Method and system for remediating heavy metal pollution in soil based on bamboo nanomaterials

Abstract

The present invention discloses a method and system for repairing heavy metal pollution in soil based on bamboo nanomaterials. The method comprises the following steps: cutting bamboo along the fiber axis and performing ultrasonic cleaning and vacuum drying, then subjecting the bamboo to energy gradient grinding, and then performing high-pressure homogenization and in-situ dispersion nano-processing to obtain porous bamboo nanomaterials; inserting a heavy metal-resistant target gene into a selected microbial expression vector, introducing the expression vector into the microorganism using electroporation, detecting the expression level of the target gene, inoculating the microorganism over-expressing the target gene into a culture medium, and obtaining microorganisms with high heavy metal tolerance and biotransformation ability based on microbial growth and heavy metal removal rate; uniformly distributing the microorganisms with the aid of a dispersing aid, performing low-temperature static cross-linking using a cross-linking agent, and adsorbing the microorganisms on the surface of the bamboo nanomaterials to obtain a composite repair agent of microorganisms and bamboo nanomaterials, which is then applied to contaminated soil for heavy metal pollution repair.

Description

Technical Field

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

[0002] Heavy metal contamination of soil is a major challenge in global environmental governance. Traditional remediation methods suffer from low efficiency, high costs, and significant risks of secondary contamination. In recent years, nanomaterials and microbial remediation technologies have garnered widespread attention, but the effectiveness of individual technologies is limited and lacks intelligent control. Therefore, there is an urgent need for efficient, intelligent, and sustainable methods for soil heavy metal remediation.

[0003] Bamboo nanomaterials are natural, renewable, and biodegradable materials with high surface area, abundant functional groups, and excellent adsorption properties. Compared with traditional synthetic nanomaterials, bamboo nanomaterials are more environmentally friendly, cost-effective, and widely available. As carriers, bamboo nanomaterials can provide a favorable attachment environment and nutrients for microorganisms, enhancing their activity.

[0004] Rhodococcus erythropolis is a Gram-positive bacterium present in the environment and is highly resistant to heavy metals.

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

[0006] In order to solve the shortcomings of existing technologies and realize the 2+ For the purpose of efficient removal and real-time monitoring, the present invention adopts the following technical solutions:

[0007] The method for remediating soil heavy metal pollution based on bamboo nanomaterials includes the following steps:

[0008] To prepare bamboo nanomaterials, bamboo is cut into sheets along the fiber axis and then ultrasonically cleaned and vacuum dried. The combination of precise cutting, ultrasonic impurity removal, and step-by-step vacuum drying can eliminate the risk of fiber damage and improve the efficiency of subsequent nano-processing. The intermediate product is then subjected to energy gradient grinding. The energy gradient grinding strategy can optimize the mechanical action mode in stages, thereby improving the fiber dissociation efficiency. High-pressure homogenization and in-situ dispersion nano-processing are then performed to obtain a solid porous bamboo nanomaterial with a high specific surface area and rich functional groups.

[0009] Microbial culture: Based on the microbial heavy metal resistance gene, which encodes a heavy metal efflux protein, an expression vector is selected for the microorganism. The target heavy metal resistance gene is inserted into the expression vector with a strong promoter and terminator downstream. The expression vector is introduced into the microorganism using electroporation, and the expression level of the target gene is detected to achieve efficient transformation and quantitative verification, ensuring stable overexpression of the target gene. The microorganisms overexpressing the target gene are inoculated into culture media containing different concentrations of heavy metals. Based on the microbial growth and heavy metal removal rate, microorganisms with high heavy metal tolerance and biotransformation ability are obtained;

[0010] Preparation of composite repair agent: using dispersing aids to evenly distribute microorganisms, using cross-linking agents 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 are able to adsorb microorganisms through the functional groups, and the high specific surface area of ​​the bamboo nanomaterials can increase the coverage area of ​​microorganisms.

[0011] 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 of 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);

[0012] The prepared composite remediation agent was applied to contaminated soil to repair heavy metal pollution. The bamboo nanomaterials were also able to quickly adsorb free heavy metal ions, reducing extracellular toxic concentrations. Microorganisms with overexpressed target genes were able to excrete intracellular heavy metals to the surface of the material, forming a cycle. This achieved a dual-path synergistic detoxification of microorganisms and bamboo nanomaterials based on adsorption and biotransformation.

[0013] For the overexpression of CzcD Rhodococcus erythropolis + 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-biotransformation" 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 remediation agent had an adsorption rate of 160 mg / L Cd 2+ The 72-hour removal rate of contaminated soil can reach 90% (synergistic enhancement coefficient 1.38), which is significantly better than traditional microbial remediation methods.

[0014] Furthermore, in the preparation of the bamboo nanomaterial, axial cutting is to cut the bamboo material into sheet samples along the fiber axis into 5mm×5mm×1mm pieces. This size design fully considers the microstructural characteristics of the bamboo fiber bundle and can improve the uniformity of subsequent grinding force.

[0015] The ultrasonic cleaning uses 40 kHz ultrasonic waves to clean and effectively remove the siliceous layer and phytolith impurities on the surface of the bamboo by using the ultrasonic cavitation effect;

[0016] Bamboo with a moisture content of less than or equal to 8% is cut along the fiber axis, cleaned, and dried under a vacuum of -0.08MPa using a step-by-step heating program (40°C for 1 hour → 50°C for 1 hour → constant temperature of 60°C) until the mass change rate is less than 0.1% / h. The moisture content is finally controlled within 2.5±0.3%. The step-by-step 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.

[0017] Furthermore, in the preparation of the bamboo nanomaterial, energy gradient grinding includes two stages. The first stage is coarse grinding. The second stage is to mix the coarse ground product with anhydrous ethanol at a certain solid-liquid ratio (1:10 g / mL), and then wet grind it at ≤40°C for 6 hours under the temperature control of a circulating water cooling system (ethylene glycol coolant temperature 5°C). Through real-time monitoring by an online temperature sensor (PT100 type) and a vibration accelerometer, the cooling flow is dynamically adjusted to make the tank temperature ≤40°C, and finally an intermediate product with a median particle size D50 (the diameter value on the abscissa corresponding to 50% of the cumulative distribution of the ordinate in the cumulative distribution diagram) of 1.2±0.3μm is obtained. Through energy gradient design (coarse grinding impact crushing 80J / g → wet grinding shear peeling 150J / g), the energy gradient grinding strategy can optimize the mechanical action mode in stages, thereby increasing the fiber dissociation efficiency by 40%, while limiting the grinding temperature rise to within 15°C. Thermogravimetric analysis verified that the thermal decomposition temperature of cellulose was maintained above 320°C.

[0018] Furthermore, in the preparation of the bamboo nanomaterial, the nano-processing 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, add 0.1wt% sodium carboxymethyl cellulose as a dispersant to the homogenate and stir to form a stable suspension with an electric potential Zeta ≤ -30mV, and after freeze-drying and dehydration, obtain a solid porous bamboo nanomaterial with a moisture content ≤ 0.5%. High-pressure homogenization is assisted by sodium carboxymethyl cellulose CMC to simultaneously achieve nano-processing and dispersion stability.

[0019] Furthermore, in the microbial culture, the microorganism is Rhodococcus erythropolis, the heavy metal is cadmium Cd, and the CzcD gene of Rhodococcus erythropolis encodes cadmium ions Cd 2+ For efflux protein, the pTip expression vector of Rhodococcus erythropolis was selected, and the target gene was inserted into the expression vector with a strong promoter and terminator downstream; the expression vector was introduced into Rhodococcus erythropolis using electroporation, and the expression level of the target gene was verified by qPCR detection, thereby achieving efficient transformation and quantitative verification, ensuring stable overexpression of the CzcD gene.

[0020] Furthermore, in the preparation of the composite repair agent, bamboo nanomaterials were combined with Rhodococcus erythropolis at a mass ratio of 1:0.3. 0.05% Tween-80 was used as a dispersant, and stirring was performed at 400 rpm for 4 hours. This allowed the hydroxyl groups on the bamboo nanomaterial's surface to physically adsorb to the peptidoglycan in the Rhodococcus erythropolis cell wall through hydrogen bonding and electrostatic interactions. The combination of Tween-80 and precise stirring (400 rpm for 4 hours) increased bacterial coverage from 50% to 83%, boosting repair efficiency by over 35%. Furthermore, the high-purity composite system, achieved through autoclaving and Tween-80 dispersion, avoided the problem of low colonization of the target strain due to competition with endogenous microorganisms.

[0021] Furthermore, in the preparation of the composite repair agent, the bamboo nanomaterials were sterilized under high pressure to avoid biological competition during subsequent microbial compounding, and then dispersed in phosphate buffered saline (PBS, pH = 7.4) to prepare a 5 mg / mL suspension. The microorganisms were inoculated into LB liquid culture medium (containing 1% glycerol) and cultured in a shaking incubator at 30°C and 180 rpm until the logarithmic growth phase (OD600 = 0.8). The microorganisms were collected by centrifuge (5000 rpm, 4°C, 10 minutes) and washed three times with sterile PBS to finally prepare a microbial suspension (concentration 1×10 8 CFU / mL).

[0022] Furthermore, in the preparation of the composite repair agent, 0.5wt% glutaraldehyde solution was added as a cross-linking agent to the mixed solution of bamboo nanomaterials and microorganisms, and the mixture was allowed to stand at 4°C for 12 hours for cross-linking, resulting in a composite electrokinetic potential Zeta of -18±2mV, and the surface potential of the composite was measured to increase from -32 mV to -18 mV, thereby increasing the microbial survival rate from 60% to 90%.

[0023] The repair system for soil heavy metal pollution based on bamboo nanomaterials includes a repair agent generating device and a repair agent applying device. The repair agent generating device includes a bamboo nanomaterial preparation unit, a microorganism cultivation unit, and a bamboo nanomaterial and microorganism composite unit. Combined with the repair agent applying device, the bamboo nanomaterial preparation, microorganism cultivation, composite repair agent preparation and repair agent application of the repair method for soil heavy metal pollution based on bamboo nanomaterials are performed in sequence.

[0024] Furthermore, the system also includes a heavy metal monitoring device, which includes a sensor, a cloud platform and a control terminal;

[0025] The sensor collects the heavy metal concentration of the soil in real time;

[0026] The cloud platform optimizes the remediation strategy based on the collected heavy metal concentrations through machine learning and generates the amount of remediation agent that needs to be increased;

[0027] The repair agent application device uses layered mixing to apply the repair agent, which increases the contact area between the repair agent and the soil while achieving uniform distribution of the repair agent in the soil profile. The repair agent application device uses a rotary tiller at 200rpm to perform layered mixing to a soil depth of 30cm, and the amount of the repair agent used is 0.5-1.5% w / w.

[0028] The advantages and beneficial effects of the present invention are:

[0029] The present invention prepares bamboo nanomaterials and cultivates Rhodococcus erythropolis to form a composite intelligent repair agent. The adsorption effect of bamboo nanomaterials and the biotransformation effect of Rhodococcus erythropolis are combined with intelligent sensor technology to achieve the removal of Cd in soil. 2+ The present invention has the advantages of high remediation efficiency, low cost, and environmental friendliness, and is suitable for large-scale soil heavy metal pollution remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a flow chart of a method in an embodiment of the present invention.

[0031] Figure 2 3 is a microstructure diagram of a bamboo fiber bundle in an embodiment of the present invention.

[0032] Figure 3This is a diagram showing the effect of bamboo nano-processing in an embodiment of the present invention (D90 < 100 nm).

[0033] Figure 4 Schematic diagram of the synergistic mechanism between bamboo nanomaterials and Rhodococcus erythropolis in an embodiment of the present invention.

[0034] Figure 5 It is an architecture diagram of the system in an embodiment of the present invention.

[0035] Figure 6 is Cd in the embodiment of the present invention 2+ Curve diagram of the effect of concentration on the growth of overexpressing bacteria.

[0036] Figure 7 3 is a graph showing the removal rate of heavy metal Cd over time in an embodiment of the present invention. DETAILED DESCRIPTION

[0037] The following describes the specific embodiments of the present invention in detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0038] like Figure 1 As shown, the method for remediating soil heavy metal pollution based on bamboo nanomaterials includes the following steps:

[0039] Step 1: Preparation of bamboo nanomaterials: bamboo nanomaterials are prepared by mechanical grinding to ensure they have high specific surface area and rich functional groups;

[0040] Bamboo nanomaterials were prepared by mechanical grinding. First, a CNC precision cutting machine was used to cut bamboo with a moisture content of ≤8% into 5mm×5mm×1mm sheet samples along the fiber axis. This size design fully considered the microstructural characteristics of bamboo fiber bundles, such as Figure 2 As shown in the figure (fiber length 1-3mm, diameter 20-50μm), the directional size design matches the microstructure of bamboo fiber, which can improve the uniformity of subsequent grinding force; the sample was then immersed in 5 times the volume of deionized water (resistivity ≥18.2MΩcm) for deep cleaning, and treated with a 40 kHz ultrasonic cleaner (power density 0.5W / cm²) for 30 minutes. The ultrasonic cavitation effect was used to effectively remove the siliceous layer and phytolith impurities on the surface of the bamboo; the cleaned sample was transferred to a vacuum drying oven and precisely dried using a step-by-step heating program (40℃ for 1 hour → 50℃ for 1 hour → constant temperature 60℃) under a vacuum degree of -0.08MPa until the mass change rate was <0.1% / h, and the moisture content was finally strictly controlled at 2.5±0.3%. The step-by-step 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.

[0041] In the existing technology, simple cutting into blocks (10-20mm) and natural drying (moisture content 5-8%) are usually adopted. The existing technology easily leaves a siliceous layer, resulting in a 30% decrease in grinding efficiency. The present invention, through the combination of 5mm×5mm×1mm precise cutting, ultrasonic impurity removal, and step vacuum drying, can eliminate the hidden danger of fiber damage and improve the subsequent nano-processing efficiency by more than 20%.

[0042] After pre-treatment, the bamboo enters the mechanical crushing stage and is ground in two stages using a QM-3SP2 planetary ball mill:

[0043] The first stage of coarse grinding was performed at a revolution speed of 300 rpm and a rotation speed of 200 rpm for 2 hours. The grinding jar was filled with 3 mm diameter zirconium oxide grinding balls (Mohs hardness 8.5, density 6.05 g / cm³) at a ball-to-material mass ratio of 20:1. Argon protective atmosphere was introduced to prevent cellulose oxidation. The ground product was graded through a 200 mesh standard sieve (pore size 75 μm) to ensure that the oversize material return rate was less than 5%.

[0044] In the second wet milling stage, the coarsely ground product was mixed with anhydrous ethanol at a solid-to-liquid ratio of 1:10 (g / mL). Milling was continued for 6 hours under a circulating water cooling system (ethylene glycol coolant temperature 5°C). The cooling flow rate was dynamically adjusted to maintain the tank temperature ≤40°C using an online temperature sensor (PT100) and a vibration accelerometer for real-time monitoring. The resulting intermediate product had a median particle size (D50) (the diameter corresponding to 50% of the cumulative distribution on the vertical axis in the cumulative distribution diagram) of 1.2±0.3μm. This energy gradient milling strategy, utilizing an energy gradient design (80 J / g impact crushing in coarse milling → 150 J / g shear exfoliation in wet milling), optimizes the mechanical action mode in stages, increasing fiber dissociation efficiency by 40% while limiting the milling temperature rise to less than 15°C. Thermogravimetric analysis confirmed that the thermal decomposition temperature of cellulose was maintained above 320°C.

[0045] In the existing technology, mechanical grinding usually uses a single ball mill, which has no energy gradient and large temperature fluctuations. The two-stage grinding (coarse 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 energy consumption by more than 25%.

[0046] Nano DeBEE high-pressure homogenizer was used in the nano-processing step. The intermediate product was processed through a diamond interaction cavity (pore size 100 μm) at a pressure of 150 MPa for 10 cycles. The particle size distribution was monitored by dynamic light scattering (DLS) after each cycle. Homogenization was terminated when D90 < 100 nm. Figure 3As shown (D90 represents the diameter on the abscissa corresponding to 90% of the cumulative distribution on the ordinate in the cumulative distribution graph). To enhance the dispersion stability of the product, 0.1 wt% sodium carboxymethyl cellulose (CMC) was added to the homogenized product as a dispersant. The product was stirred at 800 rpm for 30 minutes to form a stable suspension with a zeta potential (electrokinetic potential, i.e., the potential at the shear plane, which is used to characterize the stability of a colloidal dispersion) of -35 mV. The solid porous material obtained after freeze-drying (-50°C, 10 Pa) had a moisture content strictly controlled to ≤0.5%. This state allows for long-term storage of the end product, exhibiting a high specific surface area and a complete crystalline structure.

[0047] In the prior art, nanodispersion usually uses a single high-pressure homogenization, which is prone to agglomeration and requires subsequent separate dispersion treatment. D90 requires 15 homogenization cycles (>200nm). The present invention uses high-pressure homogenization combined with CMC in-situ dispersion (Zeta potential -35mV), and D90 can be achieved within 10 cycles <100nm. The introduction of CMC reduces the reagglomeration rate of nanofibers from 40% to below 5%, and extends the stability of the suspension by 10 times (7 days → 70 days without sedimentation). The moisture content is usually controlled by oven drying (moisture content 3-5%), while the present invention uses step vacuum drying (moisture content 2.5±0.3%).

[0048] Through the above improvements, the performance of the overall process chain of the present invention is doubled. Compared with the existing technology, the high specific surface area of ​​the final bamboo nanomaterial is increased from 120 m² / g to 180 m² / g (BET test), and Cd 2+ The adsorption capacity increased from 80 mg / g to 150 mg / g (laboratory data), and after being combined with Rhodococcus erythropolis, the remediation efficiency was enhanced. This achieved the precision, efficiency, and stability of the bamboo nanomaterial preparation process, providing an excellent carrier material for subsequent heavy metal remediation.

[0049] Step 2: Cultivation of Rhodococcus erythropolis: Isolate and screen Rhodococcus erythropolis from cadmium-contaminated soil, and expand the culture medium to obtain high-Cd 2+ tolerance and biotransformation ability of Rhodococcus erythropolis;

[0050] Rhodococcus erythropolis has CzcD gene, encoding Cd 2+ Efflux protein. Obtain the sequence of the CzcD gene from the NCBI database. Select the pTip Rhodococcus erythropolis expression vector and insert the target gene into the vector, with a strong promoter and terminator downstream. Use electroporation to introduce the expression vector into Rhodococcus erythropolis and verify the expression level of the target gene through qPCR. This achieves efficient transformation and quantitative verification, ensuring stable overexpression of the CzcD gene.

[0051] In the prior art, gene modification usually uses universal vectors (such as pET series), which have low transformation efficiency (<10³ CFU / μg DNA) and unstable gene expression due to vector compatibility issues. The present invention uses the pTip Rhodococcus erythropolis special vector combined with electroporation method (>10 5 CFU / μg DNA), can increase the transformation efficiency by 100 times and increase the CzcD protein expression to 3.2 times that of the wild type (verified by Western Blot), achieving efficient gene editing.

[0052] Rhodococcus erythropolis overexpressing the CzcD gene was inoculated into the culture medium containing different concentrations of Cd 2+ The growth curve and Cd 2+ Preparation of 0, 20, 40, 80, 160 mg / L containing different concentrations of Cd 2+ Liquid culture medium. Inoculate the overexpressed gene Rhodococcus erythropolis and culture for 24, 48, and 72 hours. Measure the optical density OD600 and Cd 2+ Concentration, calculate Cd 2+ Removal rate.

[0053] Overexpression strains at ≤80 mg / L Cd 2+ The plant could grow normally under the stress of 160 mg / L Cd (OD600>1.0 at 48 hours), indicating that its tolerance to Cd was significantly enhanced. 2+ At high concentrations of Cd, the strain can still survive and slowly proliferate (OD600 reaches 0.8 after 72 hours), while the wild-type strain is inhibited from growing at this concentration (the upper limit of tolerance of the wild-type strain is usually ≤50 mg / L Cd). 2+ ), therefore, the present invention increases the upper limit of strain tolerance to Cd from 80 mg / L to 160 mg / L, breaking through the strain's resistance to Cd 2+ Extreme tolerance.

[0054] On the other hand, Cd 2+ The removal rate is positively correlated with time. As the culture time increases, the removal rate increases significantly (the removal rate reaches 65%~90% in 72 hours). The strain of the present invention is still effective at high concentrations. Even at a high concentration of 160 mg / L, the removal rate still reaches 65% in 72 hours, indicating that the strain has strong remediation potential, thus breaking through the technical bottleneck of high-concentration pollution remediation.

[0055] The present invention combines the precision of genetic modification with the synergy of materials and organisms, and overexpresses the CzcD gene through genetic engineering technology to make the Cd 2+ The discharge capacity is increased by more than 3 times (see Example 1 for details), which solves the failure problem of traditional remediation technology in high-concentration heavy metal pollution scenarios.

[0056] Step 3: Compounding bamboo nanomaterials with Rhodococcus erythropolis to prepare a stable repair agent through physical adsorption and chemical cross-linking;

[0057] The core process of preparing the smart repair agent is to directional compound the bamboo nanomaterial obtained by mechanical grinding with Rhodococcus erythropolis. First, the bamboo nanomaterial is pretreated. The freeze-dried bamboo nanocellulose (water content ≤ 0.5%) is 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 nanomaterial and avoids biological competition during subsequent microbial compounding. It is then dispersed in phosphate buffered saline (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 bacteria are collected by centrifuge (5000 rpm, 4°C, 10 minutes) and washed three times with sterile PBS to prepare a bacterial suspension (concentration 1×10 8 CFU / mL).

[0058] In the existing technology, only surface disinfection (such as alcohol wiping) is usually used or microbial interference is ignored. However, the high-pressure sterilization (121°C, 20 minutes) of the present invention can completely inactivate endogenous microorganisms, avoid bacterial competition, and increase the colonization rate of Rhodococcus erythropolis by more than 40%.

[0059] The composite process was carried out in a biological safety cabinet. The bamboo nanomaterials were mixed with bacteria in a mass ratio of 1:0.3. 0.05% Tween-80 was added as a dispersing agent to help achieve uniform distribution of the bacteria. The mixture was reacted in a constant temperature magnetic stirrer at 25°C (400 rpm) for 4 hours. During this stage, the hydroxyl groups (-OH) on the surface of the bamboo nanomaterials and the peptidoglycan of the cell wall of Rhodococcus erythropolis were physically adsorbed through hydrogen bonds and electrostatic interactions. Observation under a laser confocal microscope (FITC-labeled bacteria) showed that the coverage rate of the bacteria on the bamboo fiber surface reached 83±5%.

[0060] Existing techniques typically employ simple mixing, which, due to the lack of a dispersant, can lead to uneven bacterial distribution. However, the present invention, through the assistance of Tween-80 and precise stirring parameters (400 rpm for 4 hours), can increase bacterial coverage from 50% to 83%, improving repair efficiency by over 35%. Furthermore, the high-purity composite system of autoclaving and Tween-80 dispersion avoids the problem of low colonization rates of target strains due to competition with endogenous microorganisms.

[0061] To enhance the stability of the composite system, 0.5 wt% glutaraldehyde solution (cross-linker) was added to the mixture and the mixture was allowed to cross-link at 4°C for 12 hours. The surface potential of the composite increased from -32 mV to -18 mV as measured by a Zeta potential meter. Low-temperature glutaraldehyde cross-linking balanced the stability of the composite with microbial activity, effectively reducing the microbial shedding rate (<5%).

[0062] In existing technologies, high-temperature crosslinking (>25°C) or sodium alginate embedding (low mechanical strength) are usually used. However, the present invention uses low-temperature (4°C) glutaraldehyde crosslinking and precise Zeta potential control, which can increase the survival rate of microorganisms from 60% to 90% and extend the stability of the complex by 3 times. In addition, the low-temperature crosslinking + bamboo fiber network structure can achieve a balance between activity and stability. The pore structure of bamboo nanofibers (pore size 1-5μm) provides physical protection for bacteria. Low-temperature crosslinking can reduce protein denaturation and maintain the function of the CzcD efflux pump, which is effective for the adsorption of Cd by bamboo materials alone. 2+ , 72 hours of 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);

[0063] The final product formed a porous network structure after freeze-drying. Scanning electron microscopy showed that Rhodococcus erythropolis was evenly embedded in the gaps between bamboo nanofibers. Real-time fluorescence quantitative PCR detection confirmed that the copy number retention rate of the bacterial 16S rRNA gene was >90%.

[0064] In the existing technology, only the morphology is observed (such as ordinary SEM), while the present invention combines SEM, fluorescent labeling, and qPCR for multi-dimensional verification. 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 bacterial load fluctuation of each batch of repair agent is less than 5%, which is far better than the existing technology (±20%).

[0065] like Figure 4 As shown, the present invention uses a synergistic optimization composite system of microbial activity protection and material structure design, based on the dual-path detoxification of overexpressed CzcD + bamboo nanomaterials, and bamboo nanomaterials quickly adsorb 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-biotransformation" dual-path synergy. Compared with the single bamboo nanomaterial for 160 mg / L Cd 2+The adsorption rate is 55%, and the removal rate of genetically engineered bacteria alone is 65%. The composite system repair agent has an adsorption rate of 160mg / L Cd 2+ The 72-hour removal rate of contaminated soil can reach 90% (synergistic enhancement coefficient 1.38), which is significantly better than traditional microbial remediation methods.

[0066] Step 4: Apply the prepared remediation agent to the contaminated soil to achieve the removal of Cd through the adsorption of bamboo nanomaterials and the biotransformation of Rhodococcus erythropolis. 2+ Efficient removal of

[0067] According to soil Cd 2+ The remediation agent dosage is determined by concentration and should be between 0.5% and 1.5% w / w. Apply the agent to the contaminated soil layer (0-30 cm) using a rotary tiller (200 rpm) by layering and mixing to ensure full soil contact. Irrigate and activate the soil (applying approximately 60% of field capacity).

[0068] Deployment of Cd in soil 2+ Concentration sensor, intelligent monitoring and control through intelligent sensors, real-time monitoring of Cd 2+ The closed-loop intelligent control system can realize dynamic and precise control of the amount of repair agent to automatically release Rhodococcus erythropolis and adsorb Cd. 2+ .

[0069] like Figure 5 As 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 and Rhodococcus erythropolis composite unit. Through the integrated remediation agent production system, continuous preparation from raw materials to finished agents is realized; the heavy metal monitoring device includes sensors, a cloud platform and a control terminal.

[0070] The bamboo nanomaterial preparation unit is used to obtain bamboo nanomaterials through nano-processing after mechanical grinding or chemical treatment of bamboo materials;

[0071] Rhodococcus erythropolis culture unit, used for high-density culture of Rhodococcus erythropolis overexpressing the CzcD gene;

[0072] Bamboo nanomaterial and Rhodococcus erythropolis composite unit, which combines bamboo nanomaterial and Rhodococcus erythropolis to prepare a stable repair agent;

[0073] Sensor for real-time collection of soil Cd 2+ concentration;

[0074] The cloud platform stores and analyzes the collected data. Based on the data-driven decision-making system, it optimizes the repair strategy through machine learning algorithms and generates automatic increases in the dosage of the repair agent.

[0075] Control terminals, generate adaptive strategies, and set alarm thresholds.

[0076] The rotary tiller adopts the layered mixing and application technology to achieve uniform distribution of the repair agent in the soil profile by optimizing the rotary tillage parameters.

[0077] In the existing technology, the application of remediation agents is usually done by broadcasting them on the soil surface or simply tilling (mixing depth ≤ 15 cm). The present invention combines a layered mixing method (0-30 cm) with optimized rotary tillage parameters (200 rpm), which can increase the contact area of ​​the remediation agent by 50% and the effective depth by 100%. Traditional monitoring and control require manual sampling and laboratory analysis (cycle > 7 days). The present invention combines in-situ sensors, real-time data transmission, and automatic control to shorten the response time from 7 days to < 1 hour, thereby improving the remediation efficiency by 40%. Existing devices have a low degree of integration and usually use decentralized equipment (materials, microbial agents, and mixing equipment are separated). The integrated remediation agent generation device of the present invention (preparation-cultivation-compound linkage) can reduce the overall cost by 35% and increase production efficiency by 3 times. Traditional decision-making usually requires empirical judgment or fixed remediation plans, while the present invention combines cloud platform data analysis with adaptive strategy generation, which is more efficient.

[0078] On the other hand, the synergistic mechanism of layered mixing and intelligent monitoring makes the remediation more accurate. Layered mixing ensures that the remediation agent covers all contaminated areas, and the real-time feedback from sensors guides the strengthening of remediation in key areas. The combination of integrated devices and cloud platforms improves operation and maintenance efficiency compared to multi-link manual operations. Dynamically regulating the collection of composite remediation agents can effectively reduce fluctuating pollution (such as Cd 2+ The long-term stability of the repair is maintained under the scenario of concentration changes. The effect of the traditional static repair solution decays by more than 50% (after 30 days). The present invention maintains a removal rate of more than 80% through dynamic adjustment (60-day test).

[0079] The present invention improves the extreme tolerance of Rhodococcus erythropolis and realizes long-term repair by combining intelligent regulation. 2+ In the pollution simulation experiment (concentration fluctuation 100-200 mg / L), the composite remediation agent was able to maintain stable remediation for 30 days (removal rate > 85%), while the traditional bacterial agent was inactivated on the 10th day.

[0080] The innovation of this invention lies in the combination of engineering application and intelligent management, which solves the three major pain points of traditional repair technology: low efficiency, high cost and poor adaptability.

[0081] The implementation of the heavy metal monitoring device is as follows:

[0082] 1. Sensor deployment:

[0083] 1. Sensor selection: Cd 2+ Ion selective electrode (ISE), performance parameters:

[0084] Detection range: 0.1-1000 mg / L Cd 2+ .

[0085] Response time: ≤30 seconds.

[0086] Working temperature: 0-50℃.

[0087] Lifespan: ≥6 months (under wild conditions).

[0088] 2. Distribution density:

[0089] One monitoring point is deployed for every 100 m² of restoration area to ensure spatial representativeness of the data.

[0090] For contaminated sites with high heterogeneity, the number of monitoring points can be increased to one per 50 m².

[0091] 3. Installation depth:

[0092] Surface sensor: buried at a depth of 10 cm, monitoring surface soil Cd 2+ Concentration changes.

[0093] Deep sensor: buried at a depth of 20 cm, monitoring Cd 2+ Downward migration situation.

[0094] 4. Installation method:

[0095] Use a soil borer (2 cm in diameter) to drill holes at designated locations to the target depth.

[0096] Insert the sensor probe vertically into the hole, ensuring full contact with the soil.

[0097] Backfill with native soil and compact it to avoid air gaps.

[0098] 2. Sensor calibration:

[0099] 1. Preparation of calibration solution:

[0100] 0.1, 1, 10, and 100 mg / L Cd was prepared using Cd(NO3)2 2+ Standard solution.

[0101] 2. Calibration steps:

[0102] Immerse the sensor in the standard solution in turn and record the potential response value (mV).

[0103] Draw the calibration curve (potential vs. Cd 2+ concentration), calculate the slope and intercept.

[0104] 3. Calibration frequency: once every 2 weeks to ensure data accuracy.

[0105] 3. Data Transmission and Cloud Platform

[0106] 1. Local hardware composition:

[0107] Data collector: Multi-channel data acquisition module, supports 16 sensor connections.

[0108] Power system: Solar panel (20 W) + lithium battery pack (12 V, 50 Ah) to ensure long-term power supply in the field.

[0109] Protective box: waterproof and dustproof box (IP67 grade), built-in temperature control module (0-40℃).

[0110] 2. Sampling frequency:

[0111] Data is collected every 10 minutes and stored in a local SD card (32 GB).

[0112] 3. Wireless transmission technology

[0113] 1) Technology selection:

[0114] LoRa: Suitable for long-distance (≤10 km), low-power transmission, and 50 kbps bandwidth.

[0115] NB-IoT: Suitable for urban areas, with a bandwidth of 250 kbps and supports high-density deployment.

[0116] 2) Transmission parameters:

[0117] Data packet size: ≤512 bytes / time.

[0118] Transmission frequency: upload data once an hour (adjustable according to demand).

[0119] 3) Network configuration:

[0120] Insert a LoRa or NB-IoT module (such as Semtech SX1276) into the data logger.

[0121] Configure the gateway address, transmission frequency, and encryption key.

[0122] 4. Cloud platform construction

[0123] 1) Platform selection:

[0124] Public cloud: such as Alibaba Cloud IoT platform and AWS IoT Core, supports large-scale data storage and analysis.

[0125] Private cloud: Built on open source platforms (such as ThingsBoard), suitable for scenarios with high data security requirements.

[0126] 2) Data storage:

[0127] Use a time series database (such as InfluxDB) to store sensor data and support efficient query and analysis.

[0128] Data retention time: ≥2 years.

[0129] 3) Visual interface:

[0130] Develop web and mobile applications to display CD in real time 2+ Concentration profiles and remediation progress.

[0131] Supports data export (CSV format) and report generation (PDF format).

[0132] 4) Data analysis and optimization:

[0133] The cloud platform pre-processes the collected data and selects a machine learning algorithm model for model training and verification, and replenishes the repair agent through the trained prediction model.

[0134] Data preprocessing includes removing outliers (such as outliers caused by sensor failure), data normalization (Cd 2+ concentrations are mapped to a 0-1 range).

[0135] Model selection is used to select regression models, classification models, etc. Regression models such as Random Forest Regression are used to predict Cd 2+ The concentration change trend and classification models such as support vector machine (SVM) are used to determine whether the remediation effect meets the standards.

[0136] Training and Validation: The model was trained using historical data (e.g., laboratory simulation data), and the model performance was evaluated through cross-validation (10-fold) (e.g., R² ≥ 0.9, RMSE ≤ 5 mg / kg).

[0137] Dynamically adjust the amount of remediation agent and collect soil Cd in real time 2+ Concentration data is uploaded to the cloud platform; the current Cd is calculated based on the latest N monitoring data 2+ Concentration decrease rate V_current; when V_current is lower than the preset target Cd 2+When the concentration decreases at a rate of V_target, the amount of repair agent added is automatically increased according to the product of the difference (V_target-V_current) and the adjustment coefficient k; the above steps are repeated periodically until Cd 2+ When the concentration of Cd 2+ When the concentration decrease rate is lower than expected, the amount of repair agent is automatically increased.

[0138] Example 1: Verification of Cd-tolerance of Genetically Engineered Bacteria

[0139] Rhodococcus erythropolis overexpressing the CzcD gene was inoculated into the culture medium containing 0, 20, 40, 80, and 160 mg / L Cd. 2+ The cells were cultured in liquid medium at 30°C and 180 rpm for 72 hours. Figure 6 、 Figure 7 As shown, the OD600 and Cd 2+ Concentration, the results show:

[0140] At 160 mg / L Cd 2+ Under stress, the 72-hour biomass of the genetically engineered bacteria (OD600=0.8) was significantly higher than that of the wild-type strain (OD600=0.1);

[0141] Genetically engineered bacteria to 160 mg / L Cd 2+ The 72-hour removal rate of the strain was 52%, while that of the wild-type strain was only 10%.

[0142] The above results showed that overexpression of CzcD gene significantly enhanced the Cd tolerance and Cd 2+ Repair efficiency.

[0143] Example 2: Intelligent Control Function

[0144] 1. Experimental design

[0145] Venue: A certain CD 2+ Contaminated farmland (area 1000 m², concentration 120 mg / kg).

[0146] Healing agent dosage: 1% w / w (initial dosage), dynamically adjusted based on sensor data.

[0147] Monitoring system:

[0148] Deploy 20 CDs 2+ Sensor (buried at a depth of 10 cm).

[0149] 2) Data is transmitted to the cloud platform via LoRa and updated every hour.

[0150] 2. Repair strategy:

[0151] Dynamic Adjustment:

[0152] If Cd 2+ If the concentration decrease rate is slower than expected (e.g., decrease <20% within 7 days), increase the amount of the repair agent by 0.2% w / w.

[0153] 2) If soil moisture is <40%, start the irrigation system (water usage ≈ 60% of field capacity).

[0154] Optimization results:

[0155]

[0156] 3. Conclusion

[0157] The intelligent control system significantly improves the remediation efficiency (72% removal rate in 60 days) by dynamically adjusting the amount of remediation agent and irrigation frequency.

[0158] Compared with traditional fixed-dose repair, it saves 20% of the amount of repair agent.

[0159] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. However, 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 remediating soil heavy metal pollution based on bamboo nanomaterials, characterized in that The steps include: Preparation of bamboo nanomaterials: bamboo is cut into sheets along the fiber axis, ultrasonically cleaned and vacuum dried, and then the intermediate product obtained after energy gradient grinding is subjected to high-pressure homogenization and in-situ dispersion nano-treatment to obtain porous bamboo nanomaterials; energy gradient grinding includes two stages: the first stage is coarse grinding, and the second stage is mixing the coarse ground product with anhydrous ethanol at a certain solid-liquid ratio, and wet grinding at a temperature of ≤40°C to finally obtain an intermediate product with a median particle size D50 of 1.2±0.3μm; nano-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 zeta potential Zeta ≤-30mV, and freeze-dry and dehydrate to obtain a solid porous bamboo nanomaterial with a moisture content of ≤0.5%; Microbial culture, wherein the microorganism is Rhodococcus erythropolis, the heavy metal is cadmium (Cd), and the CzcD gene of Rhodococcus erythropolis encodes a cadmium ion (Cd²⁺) efflux protein. The pTip expression vector of Rhodococcus erythropolis is selected, and the target gene is inserted into the expression vector so that the downstream has a strong promoter and terminator. The expression vector is introduced into Rhodococcus erythropolis by electroporation, and the expression level of the target gene is verified by qPCR detection. The microorganism overexpressing the target gene is inoculated into a culture medium containing different concentrations of heavy metals. Based on the microbial growth and heavy metal removal rate, microorganisms with high heavy metal tolerance and biotransformation ability are obtained; Preparation of the composite repair agent: using a dispersing agent to assist the uniform distribution of microorganisms, using a crosslinking agent to perform low-temperature static crosslinking, the microorganisms are adsorbed on the surface of the bamboo nanomaterial to obtain a composite repair agent of microorganisms and bamboo nanomaterials; the bamboo nanomaterials are sterilized under high pressure and then dispersed in phosphate buffer to prepare a suspension, the microorganisms are inoculated into a liquid culture medium and cultured to the logarithmic growth phase, the microorganisms are collected and sterilely washed to finally prepare a microbial suspension; the bamboo nanomaterials are composited with Rhodococcus erythropolis in a mass ratio of 1:0.3, using 0.05% Tween-80 as a dispersing agent, combined with stirring at 400 rpm for 4 hours, so that the hydroxyl groups on the surface of the bamboo nanomaterials and the peptidoglycan of the cell wall of Rhodococcus erythropolis are physically adsorbed through hydrogen bonds and electrostatic interactions; 0.5wt% glutaraldehyde solution is added as a crosslinking agent to the mixture of bamboo nanomaterials and microorganisms, and the mixture is statically crosslinked at 4°C for 12 hours, resulting in a composite with a zeta potential of -18±2mV; The prepared composite remediation agent is applied to contaminated soil to repair heavy metal pollution.

2. The method for remediating soil heavy metal pollution based on bamboo nanomaterials according to claim 1, characterized in that: In the preparation of the bamboo nanomaterial, axial cutting is to cut the bamboo material into sheet samples of 5mm×5mm×1mm along the fiber axis; The ultrasonic cleaning is performed using 40 kHz ultrasonic waves; Bamboo with a moisture content of less than or equal to 8% was cut along the fiber axis, cleaned, and dried using a step-by-step temperature increase program under a vacuum degree of -0.08 MPa until the mass change rate was less than 0.1% / h, and the moisture content was finally controlled within 2.5±0.3%.

3. A soil heavy metal contamination remediation system based on bamboo nanomaterials, comprising a remediation agent generating device and a remediation agent applying device, characterized in that: The remediation agent generating device includes a bamboo nanomaterial preparation unit, a microorganism cultivation unit, and a bamboo nanomaterial and microorganism composite unit. Combined with the remediation agent applying device, the bamboo nanomaterial preparation, microorganism cultivation, composite remediation agent preparation, and remediation agent application of the soil heavy metal pollution remediation method based on bamboo nanomaterials according to claim 1 are sequentially performed.

4. The bamboo nanomaterial-based soil heavy metal contamination remediation system according to claim 3, characterized in that: The system also includes a heavy metal monitoring device, which includes a sensor, a cloud platform and a control terminal; The sensor collects the heavy metal concentration of the soil in real time; The cloud platform optimizes the remediation strategy based on the collected heavy metal concentrations and generates the amount of remediation agent that needs to be increased; The repair agent applying device applies the repair agent by layered mixing.

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