Soil remediation agent and preparation method thereof

Abstract

The invention provides a novel soil remediation agent. The novel soil remediation agent is prepared from extracellular polymeric substances of special strains and ferrihydrite loaded with sodium carboxymethyl cellulose. The soil remediation agent not only can improve the adsorption and fixation efficiency of arsenic, but also has the advantages of remediation of arsenic-polluted soil, improvement of soil enzyme activity and reduction of bioavailability of arsenic in the soil, is environment-friendly, keeps soil fertility and the like, and is an efficient and stable soil biological-chemical in-situ remediation method for the arsenic-polluted soil.

Description

Technical Field

[0001] This invention relates to the field of soil remediation, specifically to a composite biochemical material soil remediation agent derived from microorganisms and its preparation method. Technical Background

[0002] Arsenic (As) is a highly toxic metalloid that can enter terrestrial ecosystems through atmospheric deposition, wastewater discharge, and pesticide application. Rice readily accumulates As and is a major source of As intake in the human diet. The spatial distribution of As concentration in agricultural soils is directly proportional to the risk of cancer. As migration is related to the co-precipitation and release of iron oxides. When iron oxides undergo reductive dissolution, arsenic is released from the co-precipitate surface. Furthermore, As(III) has a much lower binding capacity to iron oxides than As(V). When As(V) is reduced to As(III) from co-precipitates of iron minerals, As is released into the environment. Iron oxides can effectively reduce the bioavailability of soil arsenic as passivating agents, but large-scale application as a single agent can easily lead to a decline in soil microbial diversity and enzyme activity, resulting in degradation of soil biological function. Therefore, reducing the bioavailability of soil arsenic while maintaining the sustainable development of soil biological function is one of the main research areas urgently needed for soil passivating agents. Summary of the Invention

[0003] In view of this, the present invention provides a novel soil remediation agent, prepared by using extracellular polymers of special microbial strains and ferrous sulfate supported on sodium carboxymethyl cellulose. This soil remediation agent not only improves the adsorption and fixation efficiency of arsenic, but also remediates arsenic-contaminated soil, enhances soil enzyme activity, reduces the bioavailability of arsenic in the soil, and has advantages such as being environmentally friendly and maintaining soil fertility. It is a highly efficient and stable method for in-situ biochemical remediation of arsenic-contaminated soil.

[0004] In a first aspect, the present invention provides a soil remediation agent, comprising:

[0005] (A) Extracellular polymeric material of *Pseudomonas putida* YT-1, wherein the bacteria are deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 22872; and,

[0006] (B) Carboxymethyl cellulose-coated ferrite.

[0007] The *Pseudomonas putida* YT-1 used in this invention possesses extracellular polymers containing special proteins and polysaccharides that, when combined with surface-treated ferrous oxide, exhibit superior arsenic adsorption and fixation effects. Furthermore, compared to directly using bacteria, the use of extracellular polymers offers greater versatility and higher adsorption efficiency, enabling its application in harsher environments and rapid soil remediation.

[0008] Preferably, in the soil remediation agent, the mass ratio of component (A) to component (B) is (1-10):1, more preferably (2-6):1.

[0009] In some specific implementation methods, the mass ratio of component (A) to component (B) in the soil remediation agent can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, etc.

[0010] In a second aspect, the present invention provides a method for using the soil remediation agent described in any of the foregoing claims, comprising the following steps:

[0011] S1: Prepare a bacterial suspension of Pseudomonas putida YT-1, wherein the bacterial suspension contains wet bacterial cells of the strain and physiological saline;

[0012] S2: After heating the bacterial suspension prepared in step (S1), centrifuge to obtain the supernatant to obtain a crude extract of EPS. After dialysis of the crude extract of EPS, dry it to obtain EPS solid powder.

[0013] S3: Sodium carboxymethyl cellulose and ferric nitrate are mixed to obtain carboxymethyl cellulose-coated ferrous ore;

[0014] S4: Mix the products from steps S2 and S3 to obtain a soil remediation agent.

[0015] In some specific embodiments, the method includes the following steps:

[0016] Culture and preparation of bacterial suspension of S1 Pseudomonas putida

[0017] Inoculate *P. putida* YT-1 strain into LB medium and culture for 12-96 h. Then, inoculate onto LB solid agar plates and incubate at 20-40℃ for 12-96 h. After incubation, scrape off a portion of the strain and vortex in sterile 0.9% NaCl (w / v) solution. Wash and centrifuge the bacterial cells 2-3 times, collect the wet bacterial cells (wet cell weight: 0.9% NaCl (w / v) = 1:9-1:10), yielding approximately 10 [units of culture medium]. 6 10 11 CFU / mL bacterial suspension, store for later use.

[0018] Preparation of S2 bacterial extracellular extract P-EPS

[0019] The bacterial suspension obtained in the previous step was heated in a water bath at 40-80℃ for 0.2-1 h. After freezing and centrifugation, the supernatant was collected, which is the crude extract of EPS. The crude EPS extract was filtered, and the filtrate was dialyzed in a dialysis membrane for 12-48 h. After dialysis, the filtrate was frozen at -10℃ or below, and then freeze-dried until it became a powdery solid. This solid powder is the purified EPS solid of P. putida YT-1 strain.

[0020] Preparation of S3 carboxymethyl cellulose-coated ferrohydrate CMC-HFO

[0021] Weigh 1-5g of sodium carboxymethyl cellulose (CMC) and dissolve it in 100-2000mL of deionized water. Simultaneously, weigh 10-100g of Fe(NO3)3·9H2O and completely dissolve it in a certain amount of deionized water. Mix the two solutions thoroughly, and add KOH solution dropwise to adjust the pH to 7-8. Make up the volume to 1000mL. Place the mixture into a dialysis bag with a capacity cutoff of 2000-5000KD and dialyze for 12-48 hours to obtain a suspension of 1-10g / L CMC-HFO (based on Fe).

[0022] Preparation of S4 P-EPS-CMC-HFO Soil Remediation Agent

[0023] EPS and CMC-HFO purified from P. putida YT-1 strain were mixed and brought to a final volume of 50 mL with 0.1 mol / L NaCl solution. The concentration of EPS in the reaction system was 1-10 g / L, and the concentration of CMC-HFO was 1 g / L (based on Fe). The mixture was placed on a shaker (20-40℃, 50-300 r / min) and shaken for 1-5 h to reach adsorption equilibrium.

[0024] Thirdly, the present invention provides the application of the soil remediation agent described in any of the foregoing claims in the remediation of arsenic-contaminated soil.

[0025] Preferably, the arsenic-contaminated soil is paddy soil.

[0026] Fourthly, the present invention also provides a heavy metal treatment agent comprising a composition containing any one of the foregoing components (A) and (B). That is, the soil remediation agent of any one of the foregoing claims of the present invention can also be used as a heavy metal treatment agent.

[0027] Preferably, the heavy metal treatment agent can be used to prepare heavy metal waste treatment agents, sewage treatment agents, or air treatment agents.

[0028] Preferably, the heavy metal treatment agent can be used to purify or remediate food, cosmetic, or industrial waste.

[0029] The product of this invention is particularly suitable as a soil remediation agent, capable of not only remediating arsenic-contaminated soil but also reducing bioavailability, maintaining soil fertility, and increasing soil enzyme activity. Its mechanism lies in two aspects: First, the EPS secreted by *P. putida* YT-1 contains special proteins and polysaccharides that promote chelation reactions between Fe and numerous hydroxyl groups, facilitating secondary mineral nucleation and increasing the efficiency of Fe-As co-precipitation in the soil. Experiments have shown that the EPS secreted by *P. putida* YT-1 is superior to that of other bacterial species. Second, the carboxymethyl cellulose-coated ferrous sulfate reaches the micron level, possessing a large specific surface area and good stability. Its combination with bacterial extracellular polymers promotes the reaction of highly active sites on the iron surface. Experiments have found that the combined use of *P. putida* YT-1's EPS and carboxymethyl cellulose yields better results than other known ferrous sulfate coating agents.

[0030] Based on this, the EPS-iron oxide complex adsorbs As and forms Fe-As insoluble precipitates / secondary minerals through bio-electron transfer, improving the As fixation efficiency in contaminated soil. The EPS secreted by *P. putida* YT-1 directly acts on As-contaminated soil for remediation, without considering microbial colonization, and increases soil particle strength, mitigating soil environmental changes such as the damage to soil structure caused by alternating wet and dry conditions. It can serve as a novel bioremediation fertilizer to enhance fertility during soil remediation. Attached Figure Description

[0031] Figure 1 SEM images of soil remediation agents in the embodiments and comparative examples of the present invention: (a) P-EPS-CMC-HFO; (b) P-EPS-CMC-Fe(0); (c) P-EPS-PVP-HFO; (d) P-EPS-PVP-Fe(0).

[0032] Figure 2 Fluorescence staining characterization results of P-EPS-CMC-HFO soil remediation agent in Example 1: (A) morphology under bright field conditions; (B) protein components in P-EPS; (C) polysaccharide components in P-EPS.

[0033] Figure 3 The changes in nucleic acid, protein and polysaccharide content in the reaction solution after reacting P-EPS-CMC-HFO soil remediation agent with different concentrations of arsenic in Example 1.

[0034] Figure 4 Example 1 and Comparative Example 1: Schematic diagram showing the comparison of arsenic adsorption efficiency between two soil remediation agents.

[0035] Figure 5 Infrared spectra of soil remediation agents and arsenic in Comparative Example 1(a) and Example 1(b).

[0036] Figure 6 Schematic diagram of the adsorption efficiency of soil remediation agents for arsenic in Examples 1 and Comparative Examples 2-4.

[0037] Figure 7 Schematic diagram showing the changes in total Fe and Fe(II) concentrations in the supernatant after the reaction of the soil remediation agent and arsenic in Example 1.

[0038] Figure 8 SEM images of the soil remediation agent and arsenic before and after the reaction in Example 1: (a) before reaction; (b) after reaction.

[0039] Figure 9 X-ray electron power spectrum of the soil remediation agent and arsenic before and after the reaction in Example 1.

[0040] Figure 10 Effects of the soil remediation agent, control group, and blank group on soil enzyme activity in arsenic-contaminated paddy soil in Example 1: urease activity (a), phosphatase activity (b), and catalase activity (c).

[0041] Figure 11 Schematic diagram of the bioavailability of soil after remediation with the soil remediation agent in Example 1, and the control and blank groups. Detailed Implementation

[0042] In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described in order to avoid obscuring the invention.

[0043] Unless otherwise specified, % in this invention refers to the mass percentage (wt%).

[0044] The present invention will be described in detail below through specific embodiments.

[0045] raw material:

[0046] Pseudomonas putida YT-1 (P. putida YT-1): Accession number CGMCC: No. 22872, see Chinese patent application CN202210143484.X.

[0047] Shewanella oneidensis MR-1 (S. oneidensis MR-1): Deposited at the United States Center for Standard Microbial Culture Collection, accession number ATCC No. 700550, see Chinese patent application CN202210143484.X.

[0048] Carboxymethyl cellulose: purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0049] Polyvinylpyrrolidone (PVP): Wuxi Yatai United Chemical Co., Ltd.

[0050] All other raw materials and reagents were of analytical grade and were used directly.

[0051] instrument:

[0052] Laser confocal microscope: Model, Leica BX53.

[0053] Inductively Coupled Plasma Emission Spectrometer: Model: Agilent 5510.

[0054] Fourier transform infrared spectrometer: Model, Nicoleti S5.

[0055] X-ray photoelectron spectrometer, model: Thermo Fisher Nexsa.

[0056] Example 1

[0057] Preparation of soil remediation agents

[0058] 1.1 Culture and preparation of bacterial suspension of *Pseudomonas putida*

[0059] P. putida YT-1 strain was inoculated into LB medium and cultured at 180 rpm for 24 h. The activated strain was then streaked onto LB solid agar plates and incubated at 30°C for 48 h. After incubation, strains were scraped off using a sterile spatula and vortexed in sterile 0.9% NaCl (w / v) solution. The cells were washed and centrifuged 2–3 times. The wet cells were collected, with a wet cell weight to 0.9% NaCl (w / v) ratio of 1:9–1:10, yielding approximately 10 [units of culture medium]. 7 CFU / mL bacterial suspension, stored at 4°C for later use.

[0060] 1.2 Preparation of bacterial extracellular extract P-EPS

[0061] The *P. putida* YT-1 bacterial suspension from step 1.1 was heated in a 60°C water bath for 30 min, then centrifuged at 4°C and 5000 rpm for 15 min using a refrigerated centrifuge. The supernatant was collected, which is the crude extract of EPS. The crude EPS extract was filtered through a 0.22 μm aqueous filter membrane, and the filtrate was dialyzed in a dialysis membrane for 24 h. After dialysis, the filtrate was frozen at -20°C for 12 h, and then freeze-dried until it became a powdery solid. This solid powder is the purified EPS from *P. putida* YT-1 strain.

[0062] 1.3 Preparation of carboxymethyl cellulose-coated ferrohydrate (CMC-HFO)

[0063] Weigh 3.2 g of sodium carboxymethyl cellulose (CMC) and dissolve it in 500 mL of deionized water. Simultaneously, weigh 36.0714 g of Fe(NO3)3·9H2O and completely dissolve it in a certain amount of deionized water. Mix the two solutions thoroughly, and add 0.1 mol / L KOH solution dropwise to adjust the pH to 7-8. Make up the volume to 1000 mL. Place the mixture into a dialysis bag with a capacity cutoff of 3500 KD and dialyze for 24 hours to obtain a suspension of 5 g / L CMC-HFO (based on Fe).

[0064] 1.4 Preparation of P-EPS-CMC-HFO Soil Remediation Agent

[0065] The purified EPS and CMC-HFO from *P. putida* YT-1 strain were mixed and brought to a final volume of 50 mL with 0.1 mol / L NaCl solution. The concentration of P-EPS in the reaction system was 4 g / L, and the concentration of CMC-HFO was 1 g / L (based on Fe). The mixture was placed on a shaker (30℃, 180 r / min) and shaken for 2 h to reach adsorption equilibrium.

[0066] Comparative Example 1: Preparation of Shewanella Soil Remediation Agent

[0067] 1.1 Prepare a bacterial suspension of Shewanella oneidensis MR-1 strain as per step 1.1 of Example 1.

[0068] 1.2 Extract the extracellular polymeric substance S-EPS of Shewanella oneidensis MR-1 strain as in step 1.2 of Example 1, except that the water bath temperature was changed to 40°C.

[0069] 1.3 The S-EPS-CMC-HFO soil remediation agent was prepared according to steps 1.3 to 1.4 of Example 1.

[0070] Preparation of Comparative Example 2P-EPS-CMC-Fe(0) Soil Remediation Agent

[0071] The steps are basically the same as in Example 1, except that step 1.3 is changed to:

[0072] Nitrogen was passed through deionized water for 30 minutes to prepare deoxygenated water, which was then sealed and stored for later use. 8.34 g of FeSO4·7H2O and 0.0667 g of CMC were weighed and dissolved in 100 ml of deoxygenated water, followed by the addition of 100 ml of anhydrous ethanol. Under ultrasonic oscillation, 0.3 mol / L NaBH4 was added dropwise until the reaction ceased. Solid-liquid separation was performed using magnetic separation. A magnet was placed at the bottom of the reaction flask to collect the black precipitate, which was then washed three times with deoxygenated water and freeze-dried under vacuum. The freeze-dried black precipitate was ground through a 100-mesh nylon sieve in an anaerobic glove box to obtain CMC-Fe(0).

[0073] Preparation of Comparative 3P-EPS-PVP-HFO Soil Remediation Agent

[0074] The steps are the same as in Example 1, except that step 1.3 is changed to:

[0075] Weigh 2.5g PVP and 18.0357g Fe(NO3)3·9H2O and dissolve them in 400mL of water by sonication. After complete dissolution, adjust the pH to 7-8 with KOH and then make up the volume to 500mL. The resulting solution is a 5g / L (calculated as Fe) PVP-HFO suspension.

[0076] Preparation of Comparative Example 4P-EPS-PVP-Fe(0) Soil Remediation Agent

[0077] The steps are the same as in Example 1, except that step 1.3 is changed to:

[0078] Weigh 0.05g PVP and 13.5g FeCl3·6H2O and add them to 50ml of deoxygenated water, then sonicate to dissolve. Dissolve a certain amount of NaBH4 in the deoxygenated water and add 1mol / L NaCl solution dropwise to prepare a 1.6mol / L NaBH4 solution. Add 1.6mol / L NaBH4 dropwise to the above mixture until the color of the mixture gradually changes from orange to light green and then to black. Use magnetic separation to separate the solid and liquid to obtain a black precipitate, wash it three times with deoxygenated water, and then freeze-dry it under vacuum. After the freeze-drying, grind it through a 100-mesh nylon sieve in an anaerobic glove box to obtain PVP-Fe(0).

[0079] Example 2

[0080] Fluorescent staining characterization of P-EPS-CMC-HFO soil remediation agent

[0081] A buffer solution was prepared using 61 mL of 0.2 mol / L K₂HPO₄ and 39 mL of KH₂PO₄. After adding 0.1 mol / L NaHCO₃ and FITC staining solution, the mixture was reacted on a shaker in the dark for 1 hour. The mixture was then centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. The prepared buffer solution was added, and the mixture was washed thoroughly. The mixture was centrifuged again, and the supernatant was collected. Then, NaHCO₃ and ConA staining solution were added, and the mixture was reacted on a shaker in the dark for 30 minutes. This washing process was repeated. The fluorescence microscope was preheated for 30 minutes, and the product from Example 1 was added to a dropper slide for observation under the fluorescence microscope. The desired image was found using a bright-field microscope. The light source was turned off, and different fluorescence methods were used: blue light for DNA, green light for protein, and red light for polysaccharides. The results are as follows: Figure 2 As shown, after the equilibrium complexation reaction of P-EPS and CMC-HFO, EPS protein and polysaccharide exhibit different distribution characteristics on the surface of ferroalloy. Figure 2 A shows the morphology of the P-EPS-CMC-HFO complex observed under bright field conditions. Figure 2 The green fluorescence staining results in B represent the protein components of P-EPS. Figure 2 The red fluorescence staining result in C represents the polysaccharide component. It can be seen that the main active components of P-EPS, proteins and polysaccharides, after being balanced and complexed with CMC-HFO, are uniformly attached to the surface of the mineral, forming a P-EPS-CMC-HFO complex.

[0082] Example 3

[0083] Characterization of key components of P-EPS-CMC-HFO soil remediation agent

[0084] Weigh 1.04 g of HAsNa2O4·7H2O and dissolve it in a beaker. Transfer the solution to a 250 mL volumetric flask to prepare an As solution with a concentration of 1000 mg / L. Pipette 0 mL, 0.25 mL, 0.625 mL, 1.25 mL, 2.5 mL, and 5 mL into 50 mL centrifuge tubes, respectively, and add P-EPS-CMC-HFO soil remediation agent. Make up to 25 mL, resulting in As concentrations of 0 mg / L, 10 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L. The Fe concentration in this reaction system is 0.1 g / L. Place each reaction system in an air bath constant temperature shaking incubator at 30℃ and 180 rpm for 72 h. After the reaction, aspirate the reaction solution and use it to determine the protein, polysaccharide, and nucleic acid content in the solution.

[0085] Protein content was determined using the Coomassie Brilliant Blue method. Standard bovine serum albumin (BSA) solution was prepared as a 4 mg / mL standard protein solution and serially diluted to 2000, 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μg / mL. 20 μL of the BSA standard solution was mixed with 1980 μL of Coomassie Brilliant Blue solution in a 3 mL centrifuge tube, and the absorbance was measured at 595 nm. A standard curve was obtained by plotting protein concentration on the x-axis and absorbance on the y-axis. The above steps were repeated with 20 μL of the reaction solution, and the protein content in the P-EPS-CMC-HFO soil remediation agent after reaction with different concentrations of As was determined based on the calibrated lines.

[0086] The polysaccharide content was determined using the phenol-sulfuric acid method. 0.01 g of glucose was weighed to prepare a 100 mg / L glucose standard solution. 0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, and 1.0 mL of the glucose standard solution were respectively transferred to 10 mL colorimetric tubes. First, 1 mL of deionized water was added, followed by 1 mL of 5% phenol, and the mixture was shaken well. Then, 5 mL of concentrated sulfuric acid was added and the volume was adjusted to 10 mL. The tubes were then incubated in a 96℃ water bath for 20 min in the dark, cooled to room temperature, and the absorbance was measured at 490 nm. A standard curve was obtained by plotting polysaccharide concentration on the x-axis and absorbance on the y-axis. The above steps were repeated with 0.1 mL of the reaction solution, and the polysaccharide content in the P-EPS-CMC-HFO soil remediation agent after reaction with different concentrations of As was determined based on the calibrated lines.

[0087] DNA content determination was performed using the diphenylamine method. First, calf thymus DNA was dissolved in a small amount of 0.1 mol / L NaOH solution and then diluted with deionized water to prepare a 200 μg / mL DNA standard solution. 1.50 g of the DNA was dissolved in 100 mL of glacial acetic acid, and then 1.5 mL of concentrated sulfuric acid was added. This solution was stored in a brown bottle and was diphenylamine solution A. 1.6 mL of acetaldehyde was dissolved in 100 mL of deionized water to prepare diphenylamine solution B. Immediately before use, 20 mL of solution A and 0.1 mL of solution B were mixed to prepare diphenylamine. 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the DNA standard solution were added to 2 mL of diphenylamine reagent and diluted to 3 mL with distilled water. After mixing, the solution was incubated in a 60°C water bath for 45 min, cooled to room temperature, and measured at 595 nm. A standard curve was obtained by plotting DNA concentration on the x-axis and absorbance on the y-axis. Repeat the above operation by taking 0.1 mL of the reaction solution, and determine the DNA content in the P-EPS-CMC-HFO soil remediation agent after reacting with different concentrations of As according to the caliper.

[0088] The results are as follows Figure 3As shown, the nucleic acid content in the solution did not change significantly after the reaction under As stress, while the protein and polysaccharide contents were positively correlated with the As reaction concentration. Therefore, it is speculated that the unique proteins and polysaccharides in P-EPS are beneficial to the adsorption and fixation of As under the action of CMC-HFO.

[0089] Example 4

[0090] Comparison of arsenic adsorption efficiency of extracellular polymers in different bacterial strains

[0091] Repeat the same steps as in Example 3, adding a control experiment in which P-EPS-CMC-HFO was replaced with S-EPS-CMC-HFO from Comparative Example 1. After the 72-hour reaction, the products from both groups were centrifuged at 5000 r / min for 10 min, and the supernatant was collected, filtered through a 0.22 μm aqueous filter membrane, and the As content in the supernatant was measured. The experimental results are as follows. Figure 4 As shown, the adsorption and precipitation efficiency of P-EPS-CMC-HFO (YT-1) for As in Example 1 is higher than that of S-EPS-CMC-HFO (MR-1). This indicates that the different extracellular polymeric components of different bacteria lead to differences in the synergistic effect with ferrous sulfate.

[0092] Furthermore, the two sets of reaction precipitates were subjected to vacuum freeze-drying, and the functional groups involved in the reaction were analyzed using Fourier transform infrared spectroscopy (FTIR). The results are as follows: Figure 5 As shown, the peak heights of the indicated functional groups change with different As concentrations, indicating that these functional groups participate in the As adsorption process. The functional groups involved in the reaction of the two bacterial extracellular polymers are not entirely the same. When the As(V) concentrations are 25 mg / L and 50 mg / L, the peak positions of amide I / II are related to conformational changes in the protein, indicating that the protein participates in the adsorption of As by EPS. The absorption peak changes of COC and P=O are related to the participation of polysaccharides and nucleic acids in the adsorption process. A comparison of the infrared spectra after the reaction with different proportions of As shows a visible wavenumber of 3282 cm⁻¹. -1 3066cm -1 The increase in absorption peak intensity with increasing As concentration indicates that the special protein in P-EPS enhances the complexation of As during the adsorption process.

[0093] Example 5

[0094] Arsenic adsorption efficiency experiment of soil remediation agents coated with different ferrous ore

[0095] The experimental procedure was the same as in Example 3. The soil remediation agents were P-EPS-CMC-HFO, P-EPS-CMC-Fe(0), P-EPS-PVP-HFO and P-EPS-CMC-Fe(0). After reacting for 72 hours, each reaction system was centrifuged at 5000 r / min for 10 min and the supernatant was taken. The supernatant was filtered through a 0.22 μm aqueous filter membrane and the contents of As, total Fe and Fe(II) in the supernatant were determined.

[0096] Arsenic adsorption efficiency was calculated based on As content, and the results are as follows: Figure 6 As shown. Four soil remediation agents reacted with different concentrations of As(V) for 72 h. Among them, P-EPS-CMC-HFO achieved a removal rate of up to 98.43% for the treatment group with an initial As concentration of 100 mg / L. When the As concentration was 200 mg / L, the removal rate of CMC-HFO under P-EPS loading was significantly higher than that of the other treatment groups.

[0097] Further analysis was conducted to examine the effects of this treatment on the changes in Fe and Fe(II) content in the supernatant of the experimental group in Example 1 after the reaction. The experimental results are as follows: Figure 7 As shown, the total Fe, Fe(II) and As concentrations in the supernatant are positively correlated.

[0098] Meanwhile, the experimental data of arsenic adsorption by the soil remediation agent in Example 1 were fitted using Langmuir, Freundlich, and dual-mode adsorption isotherms, respectively, and C... e / q e -C e and lnq e -lnC e Plotting was performed, and linear fitting and regression analysis were conducted. The results are shown in Table 1. The Langmuir and Freundlich isotherm model fits the R-squared value. 2 All values ​​were below 0.8. To further investigate the effects of precipitation and adsorption, a dual-mode isothermal adsorption model was used to fit the experimental data, and the results are shown in Table 1.

[0099] The dual-mode isothermal adsorption model divides the reaction process into precipitation and adsorption processes, and fits the adsorption isotherm of As in P-EPS-CMC-HFO with a correlation coefficient R. 2 The value reached 0.8206, indicating that precipitation played a more significant role in the fixation process as the arsenic concentration in the solution increased. In the initial stage, when the arsenic concentration was low, arsenic fixation was mainly by adsorption. As the initial arsenic concentration increased, the number of adsorption sites on the surface of P-EPS-CMC-HFO gradually decreased, eventually reaching saturation. In the later stages, precipitation became the dominant process. The precipitate was freeze-dried and characterized by SEM. The results are as follows: Figure 8As shown. After P-EPS is loaded with CMC-HFO, P-EPS, as a biomacromolecule, is wrapped around the mineral surface, forming heterogeneous, smooth, large-particle material. Figure 8 a) After 72 hours of reaction with arsenic, the surface morphology of the mineral changed, exhibiting smooth, blocky, and flaky material, and the particle size became more uniform after the reaction. Figure 8 b).

[0100] Table 1. Adsorption equilibrium parameters of As(V) under the action of P-EPS-CMC-HFO

[0101]

[0102] The results of a full-spectrum X-ray electron energy dispersive spectroscopy (XPS) scan before and after the P-EPS-CMC-HFO reaction are as follows: Figure 9 As shown, the binding energies of C1s, O1s, Fe2p3, and Fe2p1 appear at 285.3 eV, 532.1 eV, 711.1 eV, and 724.6 eV, respectively. After P-EPS-CMC-HFO reacts with As, an As3d peak appears at 43.8 eV. The results show that the peak values ​​of C1s, Fe2p, and As3d change, indicating that the soil remediation agent of this invention reacts with As to produce new precipitate products.

[0103] Example 6

[0104] The impact of soil remediation agents on soil enzyme activity in arsenic-contaminated paddy soil

[0105] Using paddy soil from Yingtan, Jiangxi Province as the research object, we prepared contaminated paddy soil with As concentrations of 0, 50, 100, 200, and 400 mg / kg by exogenously adding disodium hydrogen arsenate (Na2HAsO4·7H2O). The soil was kept at 70% of its field maximum water holding capacity and incubated in a dark environment at 30℃ for 14 days. We set up control (CK), CMC-HFO treatment, and P-EPS-CMC-HFO treatment groups for different As contaminated soil concentrations. The added amount was 1% (w / w) of Fe. Each group had three replicates, for a total of 15 soil samples. The effects of different treatments on soil urease, phosphatase, and catalase activities were measured after 14 days of incubation at 30℃ in the dark. Soil urease activity was determined according to the "Determination of Soil Urease Activity - Sodium Phenolate-Sodium Hypochlorite Colorimetric Method" (T / NAIA 011-2020). Phosphatase activity was determined according to the "Determination of Soil Phosphatase Activity - Sodium Phenyl Phosphate Colorimetric Method" (T / NAIA 012-2020). Catalase activity was determined according to the "Determination of Soil Catalase Activity - Potassium Permanganate Titration Method" (T / NAIA 013-2020).

[0106] The results are as follows Figure 10As shown, the activities of soil urease, phosphatase, and catalase are negatively correlated with the concentration of As in the soil. High concentrations of As significantly inhibit catalase activity in contaminated soil. The soil remediation agent of this invention does not affect soil catalase activity at high As concentrations. Figure 10 c) Applying passivating agents CMC-HFO and P-EPS-CMC-HFO can improve the activities of urease and phosphatase to some extent. P-EPS-CMC-HFO has a more significant effect on improving the enzyme activity in As-contaminated soil. Figure 10 (a and b).

[0107] Example 7

[0108] Bioavailability of soil after remediation with soil remediation agents

[0109] This study investigated the bioavailability of heavy metals in As-contaminated soil particles using an in vitro digestion system simulating human gastric juice. As-contaminated soils with As concentrations of 0, 50, 100, 200, and 400 mg / kg (as in Example 6) were subjected to a 14-day reaction in a control group (CK), a CMC-HFO treatment group, and a P-EPS-CMC-HFO treatment group. After drying, grinding, and passing through a 100-mesh nylon sieve, 0.4 g of each sample was weighed and placed in a 50 mL polyethylene centrifuge tube. 40 mL of a pre-prepared human gastric juice simulation solution (1:100 solid-liquid ratio) was added. The centrifuge tubes were placed in a constant-temperature shaker and shaken for 1 hour to ensure thorough mixing of the soil particles and the simulated gastric juice solution. The pH of the simulated gastric juice was adjusted to 2 with 37% hydrochloric acid (v / v), and the mixture was placed in a 37°C water bath with a heated stirrer for 1 hour. After the reaction, the mixture was centrifuged at 5000 rpm for 20 minutes. The supernatant was filtered through a 0.22 μm filter membrane, and the total arsenic content in the filtrate was determined using an ICP-OES instrument.

[0110] In each group of experiments, the bioavailability of arsenic was calculated using the following formula:

[0111] BA=(C1·V1 / C s ·M s )×100% (4-1)

[0112] In the formula, BA represents the bioavailability (%) of As in the human digestive system.

[0113] C1—The soluble volume concentration of As in the reaction solution of the in vitro test (mg / L);

[0114] V1 — Volume of the reaction liquid in the digester (L);

[0115] Cs — the mass concentration of As in soil solid samples (mg / kg);

[0116] Ms — Mass of soil sample added to the digester (kg).

[0117] The results are as follows Figure 11 As shown, after 14 days of treatment with As concentrations of 0, 50, 100, 200, and 400 mg / kg in contaminated soil, the bioavailability of As was positively correlated with the As concentration. Both CMC-HFO and P-EPS-CMC-HFO significantly reduced bioavailability, with P-EPS-CMC-HFO showing better results.

[0118] All technical solutions described above that fall within the scope of this invention's conceptual framework are protected by this invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of this invention should also be considered within the scope of protection of this invention.

Claims

1. A soil remediation agent, comprising: (A) Extracellular polymeric material of *Pseudomonas putida* YT-1, wherein the bacteria are deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 22872; and, (B) Carboxymethyl cellulose-coated ferrite.

2. The soil remediation agent according to claim 1, wherein the mass ratio of component (A) to component (B) in the soil remediation agent is (1-10):

1.

3. The soil remediation agent according to claim 2, wherein the mass ratio of component (A) to component (B) in the soil remediation agent is (2-6):

1.

4. A method for preparing the soil remediation agent according to any one of claims 1-3, comprising the following steps: S1: Prepare a bacterial suspension of Pseudomonas putida YT-1, wherein the bacterial suspension contains wet bacterial cells of the strain and physiological saline; S2: After heating the bacterial suspension prepared in step (S1), centrifuge and collect the supernatant to obtain a crude extract of EPS. EPS crude extract was dialyzed and dried to obtain EPS solid powder; S3: Sodium carboxymethyl cellulose and ferric nitrate are mixed to obtain carboxymethyl cellulose-coated ferrous ore; S4: Mix the products from steps S2 and S3 to obtain a soil remediation agent.

5. The application of the soil remediation agent according to any one of claims 1-4 in the remediation of arsenic-contaminated soil.

6. The application according to claim 5, wherein the arsenic-contaminated soil is paddy soil.

7. A heavy metal treatment agent, comprising: (A) Extracellular polymeric material of *Pseudomonas putida* YT-1, wherein the bacteria are deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 22872; and, (B) Carboxymethyl cellulose-coated ferrite.

8. The heavy metal treatment agent according to claim 7, wherein the mass ratio of component (A) to component (B) is (1-10):1, preferably (2-6):

1.

9. The use of the heavy metal treatment agent according to any one of claims 7-8 in the preparation of heavy metal waste treatment agents, wastewater treatment agents, or air treatment agents.

10. The use of the heavy metal treatment agent according to any one of claims 7-8 in the purification or remediation of food, cosmetic or industrial waste.

Images