rhodococcus erythropolis for efficient heavy metal remediation is an urgent problem to be solved. Summary of the Invention

[0006] To solve the deficiencies of the prior art and achieve the purpose of efficient removal and real-time monitoring of Cd in soil 2+ The present invention adopts the following technical solutions: A method for remediating soil heavy metal pollution based on bamboo nanomaterials, comprising the following steps: Preparation of bamboo nanomaterials: Cut bamboo along the fiber axis into sheets, and perform ultrasonic cleaning and vacuum drying. Through the cooperation of precise cutting, ultrasonic impurity removal, and stepped vacuum drying, potential fiber damage can be eliminated, and the subsequent nanonization efficiency can be improved. Then, the intermediate product obtained after energy gradient grinding in sequence. The energy gradient grinding strategy can optimize the mechanical action mode in stages, improve the fiber dissociation efficiency, and then perform nanonization treatment of high-pressure homogenization and in-situ dispersion to obtain solid porous bamboo nanomaterials with high specific surface area and rich functional groups; Microbial culture, based on the heavy metal resistance gene of microorganisms, encoding heavy metal efflux proteins, selecting microbial expression vectors, inserting heavy metal resistance target genes into the expression vectors, making the downstream with strong promoters and terminators, using electroporation to introduce the expression vectors into microorganisms, and detecting the expression level of the target gene, so as to achieve efficient transformation and quantitative verification, and ensure stable overexpression of the target gene; inoculating microorganisms that overexpress the target gene into culture media containing different concentrations of heavy metals, and obtaining microorganisms with high heavy metal tolerance and biotransformation ability based on microbial growth and heavy metal removal rate; Preparation of composite repair agent, using dispersing aid to assist the uniform distribution of microorganisms, using cross-linking agent to perform low-temperature static cross-linking, adsorbing microorganisms on the surface of bamboo nanomaterials, and obtaining composite repair agent of microorganisms and bamboo nanomaterials, based on the prepared bamboo nanomaterials with high specific surface area and rich functional groups, the bamboo nanomaterials can adsorb microorganisms through functional groups, and the high specific surface area of ​​bamboo nanomaterials can increase the coverage area of ​​microorganisms; In addition, the combination of low-temperature cross-linking and the porous structure of bamboo fibers can achieve both activity and stability, effectively reducing the shedding rate of microorganisms. The pore structure of bamboo nanofibers (pore size 1-5 μm) provides physical protection for microorganisms, especially Rhodococcus erythropolis. Low-temperature cross-linking can reduce protein denaturation and maintain the function of the CzcD efflux pump, which is beneficial for the adsorption of Cd by bamboo materials alone. 2+ , 72 hours CD 2+ The removal rate was 55%, and the cross-linked bacteria alone removed Cd 2+ The removal rate of the composite repair agent is 65%, and the removal rate of the composite repair agent can reach 92% (synergy coefficient 1.42); The prepared composite repair agent is applied to contaminated soil to repair heavy metal pollution; bamboo nanomaterials can also quickly adsorb free heavy metal ions and reduce extracellular toxic concentrations. Microorganisms with overexpressed target genes can excrete intracellular heavy metals to the surface of the material, forming a cycle, realizing dual-path synergistic detoxification of microorganisms and bamboo nanomaterials based on adsorption and biotransformation. For Rhodococcus erythropolis overexpressing CzcD + bamboo nanomaterials, bamboo nanomaterials quickly adsorbed free Cd 2+ , reducing the extracellular toxic concentration, and the strain overexpressing CzcD converted intracellular Cd 2+ It is efficiently discharged to the surface of the material to form a cycle; it solves the problem of "stability and activity cannot be achieved at the same time" in traditional immobilization technology, and realizes the "adsorption-bioconversion" dual-path synergy. Compared with the single bamboo nanomaterial for 160 mg / L Cd 2+ The adsorption rate was 55%, and the removal rate of genetically engineered bacteria alone was 65%. The composite system repair agent had an adsorption rate of 160 mg / L Cd 2+The 72-hour removal rate of contaminated soil can reach 90% (synergistic coefficient of 1.38), which is significantly better than traditional microbial remediation methods.

[0007] Further, in the preparation of the bamboo nanomaterial, axial cutting means cutting the bamboo into sheet specimens of 5 mm × 5 mm × 1 mm along the fiber axis. This size design fully considers the microstructure characteristics of the bamboo fiber bundle and can improve the uniformity of the subsequent grinding force. For the ultrasonic cleaning, ultrasonic waves of 40 kHz are used for cleaning, and the ultrasonic cavitation effect is utilized to effectively peel off the silica layer and phytolith impurities on the bamboo surface. The bamboo with a water content less than or equal to 8% is cut along the fiber axis. After cleaning, it is dried under a vacuum of -0.08 MPa using a stepwise temperature increase program (holding at 40°C for 1 hour → holding at 50°C for 1 hour → constant temperature at 60°C) until the mass change rate < 0.1% / h, and finally the moisture content is controlled within 2.5 ± 0.3%. The stepwise vacuum drying method can achieve precise control of ultra-low moisture content and provide raw materials with stable physical and chemical properties for subsequent mechanical crushing.

[0008] Further, in the preparation of the bamboo nanomaterial, the energy gradient grinding includes two stages. The first stage is rough grinding. In the second stage, the rough grinding product is mixed with absolute ethanol at a certain solid-liquid ratio (1:10 g / mL) and wet ground at a temperature ≤ 40°C for 6 hours under the temperature control of a circulating water cooling system (ethylene glycol coolant temperature of 5°C). It is monitored in real time by an online temperature sensor (PT100 type) and a vibration accelerometer, and the cooling flow rate is dynamically adjusted to make the tank temperature ≤ 40°C. Finally, an intermediate product with a median particle size D50 (in the cumulative distribution diagram, the abscissa diameter value corresponding to the cumulative distribution of 50% on the ordinate) of 1.2 ± 0.3 μm is obtained. Through the energy gradient design (rough grinding impact crushing of 80 J / g → wet grinding shear peeling of 150 J / g), the energy gradient grinding strategy can optimize the mechanical action mode in stages, improve the fiber dissociation efficiency by 40%, and limit the grinding temperature rise within 15°C. It is verified by thermogravimetric analysis that the cellulose thermal decomposition temperature is maintained above 320°C.

[0009] Further, in the preparation of the bamboo nanomaterial, the nanometrization treatment is to circulate the intermediate product through a high-pressure homogenizer and detect the particle size distribution to obtain a homogenate with D90 < 100 nm. 0.1 wt% sodium carboxymethylcellulose is added to the homogenate as a dispersant and stirred to form a stable suspension with an electrokinetic potential Zeta ≤ -30 mV. After freeze-drying and dehydration, a solid porous bamboo nanomaterial with a moisture content ≤ 0.5% is obtained. Through the assistance of sodium carboxymethylcellulose CMC in high-pressure homogenization, nanometrization and dispersion stability are achieved simultaneously.

[0010] Furthermore, in the microbial culture, the microorganism is Rhodococcus erythropolis, and the heavy metal is cadmium (Cd). Through the CzcD gene possessed by Rhodococcus erythropolis, it encodes cadmium ion Cd 2+ efflux protein. Select the expression vector of pTip Rhodococcus erythropolis, insert the target gene into the expression vector, and make it have a strong promoter and terminator downstream; use the electroporation method to introduce the expression vector into Rhodococcus erythropolis, and verify the expression level of the target gene by qPCR, so as to achieve efficient transformation and quantitative verification, and ensure the stable overexpression of the CzcD gene.

[0011] Furthermore, in the preparation of the composite repair agent, bamboo nanomaterials and Rhodococcus erythropolis are compounded at a mass ratio of 1:0.3, and the dispersion aid is 0.05% Tween-80. Stir at 400 rpm for 4 hours to make the hydroxyl groups (-OH) on the surface of bamboo nanomaterials and the peptidoglycan of the cell wall of Rhodococcus erythropolis physically adsorbed through hydrogen bonds and electrostatic interactions. Through the assistance of Tween-80 + precise stirring parameters (400 rpm × 4 h), the cell coverage rate can be increased from 50% to 83%, and the repair efficiency is increased by more than 35%. In addition, through the high-purity composite system of autoclaving + Tween-80 dispersion, the problem of low colonization rate of target strains caused by endogenous microbial competition is avoided.

[0012] Furthermore, in the preparation of the composite repair agent, autoclave the bamboo nanomaterials to avoid biological competition during subsequent microbial compounding, and then disperse them in phosphate buffer solution (PBS, pH = 7.4) to prepare a 5 mg / mL suspension. Inoculate the microorganisms into LB liquid medium (containing 1% glycerol), culture them in a shaker at 30 °C and 180 rpm until the logarithmic growth phase (OD600 = 0.8), collect the microorganisms by a centrifuge (5000 rpm, 4 °C, 10 minutes) and wash them three times with sterile PBS, and finally make a microbial suspension (concentration 1×10 8 CFU / mL).

[0013] Furthermore, in the preparation of the composite repair agent, add 0.5 wt% glutaraldehyde solution as a crosslinking agent to the mixture of bamboo nanomaterials and microorganisms, and let it stand and crosslink at 4 °C for 12 hours. The Zeta potential of the obtained complex is -18 ± 2 mV, and the surface potential of the complex is measured to rise from -32 mV to -18 mV, so that the microbial survival rate can be increased from 60% to 90%.

[0014] A remediation system for heavy metal pollution in soil based on bamboo nanomaterials, comprising a remediation agent generation device and a remediation agent application device. The remediation agent generation device includes a bamboo nanomaterial preparation unit, a microorganism culture unit, and a bamboo nanomaterial and microorganism composite unit. Combining with the remediation agent application device, the bamboo nanomaterial preparation, microorganism culture, composite remediation agent preparation, and remediation agent application of the remediation method for heavy metal pollution in soil based on bamboo nanomaterials are sequentially performed.

[0015] Furthermore, the system further includes a heavy metal monitoring device, which includes a sensor, a cloud platform, and a control terminal; The sensor real-time collects the heavy metal concentration in the soil; The cloud platform, based on the collected heavy metal concentration, optimizes the remediation strategy through machine learning and generates the dosage of the remediation agent to be increased; The remediation agent application device applies the remediation agent in a stratified and mixed manner, increasing the contact area between the remediation agent and the soil while achieving uniform distribution of the remediation agent in the soil profile. The remediation agent application device uses a rotary tiller to perform stratified mixing at 200 rpm to a soil depth of 30 cm, and the dosage of the remediation agent is 0.5 - 1.5% w / w.

[0016] The advantages and beneficial effects of the present invention are as follows: The present invention prepares bamboo nanomaterials and cultivates Rhodococcus erythropolis, combines the two to form an intelligent remediation agent, and utilizes the adsorption effect of bamboo nanomaterials and the biotransformation effect of Rhodococcus erythropolis, combined with intelligent sensor technology, to achieve the efficient removal and real-time monitoring of Cd in the soil. 2+ The present invention has the advantages of high remediation efficiency, low cost, environmental friendliness, etc., and is suitable for large-scale remediation of heavy metal pollution in soil. Description of the Drawings

[0017] Figure 1 is a flowchart of the method in the embodiment of the present invention.

[0018] Figure 2 is a microstructural diagram of a bamboo fiber bundle in the embodiment of the present invention.

[0019] Figure 3 is an effect diagram of bamboo nanometerization treatment in the embodiment of the present invention (D90 < 100 nm).

[0020] Figure 4 is a schematic diagram of the synergistic action mechanism between bamboo nanomaterials and Rhodococcus erythropolis in the embodiment of the present invention.

[0021] Figure 5 is an architecture diagram of the system in the embodiment of the present invention.

[0022] Figure 6 is Cd in the embodiment of the present invention 2+The curve graph of the influence of concentration on the growth of overexpressing bacteria.

[0023] Figure 7 It is the curve graph of the removal rate of heavy metal Cd over time in the embodiment of the present invention. Detailed implementation manners

[0024] The following will describe in detail the specific implementation manners of the present invention with reference to the accompanying drawings. It should be understood that the specific implementation manners described herein are only for explaining and understanding the present invention, and are not used to limit the present invention.

[0025] As Figure 1 shown, the method for repairing soil heavy metal pollution based on bamboo nanomaterials includes the following steps: Step 1: Preparation of bamboo nanomaterials. The bamboo nanomaterials are prepared by mechanical grinding method to ensure that they have a high specific surface area and abundant functional groups; When using the mechanical grinding method to prepare bamboo nanomaterials, first use a numerically controlled precision cutting machine to cut bamboo with a moisture content ≤ 8% along the fiber axis into sheet specimens of 5mm×5mm×1mm. This size design fully considers the microstructural characteristics of bamboo fiber bundles such as Figure 2 shown (fiber length 1 - 3mm, diameter 20 - 50μm). The directional size design matches the bamboo fiber microstructure, which can improve the uniformity of the subsequent grinding force; then immerse the specimens in 5 times the volume of deionized water (resistivity ≥ 18.2MΩcm) for deep cleaning, and treat them with a 40 kHz ultrasonic cleaner (power density 0.5W / cm²) for 30 minutes. Utilize the ultrasonic cavitation effect to effectively peel off the silica layer and phytolith impurities on the surface of bamboo; transfer the cleaned specimens to a vacuum drying oven, and use a stepwise heating program (-0.08MPa vacuum degree, 40℃ for 1 hour → 50℃ for 1 hour → 60℃ constant temperature) for precise drying until the mass change rate < 0.1% / h. Finally, strictly control the moisture content at 2.5 ± 0.3%. The stepwise vacuum drying method can achieve precise control of ultra-low moisture content, providing raw materials with stable physical and chemical properties for subsequent mechanical crushing.

[0026] In the prior art, it is usually simply cut into blocks (10 - 20mm) and naturally dried (moisture content 5 - 8%). The prior art is likely to leave residual silica layer, resulting in a 30% decrease in grinding efficiency; while the present invention can eliminate the hidden danger of fiber damage and improve the subsequent nanometerization efficiency by more than 20% through the cooperation of 5mm×5mm×1mm precise cutting, ultrasonic impurity removal, and stepwise vacuum drying.

[0027] The pretreated bamboo enters the mechanical crushing stage, and a QM-3SP2 type planetary ball mill is used for two-stage grinding: In the first stage of rough grinding, it runs at a revolution speed of 300 rpm and a rotation speed of 200 rpm for 2 hours. The grinding jar is filled with zirconia grinding balls with a diameter of 3 mm (Mohs hardness 8.5, density 6.05 g / cm³), loaded according to a ball-to-material mass ratio of 20:1, and an argon protective atmosphere is introduced to prevent cellulose oxidation. The product after grinding is classified by a 200-mesh standard sieve (aperture 75 μm) to ensure that the return rate of the oversize material is <5%; In the second stage of wet grinding, the rough grinding product is mixed with absolute ethanol at a solid-to-liquid ratio of 1:10 (g / mL), and grinding is continued for 6 hours under temperature control of a circulating water cooling system (ethylene glycol coolant temperature 5°C). It is monitored in real time by an online temperature sensor (PT100 type) and a vibration accelerometer, and the cooling flow is dynamically adjusted to keep the tank temperature ≤40°C. Finally, an intermediate product with a median particle size D50 (in the cumulative distribution graph, the abscissa diameter value corresponding to a cumulative distribution of 50% on the ordinate) of 1.2 ± 0.3 μm is obtained. In this stage, through the energy gradient design (rough grinding impact crushing 80 J / g → wet grinding shear peeling 150 J / g), the energy gradient grinding strategy can optimize the mechanical action mode in stages, increase the fiber dissociation efficiency by 40%, and limit the grinding temperature rise within 15°C. It is verified by thermogravimetric analysis that the cellulose thermal decomposition temperature remains above 320°C.

[0028] In the prior art, mechanical grinding usually adopts single ball milling, and single ball milling has no energy gradient and large temperature fluctuations. However, the two-stage grinding (rough grinding + wet grinding) and real-time temperature control (≤40°C) adopted in the present invention can increase the cellulose retention rate by 35% and reduce the energy consumption by more than 25%.

[0029] In the nanosizing treatment step, a Nano DeBEE high-pressure homogenizer is used. The intermediate product is subjected to 10 cycles of treatment through a diamond interaction chamber (aperture 100 μm) at a pressure of 150 MPa. After each cycle, the particle size distribution is monitored by a dynamic light scattering instrument (DLS). Homogenization is terminated when D90 < 100 nm, as Figure 3 shown (D90 represents the abscissa diameter value corresponding to a cumulative distribution of 90% on the ordinate in the cumulative distribution graph). To enhance the dispersion stability of the product, 0.1 wt% sodium carboxymethyl cellulose (CMC) is added to the homogenized product as a dispersant and treated under magnetic stirring at 800 rpm for 30 minutes to form a stable suspension with a Zeta potential (electrokinetic potential, that is, the potential of the shear plane, used to characterize the stability of the colloidal dispersion system) of -35 mV. The solid porous material obtained after freeze-drying (-50°C, 10 Pa) dehydration has a moisture content strictly controlled at ≤0.5%. This state is the end product that can be stored for a long time and has a high specific surface area and a complete crystal structure.

[0030] In the prior art, single high-pressure homogenization is usually adopted for nano-dispersion. However, single high-pressure homogenization is prone to agglomeration and requires subsequent separate dispersion treatment. The D90 requires 15 homogenization cycles (>200 nm). In contrast, the present invention adopts high-pressure homogenization combined with in-situ dispersion of CMC (Zeta potential -35 mV). After 10 cycles, D90 < 100 nm can be achieved. Moreover, the introduction of CMC reduces the re-agglomeration rate of nanofibers from 40% to less than 5%, and extends the suspension stability by 10 times (from 7 days to 70 days without sedimentation). For the control of moisture content, oven drying (moisture content 3 - 5%) is usually adopted, while the present invention adopts step vacuum drying (moisture content 2.5 ± 0.3%).

[0031] Through the above improvements, the performance of the overall process chain of the present invention has doubled. Compared with the prior art, the high specific surface area of the final bamboo nano-material has increased from 120 m² / g to 180 m² / g (BET test), and the Cd 2+ adsorption capacity has increased from 80 mg / g to 150 mg / g (laboratory data). After being combined with Rhodococcus erythropolis, the remediation efficiency has been improved. Thus, the precision, efficiency, and stability of the bamboo nano-material preparation process have been realized, providing a carrier material with excellent performance for subsequent heavy metal remediation.

[0032] Step 2: Cultivation of Rhodococcus erythropolis. Isolate and screen Rhodococcus erythropolis from cadmium (Cd)-contaminated soil, and expand the culture through a culture medium to obtain Rhodococcus erythropolis with high Cd 2+ tolerance and biotransformation ability. Rhodococcus erythropolis has the CzcD gene, which encodes a Cd 2+ efflux protein. Obtain the sequence of the CzcD gene of Rhodococcus erythropolis from the NCBI database. Select the pTip expression vector for Rhodococcus erythropolis, insert the target gene into the vector, and make it have a strong promoter and terminator downstream. Use the electroporation method to introduce the expression vector into Rhodococcus erythropolis, and verify the expression level of the target gene through qPCR, so as to achieve efficient transformation and quantitative verification, and ensure the stable overexpression of the CzcD gene.

[0033] In the prior art, gene modification usually adopts a general vector (such as the pET series), and the transformation efficiency is low (<10³ CFU / μg DNA). Due to vector compatibility problems, gene expression is unstable. In contrast, the pTip special vector for Rhodococcus erythropolis combined with the electroporation method adopted in the present invention (>10 5 CFU / μg DNA) can increase the transformation efficiency by 100 times, and make the expression level of the CzcD protein reach 3.2 times that of the wild type (verified by Western Blot), realizing efficient gene editing.

[0034] Inoculate Rhodococcus erythropolis overexpressing the CzcD gene into a culture medium containing different concentrations of Cd 2+ and measure its growth curve and Cd2+ Removal rate. Liquid media containing different concentrations of Cd at 0, 20, 40, 80, and 160 mg / L were prepared. 2+ Rhodococcus erythropolis with overexpressed genes was inoculated and cultured for 24, 48, and 72 hours. The optical density OD600 of the bacterial solution and the Cd 2+ concentration were measured, and the Cd 2+ removal rate was calculated.

[0035] The overexpressed strain could grow normally under the stress of ≤80 mg / L Cd 2+ (OD600 > 1.0 at 48 hours), indicating that its Cd tolerance was significantly enhanced. At a high concentration of 160 mg / L Cd 2+ , the strain could still survive and slowly proliferate (OD600 reached 0.8 at 72 hours), while the wild-type strain was inhibited from growing at this concentration (the upper limit of wild-type strain tolerance is usually ≤50 mg / L Cd 2+ ). Therefore, the present invention increased the upper limit of strain Cd tolerance from 80 mg / L to 160 mg / L, breaking through the extreme tolerance of the strain to Cd 2+ .

[0036] On the other hand, the Cd 2+ removal rate was positively correlated with time. As the culture time extended, the removal rate increased significantly (the removal rate reached 65% - 90% at 72 hours). Moreover, the strain of the present invention was still effective at high concentrations. Even at a high concentration of 160 mg / L, the removal rate at 72 hours still reached 65%, indicating that the strain had strong repair potential, thus breaking through the technical bottleneck of high-concentration pollution repair.

[0037] The present invention combines gene modification precision and material-biology synergy. By overexpressing the CzcD gene through genetic engineering technology, the Cd 2+ efflux ability of Rhodococcus erythropolis was increased by more than 3 times (see Example 1 for details), solving the problem of the failure of traditional repair technologies in high-concentration heavy metal pollution scenarios.

[0038] Step 3: Composite bamboo nanomaterials with Rhodococcus erythropolis and prepare a stable repair agent through physical adsorption and chemical cross-linking; The core process for preparing the intelligent repair agent is to directionally compound bamboo nanomaterials obtained by mechanical grinding method with Rhodococcus erythropolis. First, the bamboo nanomaterials are pretreated. The freeze-dried bamboo nanofibrils (water content ≤ 0.5%) are placed in an autoclave (121 °C, 0.15 MPa) for 20 minutes to eliminate endogenous microbial interference. The autoclave pretreatment ensures the sterility of the bamboo nanomaterials and avoids biological competition during subsequent microbial compounding. Subsequently, they are dispersed in phosphate buffer solution (PBS, pH = 7.4) to prepare a 5 mg / mL suspension. At the same time, the Rhodococcus erythropolis strain is inoculated into LB liquid medium (containing 1% glycerol) and cultured in a shaker at 30 °C and 180 rpm until the logarithmic growth phase (OD600 = 0.8). The cells are collected by a centrifuge (5000 rpm, 4 °C, 10 minutes) and washed three times with sterile PBS, and finally a cell suspension (concentration 1×10 8 CFU / mL) is prepared.

[0039] In the prior art, usually only surface disinfection (such as wiping with alcohol) is adopted or microbial interference is ignored. However, the autoclaving (121 °C, 20 minutes) of the present invention can completely inactivate endogenous microorganisms, avoid strain competition, and the colonization rate of Rhodococcus erythropolis is increased by more than 40%.

[0040] The compounding process is carried out in a biosafety cabinet. They are mixed according to the mass ratio of bamboo nanomaterials to cells of 1:0.3, and 0.05% Tween-80 is added as a dispersion aid to assist in achieving uniform distribution of the cells. The reaction is carried out in a constant temperature magnetic stirrer (rotation speed 400 rpm) at 25 °C for 4 hours. At this stage, the hydroxyl groups (-OH) on the surface of the bamboo nanomaterials and the peptidoglycan of the cell wall of Rhodococcus erythropolis achieve physical adsorption through hydrogen bonding and electrostatic interaction. Observed by a laser confocal microscope (FITC-labeled cells), it shows that the coverage rate of the cells on the surface of the bamboo fibers reaches 83 ± 5%.

[0041] In the prior art, usually a simple mixing method is adopted. Due to the absence of a dispersant, it will lead to uneven distribution of the cells. However, in the present invention, through the assistance of Tween-80 + precise stirring parameters (400 rpm × 4 h), the cell coverage rate can be increased from 50% to 83%, and the repair efficiency is increased by more than 35%. In addition, through the high-purity compounding system of autoclaving + Tween-80 dispersion, the problem of low colonization rate of the target strain caused by endogenous microbial competition is avoided.

[0042] To enhance the stability of the compounding system, 0.5 wt% glutaraldehyde solution (crosslinking agent) is added dropwise to the mixed solution and left to crosslink at 4 °C for 12 hours. The surface potential of the complex measured by a Zeta potentiometer rises from -32 mV to -18 mV. Through low-temperature glutaraldehyde crosslinking, the stability of the complex and the microbial activity are balanced, and the microbial shedding rate is effectively reduced (< 5%).

[0043] In the prior art, high-temperature crosslinking (>25°C) or sodium alginate embedding (low mechanical strength) is usually adopted. In the present invention, low-temperature (4°C) glutaraldehyde crosslinking and precise Zeta potential regulation are used, which can increase the microbial survival rate from 60% to 90% and extend the stability of the complex by 3 times. In addition, the combination of low-temperature crosslinking and bamboo fiber network structure can balance activity and stability. The pore structure of bamboo nanofibers (pore diameter 1-5 μm) provides physical protection for the bacteria. Low-temperature crosslinking can reduce protein denaturation and maintain the CzcD efflux pump function, for the adsorption of Cd by bamboo materials alone 2+ , the removal rate of Cd for 72 hours 2+ is 55%. The removal rate of Cd by crosslinked bacteria alone 2+ is 65%, and the removal rate of the composite repair agent can reach 92% (synergy coefficient 1.42); The final product forms a porous network structure after freeze-drying. Scanning electron microscopy shows that Rhodococcus erythropolis is evenly embedded in the gaps of bamboo nanofibers. Real-time fluorescence quantitative PCR detection confirms that the retention rate of the 16S rRNA gene copy number of the bacteria is >90%.

[0044] In the prior art, only the morphology is observed (such as ordinary SEM), while in the present invention, multi-dimensional verification is carried out by combining SEM, fluorescence labeling, and qPCR. The quality controllability is improved through multi-dimensional characterization + process linkage. The triple verification of Zeta potential (-18 mV), qPCR (16S rRNA > 90%), and SEM morphology ensures that the fluctuation of the bacterial load of each batch of repair agent is <5%, far better than the prior art (±20%).

[0045] Such as Figure 4 shown, the present invention optimizes the composite system synergistically through the protection of microbial activity and the design of material structure. Based on the dual-path detoxification of overexpressed CzcD + bamboo nanomaterials, the bamboo nanomaterials rapidly adsorb free Cd 2+ , reducing the extracellular toxicity concentration. The strain overexpressing CzcD effluxes intracellular Cd 2+ efficiently to the material surface to form a cycle; solving the problem of "incompatibility between stability and activity" in traditional immobilization technologies, realizing the dual-path synergistic effect of "adsorption-biotransformation". Compared with the adsorption rate of 55% of 160 mg / L Cd by bamboo nanomaterials alone 2+ , and the removal rate of 65% by genetically engineered bacteria alone, the repair agent of the composite system can achieve a 72-hour removal rate of 90% for 160 mg / L Cd 2+ contaminated soil (synergistic enhancement coefficient 1.38), significantly superior to traditional microbial remediation methods.

[0046] Step 4: Apply the prepared repair agent to the contaminated soil, and through the adsorption of bamboo nanomaterials and the biotransformation of Rhodococcus erythropolis, achieve the efficient removal of Cd 2+ ; Based on the Cd concentration in the soil 2+ Determine the dosage of the remediation agent, and the dosage of the remediation agent is 0.5% - 1.5% w / w. Apply it to the 0 - 30 cm contaminated soil layer by a rotary tiller (rotating speed 200 rpm) using the layered mixing method to ensure full contact between the remediation agent and the soil. Irrigate and activate (water consumption ≈ 60% of the field water holding capacity).

[0047] Deploy Cd concentration sensors in the soil, and conduct intelligent monitoring and control through intelligent sensors. Real - time monitor the Cd concentration, and automatically adjust the release rate of the remediation agent and the remediation strategy according to the monitoring results. This closed - loop intelligent control system can achieve dynamic and precise control of the dosage of the remediation agent to automatically release Rhodococcus erythropolis and adsorb Cd 2+ 2+ 2+ 2+

[0048] As Figure 5 shown, the soil heavy metal pollution remediation system based on bamboo nanomaterials includes a remediation agent generation device, a heavy metal monitoring device, and a rotary tiller. The remediation agent generation device includes a bamboo nanomaterial preparation unit, a Rhodococcus erythropolis culture unit, and a bamboo nanomaterial - Rhodococcus erythropolis composite unit, and realizes continuous preparation from raw materials to finished agents through an integrated remediation agent production system; the heavy metal monitoring device includes sensors, a cloud platform, and a control terminal.

[0049] The bamboo nanomaterial preparation unit obtains bamboo nanomaterials through nanometerization after mechanical grinding or chemical treatment of bamboo. The Rhodococcus erythropolis culture unit is used for high - density cultivation of Rhodococcus erythropolis overexpressing the CzcD gene. The bamboo nanomaterial - Rhodococcus erythropolis composite unit composites bamboo nanomaterials and Rhodococcus erythropolis to prepare a stable remediation agent. The sensor is used to collect the Cd concentration in the soil in real - time 2+ The cloud platform stores and analyzes the collected data. Based on a data - driven decision - making system, it optimizes the remediation strategy through machine learning algorithms and generates an automatic increase in the dosage of the remediation agent. The control terminal generates an adaptive strategy and sets an alarm threshold.

[0050] The rotary tiller adopts a layered mixing application technology to achieve uniform distribution of the remediation agent in the soil profile by optimizing the rotary tiller parameters.

[0051] ​​​In the prior art, the application of remediation agents is usually carried out by spreading on the soil surface or simple tillage (mixing depth ≤ 15 cm). In contrast, the present invention combines the layered mixing method (0 - 30 cm) with the optimization of rotary tillage parameters (200 rpm), which can increase the contact area of the remediation agent by 50% and the action depth by 100%. For traditional monitoring and regulation, manual sampling and laboratory analysis are required (cycle > 7 days), while the present invention combines in-situ sensors, real-time data transmission, and automatic regulation, which can shorten the response time from 7 days to < 1 hour and improve the remediation efficiency by 40%. The integration level of existing devices is relatively low, and usually, decentralized equipment is adopted (materials, bacteria agents, and mixing equipment are separated), while the integrated remediation agent generation device of the present invention (preparation - cultivation - compound linkage) can reduce the comprehensive cost by 35% and increase the production efficiency by 3 times. Traditional decision-making usually requires empirical judgment or fixed remediation plans, while the present invention combines data analysis on the cloud platform with the generation of adaptive strategies, resulting in higher efficiency.

[0052] On the other hand, the synergistic mechanism of layered mixing and intelligent monitoring makes the remediation more precise. Layered mixing ensures that the remediation agent covers all contaminated areas, and the sensors provide real-time feedback to guide enhanced remediation in key areas. The combination of the integrated device and the cloud platform improves the operation and maintenance efficiency compared to multi-step manual operations. The dynamic regulation combined with the composite remediation agent can maintain the long-term stability of remediation in scenarios of fluctuating pollution (such as changes in Cd concentration caused by rainfall). The effect of traditional static remediation plans decays by > 50% (after 30 days), while the present invention can maintain a removal rate of over 80% through dynamic adjustment (60-day test). 2+ In the present invention, by enhancing the extreme tolerance of Rhodococcus erythropolis and combining intelligent regulation, long-term remediation is achieved. In the pulsed Cd pollution (concentration fluctuation of 100 - 200 mg / L) simulation experiment, the composite remediation agent can maintain stable remediation for 30 days (removal rate > 85%), while traditional bacteria agents become inactivated on the 10th day.

[0053] The innovation of the present invention lies in the combination of engineering application and intelligent management, which solves the three major pain points of traditional remediation technologies, namely low efficiency, high cost, and poor adaptability. 2+ The realization of the heavy metal monitoring device is as follows:

[0054] I. Sensor deployment:

[0055] 1. Sensor selection: Cd ion-selective electrode (ISE), performance parameters: Detection range: 0.1 - 1000 mg / L Cd 2+ Response time: ≤ 30 seconds. 2+

[0056]

[0057] ​​​​Operating temperature: 0 - 50 °C.

[0058] Lifespan: ≥ 6 months (under field conditions).

[0059] 2. Sampling point density: Deploy 1 monitoring point per 100 m² of the remediation area to ensure the spatial representativeness of the data.

[0060] For contaminated sites with high heterogeneity, it can be increased to 1 monitoring point per 50 m².

[0061] 3. Installation depth: Surface sensor: Buried at a depth of 10 cm to monitor the Cd concentration change in the surface soil. 2+ Concentration change.

[0062] Deep sensor: Buried at a depth of 20 cm to monitor the downward migration of Cd. 2+ Downward migration situation.

[0063] 4. Installation method: Use a soil drill (2 cm in diameter) to drill to the target depth at the specified location.

[0064] Vertically insert the sensor probe into the hole to ensure full contact with the soil.

[0065] Backfill with the original soil and compact it to avoid forming air gaps.

[0066] II. Sensor calibration: 1. Preparation of calibration solution: Prepare 0.1, 1, 10, 100 mg / L Cd standard solutions using Cd(NO3)2. 2+ Standard solution.

[0067] 2. Calibration steps: Immerse the sensor in the standard solutions in sequence and record the potential response values (mV).

[0068] Plot the calibration curve (potential vs. Cd concentration) and calculate the slope and intercept. 2+ Concentration), and calculate the slope and intercept.

[0069] 3. Calibration frequency: Once every 2 weeks to ensure data accuracy.

[0070] III. Data transmission and cloud platform 1. Local hardware components: Data collector: Multichannel data acquisition module, supporting the access of 16 sensors.

[0071] Power supply system: Solar panel (20 W) + lithium battery pack (12 V, 50 Ah) to ensure long-term power supply in the field.

[0072] Protection box: Waterproof and dustproof box (IP67 rating), with a built-in temperature control module (0 - 40 °C).

[0073] 2. Sampling frequency: Data is collected every 10 minutes and stored in a local SD card (32 GB).

[0074] 3. Wireless transmission technology 1) Technology selection: LoRa: Suitable for long-distance (≤10 km), low-power transmission, with a bandwidth of 50 kbps.

[0075] NB-IoT: Suitable for urban areas, with a bandwidth of 250 kbps, supporting high-density deployment.

[0076] 2) Transmission parameters: Packet size: ≤512 bytes per time.

[0077] Transmission frequency: Data is uploaded once an hour (can be adjusted according to requirements).

[0078] 3) Network configuration: Insert a LoRa or NB-IoT module (such as Semtech SX1276) into the data collector.

[0079] Configure the gateway address, transmission frequency, and encryption key.

[0080] 4. Cloud platform construction 1) Platform selection: Public cloud: Such as Alibaba Cloud IoT Platform, AWS IoT Core, supporting large-scale data storage and analysis.

[0081] Private cloud: Built based on an open-source platform (such as ThingsBoard), suitable for scenarios with high data security requirements.

[0082] 2) Data storage: Use a time series database (such as InfluxDB) to store sensor data, supporting efficient query and analysis.

[0083] Data retention time: ≥2 years.

[0084] 3) Visualization interface: Develop Web and mobile applications to display the Cd 2+ Concentration distribution map and remediation progress in real-time.

[0085] Support data export (CSV format) and report generation (PDF format).

[0086] 4) Data analysis and optimization: The cloud platform preprocesses the collected data and selects machine learning algorithm models for model training and verification, and supplements the repair agent through the trained prediction model.

[0087] Data preprocessing includes removing outliers (such as outliers caused by sensor failures) and data normalization (mapping the Cd 2+ concentration to the range of 0-1).

[0088] Model selection is used to select regression models, classification models, etc. Regression models such as RandomForest Regression are used to predict the Cd 2+ concentration change trend, and classification models such as Support Vector Machine (SVM) are used to judge whether the repair effect meets the standard.

[0089] Training and verification use historical data (such as laboratory simulation experiment data) to train the model, and evaluate the model performance (such as R²≥0.9, RMSE≤5 mg / kg) through cross-validation (10-fold).

[0090] Dynamically adjust the dosage of the repair agent, collect the soil Cd 2+ concentration data in real time and upload it to the cloud platform; calculate the current Cd 2+ concentration decline rate V_current based on the recent N monitoring data; when V_current is lower than the preset target Cd 2+ concentration decline rate V_target, automatically increase the dosage of the repair agent according to the product of the difference (V_target - V_current) and the adjustment coefficient k; periodically repeat the above steps until the Cd 2+ concentration reaches the standard. When the Cd 2+ concentration decline rate is lower than expected, automatically increase the dosage of the repair agent.

[0091] Example 1: Verification of the Cd tolerance of genetically engineered bacteria Rhodococcus erythropolis overexpressing the CzcD gene was inoculated into liquid media containing 0, 20, 40, 80, 160 mg / L Cd 2+ and cultured at 30°C and 180 rpm for 72 hours. As Figure 6 、 Figure 7 shown, by measuring the OD600 of the bacterial solution and the Cd 2+ concentration, the results showed that: Under the stress of 160 mg / L Cd 2+ , the biomass of the genetically engineered bacteria after 72 hours (OD600 = 0.8) was significantly higher than that of the wild-type strain (OD600 = 0.1); The genetically engineered bacteria had a tolerance to 160 mg / L Cd 2+The 72-hour removal rate reached 52%, while that of the wild-type strain was only 10%.

[0092] The above results indicate that overexpression of the CzcD gene significantly enhanced the Cd tolerance and Cd 2+ remediation efficiency of Rhodococcus erythropolis.

[0093] Example 2: Intelligent regulation function 1. Experimental design: Site: A Cd 2+ contaminated farmland (area 1000 m², concentration 120 mg / kg).

[0094] Dosage of the remediation agent: 1% w / w (initial dosage), dynamically adjusted according to sensor data.

[0095] Monitoring system: Deploy 20 Cd 2+ sensors (buried depth 10 cm).

[0096] 2) Data is transmitted to the cloud platform via LoRa and updated hourly.

[0097] 2. Remediation strategy: Dynamic adjustment: If the Cd 2+ concentration decline rate is lower than expected (e.g., <20% decline within 7 days), increase the dosage of the remediation agent by 0.2% w / w.

[0098] 2) If the soil humidity <40%, activate the irrigation system (water consumption ≈ 60% of the field capacity).

[0099] Optimization results:

[0100] 3. Conclusion: The intelligent regulation system significantly improved the remediation efficiency (72% removal rate in 60 days) by dynamically adjusting the dosage of the remediation agent and the irrigation frequency.

[0101] Compared with traditional remediation with a fixed dosage, the dosage of the remediation agent was saved by 20%.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for repairing soil heavy metal pollution based on bamboo nanomaterials, characterized in that It includes the following steps: Preparation of bamboo nanomaterials: Cut bamboo along the fiber axis into flakes, perform ultrasonic cleaning and vacuum drying, then, successively obtain intermediate products through energy gradient grinding, and then perform high-pressure homogenization and in-situ dispersion nanometrization treatment to obtain porous bamboo nanomaterials; Microbial culture: Based on the heavy metal resistance gene of microorganisms, encoding heavy metal efflux proteins, select the expression vector of microorganisms, insert the target gene for heavy metal resistance into the expression vector, use electroporation to introduce the expression vector into microorganisms, and detect the expression level of the target gene; Inoculate the microorganisms overexpressing the target gene into a medium containing different concentrations of heavy metals, and based on the microbial growth and heavy metal removal rate, obtain microorganisms with high heavy metal tolerance and biotransformation ability; Preparation of composite repair agent: Assist the uniform distribution of microorganisms through a dispersion aid, perform low-temperature static cross-linking with a cross-linking agent, and adsorb the microorganisms on the surface of bamboo nanomaterials to obtain a composite repair agent of microorganisms and bamboo nanomaterials; Apply the prepared composite repair agent to the contaminated soil for the repair of heavy metal pollution.

2. The remediation method for soil heavy metal pollution based on bamboo nanomaterials according to claim 1, characterized in that: In the preparation of the bamboo nanomaterials, axial cutting is to cut the bamboo along the fiber axis into sheet specimens of 5mm×5mm×1mm; For the ultrasonic cleaning, ultrasonic waves of 40 kHz are used for cleaning; Cut the bamboo with a water content less than or equal to 8% along the fiber axis, after cleaning, dry it using a stepwise temperature increase program under a vacuum of -0.08 MPa until the mass change rate < 0.1% / h, and finally control the moisture content within 2.5±0.3%; 3. The method for repairing soil heavy metal pollution based on bamboo nanomaterials according to claim 1, characterized in that: In the preparation of the bamboo nanomaterials, the energy gradient grinding includes two stages. The first stage is rough grinding, and the second stage is to mix the rough grinding product with absolute ethanol at a certain solid-liquid ratio and perform wet grinding at a controlled temperature ≤40°C to finally obtain an intermediate product with a median particle size D50 of 1.2±0.3μm.

4. The remediation method for soil heavy metal pollution based on bamboo nanomaterials according to claim 1, characterized in that: In the preparation of the bamboo nanomaterials, the nanometrization treatment is to circulate the intermediate product through a high-pressure homogenizer and detect the particle size distribution to obtain a homogenate with D90 < 100nm. Add 0.1wt% sodium carboxymethyl cellulose as a dispersant to the homogenate and stir to form a stable suspension with an electrokinetic potential Zeta ≤ -30mV. After freeze-drying and dehydration, obtain a solid porous bamboo nanomaterial with a moisture content ≤ 0.5%.

5. The method for repairing soil heavy metal pollution based on bamboo nanomaterials according to claim 1, wherein: In the microbial culture, the microorganism is Rhodococcus erythropolis, and the heavy metal is cadmium Cd. Through the CzcD gene possessed by Rhodococcus erythropolis, it encodes a cadmium ion Cd 2+ efflux protein. Select the expression vector of pTip Rhodococcus erythropolis, insert the target gene into the expression vector, and make a strong promoter and a terminator downstream; use the electroporation method to introduce the expression vector into Rhodococcus erythropolis, and verify the expression level of the target gene by qPCR detection.

6. The remediation method for soil heavy metal pollution based on bamboo nanomaterials according to claim 5, characterized in that: In the preparation of the composite repair agent, the bamboo nanomaterials and Rhodococcus erythropolis are compounded at a mass ratio of 1:0.

3. The dispersion aid is 0.05% Tween-80, and stir at 400 rpm for 4 hours to physically adsorb the hydroxyl -OH on the surface of the bamboo nanomaterials and the peptidoglycan in the cell wall of Rhodococcus erythropolis through hydrogen bonding and electrostatic interactions.

7. The remediation method for soil heavy metal pollution based on bamboo nanomaterials according to claim 1, characterized in that: In the preparation of the composite repair agent, perform high-pressure sterilization on the bamboo nanomaterials and disperse them in phosphate buffer to prepare a suspension. Inoculate the microorganisms into a liquid medium and culture them to the logarithmic growth phase, collect the microorganisms and wash them aseptically, and finally prepare a microbial suspension.

8. The remediation method for soil heavy metal pollution based on bamboo nanomaterials according to claim 1, characterized in that: In the preparation of the composite repair agent, 0.5 wt% glutaraldehyde solution is added as a cross-linking agent to the mixture of bamboo nanomaterials and microorganisms, and the mixture is allowed to stand and cross-link at 4 °C for 12 hours to obtain a composite with an electrokinetic potential Zeta of -18 ± 2 mV.

9. A remediation system for heavy metal pollution in soil based on bamboo nanomaterials, comprising a remediation agent generating device and a remediation agent applying device, characterized in that: The repair agent generating device includes a bamboo nanomaterial preparation unit, a microorganism culture unit, and a bamboo nanomaterial and microorganism composite unit. In combination with the repair agent application device, the preparation of bamboo nanomaterials, microorganism culture, preparation of composite repair agent, and application of repair agent in the soil heavy metal pollution repair method based on bamboo nanomaterials described in claim 1 are sequentially performed.

10. The remediation system for soil heavy metal pollution of bamboo-based nanomaterials according to claim 9, characterized in that: The system further includes a heavy metal monitoring device, which includes a sensor, a cloud platform, and a control terminal; The sensor collects the heavy metal concentration in the soil in real time; The cloud platform optimizes the repair strategy and generates the dosage of the repair agent to be increased based on the collected heavy metal concentration; The repair agent application device applies the repair agent by layering and mixing evenly.

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