Method for realizing natural removal of arsenic pollution in soil by constructing liquid surface iron film based on microorganism-driven iron circulation

Abstract

The invention discloses a method for realizing natural removal of soil arsenic pollution by constructing a liquid surface iron film based on microorganism-driven iron circulation. A layer of hydrophobic biological membrane is formed on a water-gas interface by introducing a carbon source into the flooded soil polluted by arsenic containing microorganisms and iron, so that iron oxide which is reduced in the soil and then diffused to the gas-liquid interface and secondarily precipitated floats on the surface of a water body and does not precipitate into the soil again, and natural migration of the iron oxide in the soil to the water-gas interface is realized; arsenic is enriched on a gas-liquid interface by utilizing the adsorption effect of newly generated ferric oxide on arsenic, so that natural removal of soil arsenic pollution can be realized through simple fishing. Furthermore, a series of complex processes of reducing iron by iron reducing bacteria in nature and the like are accelerated by virtue of iron reduction catalysts such as electron shuttles and the like. The method is different from a traditional adsorption fixing technology for treating symptoms but not root causes in soil arsenic pollution prevention and control, directional removal of arsenic in the soil environment is achieved through element circulation, and the total amount of arsenic in soil is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of remediation of heavy metal contaminated soil, in particular, the present application relates to a method for realizing natural removal of arsenic contaminated soil based on microbial-driven iron cycle and construction of liquid iron membrane. BACKGROUND

[0002] Arsenic (As) is a widely distributed metalloid element in nature, which has significant toxicity and carcinogenicity. Globally, arsenic pollution is mainly concentrated in rice-growing areas, especially in South and Southeast Asia. Due to geological enrichment and groundwater irrigation, the arsenic content in the soil of these areas is significantly over-standard, resulting in the arsenic content in rice far exceeding the safety standard (0.2 mg / kg of inorganic arsenic) of the World Health Organization (WHO). The existence form of arsenic in soil is complex, mainly including inorganic arsenic and organic arsenic. Inorganic arsenic is mainly trivalent arsenic (As(III)) and pentavalent arsenic (As(V)), and the toxicity of As(III) is 25-60 times that of As(V). The migration and bioavailability of arsenic are affected by the adsorption of iron, aluminum oxide and other substances in soil. The dry-wet alternating environment of paddy field significantly affects the form and migration of arsenic. Under flooding conditions, arsenic mainly exists in the form of As(III), while under drought conditions, it is re-oxidized to As(V) and adsorbed and fixed by iron oxide. The threat of arsenic pollution to the ecological environment and human health mainly manifests in the following aspects: 1) ecological toxicity, inhibiting the growth and metabolic activity of soil microorganisms; 2) phytotoxicity, leading to crop yield reduction and quality decline; 3) human health risk, long-term intake of arsenic-contaminated food and drinking water can cause various diseases, including skin cancer, lung cancer and cardiovascular disease, etc.

[0003] The remediation technologies for arsenic-contaminated soil mainly include physical remediation, chemical remediation and biological remediation. Physical remediation technologies such as soil replacement can reduce the concentration of arsenic in soil, but the cost is very high. Chemical remediation technologies such as chemical leaching and chemical fixation use acid, alkali and chelating agent solutions, which can easily lead to soil acidification and may cause secondary pollution. Biological remediation technologies use plants, animals and microorganisms to remediate arsenic-contaminated soil, which has the advantages of low cost and environmental friendliness, but has the disadvantages of long remediation period, low efficiency and high dependence on environmental conditions. It is of great significance to use microbial-driven iron cycle coupled with arsenic form transformation for the remediation of arsenic-contaminated soil.

[0004] Iron is one of the most abundant metal elements in paddy soil, and its redox behavior, mineral transformation and interaction with arsenic significantly affect the environmental processes of paddy soil and the growth of rice. Because arsenic often exists in the form of anion of arsenate and arsenite, positively charged iron oxide has strong adsorption capacity for arsenic. Dissimilatory iron-reducing bacteria (DIRB) can reduce Fe(III) to Fe(II) through outer membrane cytochrome under anaerobic conditions, resulting in the dissolution of iron minerals and the release of adsorbed arsenic. Therefore, in the environment of paddy field with dry-wet alternation, the reduction and oxidation of iron can drive the dissolution and release of arsenic, and the microbial-driven iron cycle has a significant impact on the activity of arsenic and the accumulation of arsenic in rice.

[0005] Because the environmental behavior of arsenic is closely related to iron, a series of arsenic remediation technologies based on iron oxide have been reported in the prior art, especially arsenic adsorption and fixation technologies based on iron oxide. For example, nano zero-valent iron is prepared, which has high reactivity and reducing property, and its iron oxide shell and corrosion products have adsorption effect on arsenic ions, which can fix a large amount of dissolved arsenic in soil; for example, iron membrane is formed on the surface of rice roots, which can adsorb a large amount of arsenic on the root surface and act as a barrier to block the absorption of arsenic in soil into the inside of rice plants. Although iron oxide is widely used for arsenic pollution control, it is mainly used for arsenic fixation technology to reduce the activity of arsenic, and cannot completely remove arsenic in soil. At present, there is no report on the technology for completely removing arsenic in soil by microbial iron cycle. SUMMARY

[0006] The purpose of the present application is to overcome the above-mentioned defects and deficiencies in the prior art, and to provide a method for natural removal of soil arsenic pollution. A carbon source is added to the overlying water body of the flooded soil containing microorganisms and iron contaminated by arsenic, a bacteria-iron-arsenic composite membrane is formed at the water-air interface, and the bacteria-iron-arsenic composite membrane is fished out to achieve natural removal of soil arsenic pollution.

[0007] The above-mentioned purpose of the present application is realized by the following technical scheme: The present application provides a method for natural removal of soil arsenic pollution. A carbon source is added to the overlying water body of the flooded soil containing microorganisms and iron contaminated by arsenic, a bacteria-iron-arsenic composite membrane is formed at the water-air interface, and the bacteria-iron-arsenic composite membrane is fished out to achieve natural removal of soil arsenic pollution.

[0008] This invention comprehensively utilizes complex knowledge of soil microorganisms, elemental geochemistry, and physicochemistry to create a low-cost natural removal technology for soil arsenic pollution. This technology includes a series of principles such as arsenic adsorption by iron oxide, iron reduction and dissolution of iron, the diffusion of dissolved arsenic and iron in water, secondary precipitation of iron oxidation to re-fix arsenic, the inability of surface tension at the water-air interface to support the iron oxide generated by secondary precipitation, and the ability of hydrophobic biofilms to float at the water-air interface through surface tension. Specifically, based on the principle of microbial-driven iron cycling, this invention cleverly introduces a carbon source into arsenic-contaminated flooded soil. The addition of the carbon source affects the active bacterial communities in the soil (such as iron-reducing bacteria), promoting the reduction and dissolution of iron minerals in the soil to generate Fe. 2+ The generated Fe 2+ Gradient migration to the oxidation interface leads to an oxidation reaction, constructing a hydrophobic microbial-iron-arsenic complex membrane at the water-air interface, namely the water-air interface iron membrane. The microorganisms in this composite membrane are highly hydrophobic, allowing them to float on the water surface and support iron oxide precipitates diffused from the solution to the water surface. This facilitates the natural migration of soil iron oxide to the water-air interface. Utilizing the adsorption of arsenic by the newly generated iron oxide, arsenic is enriched at the gas-liquid interface, thus achieving a series of complex processes including soil iron reduction, arsenic release, arsenic-iron diffusion and migration, and secondary precipitation of arsenic and iron. Ultimately, arsenic in the soil naturally migrates to the water surface, and the natural removal of soil arsenic pollution can be achieved by capturing the precipitates on the liquid surface. This invention differs from traditional adsorption and fixation technologies in soil arsenic pollution control, which only treat the symptoms and not the root cause. Through element cycling, it achieves targeted removal of arsenic in complex soil environments, reducing the total amount of arsenic in the soil.

[0009] Furthermore, the final concentration of the added carbon source is 8–35 mM.

[0010] Preferably, the final concentration of the added carbon source is 10 mM. For a system of 4 g soil (water-to-soil ratio 20:1), applying a final concentration of 10 mM ethanol is sufficient to achieve a good remediation effect. In subsequent engineering applications, the amount of ethanol applied can be increased proportionally according to this concentration to avoid waste and reduce remediation costs.

[0011] Furthermore, the carbon source is one or more of ethanol, acetic acid, lactic acid, or glucose. The addition of ethanol as a carbon source affects active microorganisms in the soil (such as iron-reducing bacteria); acetic acid (a short-chain fatty acid) can be rapidly utilized by electrogenic and iron-reducing bacteria through direct oxidation; the metabolism of lactic acid (a hydroxy acid) depends on a dehydrogenase system, and its decomposition may be accompanied by a local decrease in pH; while glucose (a polysaccharide) needs to be gradually converted into pyruvate through hydrolysis and glycolysis, which may preferentially stimulate the proliferation of fermenting microorganisms, further promoting the reduction and dissolution of iron minerals in the soil to generate Fe. 2+ The generated Fe 2+Gradient migration to the oxidation interface causes an oxidation reaction, generating a complex gas-liquid interface film (possibly composed of iron oxides and organic matter).

[0012] Furthermore, the method also includes the addition of an iron reduction catalyst. By utilizing iron reduction catalysis to enhance soil microbial iron reduction, iron is dissolved through iron reduction, accelerating the release of arsenic adsorbed on the surface of iron oxide in the soil. This, in turn, accelerates the enrichment of reduced iron oxide that diffuses to the gas-liquid interface and undergoes secondary precipitation on the iron film at the water-air interface, thus accelerating the adsorption of arsenic by iron oxide and achieving natural purification of arsenic pollution in the soil.

[0013] Furthermore, the iron reduction catalyst is an electron shuttle, which accelerates the iron reduction process promoted by iron-reducing bacteria in nature. By utilizing electron shuttles with different properties, the transfer of as many electrons provided by the carbon source as possible to the iron reduction process is mediated, thereby maximizing the efficiency of natural purification.

[0014] Preferably, the electron shuttle is selected from one or more of riboflavin (RF), phenazine-1-carboxylic acid (PCA), anthraquinone 2,6-disulfonic acid disodium (AQDS), 1-hydroxyphenazine (1-OHP), 2-hydroxy-1,4-naphthoquinone (2-HNQ), anthraquinone 2-sulfonic acid sodium (AQS), phenazine (PHZ), and flavonoids (FMN).

[0015] More preferably, the final concentration of the electron shuttle is 10–500 μM.

[0016] Specifically, the final concentration of the added electron shuttle is 100 μM.

[0017] Furthermore, the microorganisms include arsenic-resistant microorganisms and iron-reducing bacteria. This invention activates arsenic-resistant and iron-reducing microorganisms in the soil through an exogenous carbon source and electron shuttle. The arsenic-resistant microorganisms primarily play a role in the reduction and activation of arsenic, while the iron-reducing bacteria promote the reduction and dissolution of iron. TEM results show that iron minerals and microorganisms coexist in the gas-liquid interface membrane, and a large number of microbial cells can be clearly observed. Hydrophilic / hydrophobic analysis indicates that the iron membrane containing microorganisms is highly hydrophobic. It is precisely the presence of these microorganisms that allows the iron membrane to maintain a relatively hydrophobic state overall, enabling it to be supported and float at the gas-liquid interface by the surface tension of water. In addition, extracellular polymers secreted by bacteria and the hyphal network of fungi may further promote the formation of the biofilm, which in turn provides support and stability for the iron membrane floating on the water surface.

[0018] Furthermore, the method also includes removing water, re-adding a carbon source, forming a bacteria-iron-arsenic composite film at the water-air interface, and then retrieval the bacteria-iron-arsenic composite film, repeating the above steps. After draining the water from the flooded soil, water, carbon source, and iron reduction catalyst are added again, and the cycle is repeated multiple times to effectively remove arsenic pollution from flooded paddy fields.

[0019] Furthermore, the flooded soil mainly refers to flooded paddy fields.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for the natural removal of arsenic pollution from soil by constructing a liquid-surface iron film based on microbial-driven iron cycling. The invention introduces a carbon source into flooded soil contaminated with arsenic and containing microorganisms and iron, forming a hydrophobic biofilm at the air-water interface. This prevents iron oxide, which has dissolved in the soil and diffused to the air-liquid interface before secondary precipitation, from re-precipitating back into the soil. Instead, it floats on the water surface, thus achieving the natural migration of soil iron oxide to the air-water interface. Utilizing the adsorption of arsenic by the newly generated iron oxide, arsenic is enriched at the air-liquid interface, allowing for natural removal of soil arsenic pollution through simple retrieval. Furthermore, iron reduction catalysts such as electron shuttles are used to accelerate a series of complex processes in nature, including iron reduction by iron-reducing bacteria, arsenic release, arsenic-iron diffusion and migration, and secondary precipitation of arsenic-iron. This invention differs from traditional adsorption and fixation technologies in soil arsenic pollution control, which only address the symptoms. Through element cycling, it achieves targeted removal of arsenic in complex soil environments, reducing the total amount of arsenic in the soil. In particular, it is a low-cost natural remediation technology that relies solely on the action of microorganisms and physicochemical processes such as diffusion and migration, without requiring external forces. Attached Figure Description

[0021] Figure 1 The experimental system was constructed with effect diagrams and functional illustrations.

[0022] Figure 2 This is a diagram demonstrating the effectiveness of the iron-arsenic composite membrane. Figure 2 In the diagram, 'a' represents the effect of natural purification; 'b' represents the test results of iron content on the membrane; and 'c' represents the test results of arsenic content.

[0023] Figure 3 The contents of water-soluble arsenic (a), total arsenic remaining in soil (b), arsenic phosphate remaining in soil (c), and arsenic oxalate remaining in soil (d) were determined after natural purification.

[0024] Figure 4 The results are XRD and mapping tests of the gas-liquid interface membrane.

[0025] Figure 5 Gas-liquid interface film formed after adding different carbon sources.

[0026] Figure 6 The graph shows the iron content (a), arsenic content (b), and arsenic-iron relationship on the gas-liquid interface membrane under different carbon source treatments.

[0027] Figure 7 Total arsenic (a), arsenic phosphate (b), and arsenic oxalate (c) remaining in soil under different carbon source treatments.

[0028] Figure 8 Iron content (a) and arsenic content (b) on the gas-liquid interface membrane under different shuttle treatments.

[0029] Figure 9 Total arsenic (a), arsenic phosphate (b), and arsenic oxalate (c) remaining in soil under different shuttle treatments.

[0030] Figure 10 TEM images of biological sections with different carbon sources and RF-added membranes.

[0031] Figure 11 The water contact angle results for different carbon sources and membranes with added RF components are shown in the figure.

[0032] Figure 12 The iron (a) and arsenic (b) contents on the gas-liquid interface membrane under the influence of the ethanol concentration gradient are shown.

[0033] Figure 13 The values ​​represent the total arsenic content (a), phosphate arsenic content (b), and oxalate arsenic content (c) in the soil under the influence of ethanol concentration gradient.

[0034] Figure 14 The iron content (a) and arsenic content (b) on the gas-liquid interface film under different water-soil ratio systems.

[0035] Figure 15 The total arsenic content (a), phosphate arsenic content (b), and oxalate arsenic content (c) in soils under different water-to-soil ratio systems are shown.

[0036] Figure 16 The 16S genus-level species diversity (a), absolute 16S quantification (b), and proportion of iron-reducing bacteria (c) of the soil after natural purification.

[0037] Figure 17 The effect of adding dissolved ferrous iron to sterilized soil.

[0038] Figure 18 A rendering of the soil column (a) and a custom sampler (b).

[0039] Figure 19 This is the result after 5 cycles of soil column rotation. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0041] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0042] Example 1: Performance Verification of Iron-Arsenic Composite Membrane I. Experimental Methods 1. Construction of the experimental system This experiment used arsenic-contaminated soil from Lianhuashan, Shantou, and a 100 mL tall beaker as the system container. The beaker contained 4 g of contaminated soil and 80 mL of water. After sealing the mouth of the beaker with an oxygen-permeable membrane, it was strictly statically cultured in a 28℃ incubator for 21 days. Figure 1 To ensure accuracy, each process consists of 5 parallels.

[0043] 2. Preparation of iron-arsenic composite membranes In the shallow flooding system of the above experimental system, 34 mM ethanol was added to the system as the experimental group, and no ethanol was added as the blank control group. The working principle of the iron-arsenic composite membrane is as follows: Figure 1 As shown, the addition of ethanol as a carbon source may affect the active microbial community in the soil (such as iron-reducing bacteria), promoting the reduction and dissolution of iron minerals in the soil to generate Fe. 2+ The generated Fe 2+ Gradient migration to the oxidation interface causes an oxidation reaction, generating a complex gas-liquid interface film (possibly composed of iron oxides and organic matter).

[0044] 3. Extraction and testing of iron-arsenic composite membranes (gas-liquid interface membranes, bacteria-iron-arsenic composite membranes, or water-gas interface iron membranes). The collection and processing of the iron-arsenic composite membrane was a crucial step in the experiment. The iron-arsenic composite membrane was manually retrieved from the reaction system using a glass slide. Subsequently, the collected membrane was transferred entirely to 10 mL of 6 M hydrochloric acid solution to allow for complete dissolution. To accelerate the dissolution process and ensure complete release of the membrane components into the solution, the solution was subjected to ultrasonic treatment for 15 minutes.

[0045] After dissolution, the solution is filtered through a 0.22 μm pore size aqueous filter to remove any particulate impurities. The filtered solution is then used for subsequent analysis. This method allows for the precise determination of arsenic and iron content in the iron-arsenic composite membrane, providing reliable data support for the study of membrane composition and performance.

[0046] 4. Extraction and testing of soil after natural purification The naturally purified soil was used for the extraction of total arsenic, arsenic phosphate, and arsenic oxalate. Arsenic extracted with arsenic phosphate and arsenic extracted with oxalate represent specifically adsorbed arsenic and amorphous iron oxide-bound arsenic in the soil, respectively. The specific steps are as follows: (1) Each batch of soil samples was centrifuged at 7000×g for 5 minutes in a centrifuge. After centrifugation, the supernatant was discarded and the soil was stored at -40℃ for subsequent analysis of the speciation of arsenic and iron in the soil solid phase.

[0047] (2) The speciation of arsenic in the soil was analyzed by a stepwise extraction method. First, phosphate-extracted arsenic was extracted using 1 M KH2PO4 + 0.1 M ascorbic acid (pH adjusted to 5.0 with phosphoric acid). 1.0 g of soil was accurately weighed and placed in a 50 mL capped centrifuge tube. After adding 25 mL of KH2PO4 extraction solution, the sample was placed in a 30℃ constant temperature shaker and shaken at 180 rpm in the dark for 4 hours. Subsequently, the sample was centrifuged at 8000 r / min for 10 minutes, and the supernatant was collected for the determination of phosphate-extracted arsenic (PO4-As(III) / As(V)) (Wang et al., 2012).

[0048] (3) Next, the soil precipitate after centrifugation was subjected to extraction of oxalate-extractable arsenic. Then, 25 mL of oxalate-ammonium oxalate buffer (pH 3.0) was added to the precipitate (1 L system contains 16.15 g ammonium oxalate and 10.90 g oxalate, and the pH was adjusted to 3.0 with ammonia). The sample was also placed in a constant temperature shaker at 30 °C and shaken at 180 rpm in the dark for 4 hours, and then centrifuged at 8000 r / min for 10 minutes. The supernatant was collected for the determination of oxalate-extractable arsenic (oxalate-As(III) / As(V)) (Wenzel et al., 2001).

[0049] (4) The extraction and determination of total arsenic in soil were performed in accordance with the People's Republic of China National Standard GB / T 22105.2-2008. Weigh 0.5 g (accurate to 0.0002 g) of soil sample after centrifugation to remove the supernatant, and place it in a 50 mL stoppered colorimetric tube. Add 10 mL of (1+1) aqua regia (made by mixing 1 part nitric acid and 3 parts hydrochloric acid, then diluting with water by half), stopper, shake well, and place in a boiling water bath for digestion for 2 hours, shaking several times during the process. After digestion, remove and cool, dilute with water to the mark, and shake well. Finally, filter the supernatant through a 0.22 μm filter membrane for atomic fluorescence spectrometry concentration determination.

[0050] The naturally purified soil is used for the extraction of ferrous iron and total iron. The specific steps are as follows: (1) Determination of ferrous iron Take a 5 mL centrifuge tube, add 2.5 mL of NaAC-HAC buffer (pH≈5.0), 1 mL of iron-containing sample (the suspension was filtered through a 0.22 μm aqueous filter and diluted to an appropriate concentration), and add 1 mL of o-phenanthroline (0.1%). Incubate for 15 min. Spot the incubated sample into a 96-well plate, 200 μL per well, with three replicates for each concentration. Measure the absorbance at 510 nm using spectrophotometry and calculate the iron ion concentration.

[0051] (2) Determination of total iron Before adding the iron-containing sample, add 1 mL of hydroxylamine hydrochloride (10%) as a reducing agent, let it stand for half an hour to allow all the iron to be completely reduced to ferrous iron, and then perform a colorimetric test on the absorbance according to the ferrous iron determination procedure to obtain the total iron concentration.

[0052] 5. Composite film XRD The iron-arsenic composite membrane was prepared using method 2 described above. Then, it was carefully removed from the reaction system entirely by hand using a glass slide. Extreme care was required during the process to ensure the membrane's integrity and surface smoothness, avoiding any damage. The removed membrane was then allowed to air dry at room temperature to maintain its original state. After complete drying, X-ray diffraction was used to examine its internal crystal structure to obtain microstructural information.

[0053] II. Experimental Results 1. In the aforementioned shallow flooding system, the addition of 34 mM ethanol (i.e., the final concentration of ethanol in the flooding system) resulted in the formation of a complex gas-liquid interface film, while no obvious film formation was observed in the control group. Figure 2 a) Total iron and total arsenic content extraction of the gas-liquid interface membrane revealed no ferrous residue on the membrane, and the HCl-total Fe content was significantly increased in the ethanol group, indicating that the iron existed in the form of Fe. 3+ ( Figure 2 b). At the same time Figure 2 c shows that the HCl-total As content of the gas-liquid interface membrane in the ethanol group was significantly higher than that in the control group, indicating that the membrane enriches arsenic on the liquid surface through physical adsorption or chemical binding, blocking the migration pathway of arsenic to the environment. Its maximum adsorption capacity for As can reach 491 μg ( Figure 2 c), the As / Fe ratio is 67.5-101.1 g / kg ( Figure 2 (b and c).

[0054] 2. The release of arsenic from soil mainly originates from the diffusion process after the dissolution of iron minerals. Through... Figure 3A comparison of water-soluble arsenic concentrations shows that the residual arsenic leaching in the blank group was higher (58.2 μg / L), while that in the ethanol group was 43.5 μg / L. Although the decrease was 25.3%, this was not because the ethanol group dissolved less arsenic, but because the arsenic dissolved in the ethanol group diffused to the surface iron film and was re-fixed by the iron film. Figure 2 ).

[0055] according to Figure 3 b indicates that the addition of ethanol effectively removed total arsenic from the soil, reducing the remaining arsenic content by 30 mg / kg compared to the control group. Compared to total arsenic, the removal effect on arsenic phosphate was the most significant, meaning that ethanol promoted the conversion or release of arsenic phosphate. Figure 3 c. Compared with the control group, the addition of ethanol reduced the content of arsenic phosphate in the soil by 56.0%. Figure 3 As can be seen from d, ethanol has a significant effect on reducing oxalate arsenic in the soil, with a reduction of 22.7% compared to the control group.

[0056] 3. Through XRD patterns ( Figure 4 a) The crystal structure and composition information of the gas-liquid interface film can be analyzed to generate amorphous iron hydroxide with a high specific surface area. This secondary mineral exhibits strong adsorption of dissolved arsenic through mechanisms such as ligand exchange and surface complexation. X-ray diffraction analysis confirms that its main component is likely ferrihydrite. From the iron, arsenic, and oxygen distribution mapping diagram ( Figure 4 (b) The distribution of iron (red area), arsenic (green area), and oxygen (blue area) in the membrane can be visually observed. The high concentration areas of iron and the enriched areas of arsenic show a certain correlation, indicating a possible chemical bond or interaction between the two. The distribution of oxygen, along with that of iron and arsenic, reflects the chemical composition and structural characteristics of the membrane. Its uniform distribution supports the extensive formation of iron oxides, and is consistent with Fe... 2+ The oxidation process was consistent, further confirming the role of the membrane in the adsorption of arsenic.

[0057] Example 2: The effect of different carbon sources on natural purification effect In Example 1, it was clearly demonstrated that ethanol, when added as a carbon source to a flooding system, exhibited significant effects in the natural purification of arsenic-contaminated soil, including the formation of a special gas-liquid interface film, reduction of arsenic residues in the solution, and effective removal of total and speciation of arsenic from the soil. However, there are many types of carbon sources, and the differences in characteristics of different carbon sources may have different impacts on the soil ecosystem. The activity and community structure of functional microorganisms in the soil are closely related to the types and metabolic pathways of their available carbon sources. As the core substrate for microbial energy metabolism, carbon sources not only drive the speciation of arsenic by altering soil redox conditions (Eh), but also influence the mineral dissolution-reprecipitation process and the arsenic fixation efficiency by regulating the abundance and activity of specific functional bacterial groups (such as iron-reducing bacteria and sulfate-reducing bacteria). Previous studies have shown that ethanol, as a carbon source, can accelerate the Fe2+ metabolism by promoting the metabolic activity of iron-reducing bacteria. 3+ The reducing dissolution of oxides leads to arsenic fixation at the gas-liquid interface. However, different carbon sources, due to differences in molecular structure, metabolic rate, and intermediate products, may significantly differentiate microbial community succession and arsenic migration and transformation pathways.

[0058] Acetic acid, lactic acid, and glucose, as typical small-molecule carbon sources, exhibit significant differences in bioavailability and metabolic pathways: acetic acid (a short-chain fatty acid) can be rapidly utilized by electrogenic and iron-reducing bacteria via direct oxidation; lactic acid (a hydroxy acid) metabolism relies on a dehydrogenase system, and its decomposition may be accompanied by a local decrease in pH; while glucose (a polysaccharide) requires hydrolysis and glycolysis to be gradually converted into pyruvate, potentially preferentially stimulating the proliferation of fermenting microorganisms. This differentiation in metabolic pathways may lead to competitive enrichment of functional microorganisms (such as Bacillus, Geobacter, and Shewanella), further affecting Fe... 3+ Mineral reduction kinetics and the formation efficiency of secondary minerals (such as magnetite and goethite). Furthermore, the type of carbon source may also regulate the arsenic complexation-desorption balance by altering the composition of soil dissolved organic matter (DOM). For example, low molecular weight organic acids (acetic acid, lactic acid) can promote arsenic release through ligand competition, but their metabolites (such as CO3) can also contribute to arsenic release. 2- S 2- This may promote the co-precipitation of arsenic. Therefore, exploring the mechanism of action of different carbon sources on the microbial-mineral-arsenic ternary system is of great significance for optimizing the selection of carbon sources for bioremediation.

[0059] I. Experimental Methods 1. Construction of biomembranes An iron-arsenic composite membrane was constructed using method 2 of Example 1, while replacing the carbon source ethanol with acetic acid, lactic acid, and glucose. The effects of different carbon sources on the natural purification effect were compared.

[0060] II. Experimental Results Depend onFigure 5 It can be seen that the gas-liquid surface films of the flooded systems after natural purification with the addition of different carbon sources show visible differences in bacterial communities and iron content. This suggests that different carbon sources directionally regulate the bacterial communities in the soil, thus affecting the natural purification effect.

[0061] Gas-liquid interface films formed after adding different carbon sources ( Figure 5 Extraction reveals that, Figure 6 The data points shown in c are distributed roughly along a straight line, indicating that under these experimental conditions, there is a significant linear positive correlation between the iron content and arsenic content on the liquid surface iron film. That is, as the iron content increases, the arsenic content also increases accordingly, indicating a synergistic accumulation of the two. This further illustrates that the gas-liquid interface film fixes arsenic migrating from the soil through the co-precipitation of iron and arsenic. Figure 6 From a and b, it can be seen that the gas-liquid interface membrane produced by the ethanol treatment group has the best adsorption effect on arsenic.

[0062] Figure 7 'a' represents the residual arsenic in the soil. The glucose group showed a relatively low residual total arsenic content, consistent with the higher arsenic residue levels in this group's solution. Furthermore, the glucose treatment may have promoted a change in the form of arsenic. According to... Figure 7 Compared with the control group, the glucose group showed a 64.6% decrease in phosphate arsenic content and a 73.4% increase in oxalate arsenic content, suggesting that a transformation from specifically adsorbed arsenic to amorphous iron oxide-bound arsenic may have occurred.

[0063] Example 3: The Influence of Different Shuttles on Natural Purification Effects By using an iron reduction catalyst to enhance soil microbial iron reduction, iron is dissolved through iron reduction, which accelerates the release of arsenic adsorbed on the surface of iron oxide in the soil. This, in turn, accelerates the enrichment of iron oxide that has diffused to the gas-liquid interface and precipitated again after reduction on the iron film at the water-air interface, thus accelerating the adsorption of arsenic by iron oxide and achieving natural purification of arsenic pollution in the soil. At the same time, the above experimental results also confirm that the content of iron and arsenic on the gas-liquid interface film formed during the natural purification process is positively correlated.

[0064] Based on the above research findings, it is crucial to explore the impact of electron shuttles (iron reduction catalysts) with different properties on the natural purification effect of arsenic-contaminated soil. This part of the study aims to utilize electron shuttles with different properties to mediate the transfer of as many electrons provided by the carbon source as possible to the iron reduction process, thereby maximizing the natural purification efficiency.

[0065] I. Experimental Methods 1. Construction of biomembranes As in Method 2 of Example 1, an iron-arsenic composite membrane was constructed, with different electron shuttles added (i.e., a final concentration of 100 μM electron shuttles in the flooded system): RF, PCA, AQDS, 1-OHP, 2-HNQ, AQS, PHZ, and FMN. The effects of different shuttles on the natural purification effect were compared.

[0066] 2. High-resolution and biological TEM testing The biofilm sampling method was consistent with that described above. The biological TEM samples were promptly fixed in 1.5 mL of 2.5% glutaraldehyde solution. The high-resolution TEM samples were dried and fixed on a copper grid for imaging.

[0067] II. Experimental Results 1. Eight types of electron shuttles were selected, and 34 mM ethanol was chosen as the carbon source, indicating the best performance. The effects of different electron shuttles on the natural purification effect were investigated. For example... Figure 8 After the addition of electron shuttles, the arsenic content on the membrane increased compared to the treatment group without shuttles.

[0068] according to Figure 9 The total arsenic remaining in the soil after natural purification with different shuttles shows that its concentration generally follows the pattern of increasing arsenic adsorption. According to... Figure 9 As shown in b and c, the addition of electron shuttles may have promoted the transformation of arsenic forms, with a phenomenon of transformation into amorphous iron oxide-bound arsenic.

[0069] 2. From Figure 10 TEM results show that iron minerals and microorganisms coexist in the gas-liquid interface membrane. Numerous microbial cells can be observed. It is the presence of these microorganisms that supports the iron membrane, allowing it to float at the gas-liquid interface.

[0070] When the water contact angle is less than 90°, the membrane surface is hydrophilic, and water can spread well on its surface, resulting in good wettability; when the contact angle is greater than 90°, the membrane surface is hydrophobic, and water will form spherical droplets on the surface, resulting in poor wettability. According to... Figure 11 It can be seen that the contact angle of the gas-liquid interface film generated in all treatment groups is greater than 90°, therefore they are all hydrophobic.

[0071] This gas-liquid interface membrane may form a floating biofilm on the water surface through the extracellular polymers of bacteria and the hyphal network of fungi, and this biofilm may support the floating iron. The lipids (Stromberg et al., 2017) and some proteins in the extracellular polymers (EPS) formed by some bacteria may be the key components that give the biofilm its hydrophobicity. The hyphae and extracellular matrix (ECM) of some fungi are also hydrophobic, giving this gas-liquid interface membrane hydrophobic properties.

[0072] Example 4: Effect of different carbon source concentrations on natural purification effect I. Experimental Methods Under the premise that the riboflavin system concentration (i.e., the final concentration of riboflavin in the flooded system) (100 μM), 4 g of soil (water-soil ratio 20:1) and other experimental conditions remain the same as before, the effects of different ethanol system concentration gradients on the natural purification effect were investigated.

[0073] II. Experimental Results 1. Under the premise that the riboflavin concentration and other experimental conditions remained consistent with the previous results, based on the application experiments with different ethanol system concentration gradients, it was found that in the extraction results of the generated gas-liquid interface membrane, the iron and arsenic content in the 5 mM ethanol system was significantly lower than that in the other four groups. However, when the ethanol system concentration was doubled, reaching 10 mM, the iron content on the membrane increased by approximately 3.3 times. Figure 12 a), while the arsenic content increased by approximately 3.2 times ( Figure 12 b). When the concentration of the ethanol system was doubled again, reaching 20 mM, the iron content on the membrane remained almost unchanged, but the arsenic content decreased by approximately 48.4 μg. Figure 12 (b) When the concentration of ethanol applied was further increased, the amount of adsorption on the membrane did not change significantly.

[0074] The same conclusion was reached by extracting from naturally purified soil. When the concentration of ethanol in the application system was increased from 5 mM to 10 mM, total arsenic in the soil could be observed (…). Figure 13 a) Arsenic phosphate ( Figure 13 b) Arsenic oxalate ( Figure 13 c) All showed a significant decrease. However, further increasing the dosage did not significantly change the effect. This indicates that for a system of 4 g soil (soil-to-water ratio 20:1), applying 10 mM ethanol is sufficient to achieve a good remediation effect. In subsequent engineering applications, the amount of ethanol applied can be increased proportionally according to this concentration to avoid waste and reduce remediation costs.

[0075] Example 5: The effect of different water-to-soil ratios on natural purification effects I. Experimental Methods The soil-water ratio not only affects the rationality of remediation concentrations but also significantly influences their ability to ensure sufficient contact and promote efficient reactions. This study investigated the permeability and effectiveness of different soil-water ratios under the premise that the concentrations of the riboflavin system (100 μM), the ethanol system (34 mM), and other experimental conditions remained consistent with previous findings. The experiment was conducted with a fixed water volume of 80 mL, and different soil-water ratios were compared by varying the weight of the soil.

[0076] II. Experimental Results according to Figure 14 a and b found that increasing the amount of soil did not increase the absorption of arsenic and iron on the membrane when the overlying water volume remained constant. This suggests that there may be a saturation point for the absorption of iron by the membrane. That is, in the flooded system, a final concentration of 34 mM ethanol and 100 μM RF can transfer approximately 4306.1 ± 324.5 μg of iron and 293.61 ± 31.63 μg of arsenic.

[0077] In contrast, with naturally purified soil, the amount of arsenic added varies; the more soil added, the higher the total amount of arsenic in the system, and therefore the more arsenic remains in the soil after treatment. According to... Figure 15 It can be seen that the less soil there is, the less residual arsenic there is in all three different forms of arsenic.

[0078] Example 6: The Influence of Microorganisms on Natural Purification Effects I. Experimental Methods 1. Determination of total bacterial count in soil after natural purification Approximately 250 mg of the naturally purified soil was accurately weighed and the weight was recorded. DNA was extracted from the sample using the QIAGEN DNeasyPowerSoil Pro kit. After testing the purity and concentration of the DNA, the 16S rRNA gene in the extracted DNA was quantified by qPCR. At the same time, 16S amplicon sequencing was performed on the sample to analyze the changes in the microbial population. The primers used are shown in Table 1.

[0079] Table 1 Primers for 16S Real-Time PCR

[0080] II. Experimental Results Arsenic release from soil is significantly influenced by the structure and function of the microbial community. The input of different carbon sources alters the composition and metabolic activities of soil microorganisms, thereby affecting iron reduction and arsenic release. Azospirillum Arsenic-resistant microorganisms and Bacillus and Clostridium Iron-reducing bacteria play an important role in this process, promoting the migration and transformation of iron and arsenic through biochemical reactions. Figure 16 ). Figure 16 The quantitative PCR results for b showed that the total microbial population was significantly increased after the addition of a carbon source. This indicates that the soil microorganisms were activated, which may be an important reason for the enhanced iron reduction.

[0081] To verify the crucial role of microorganisms in the natural purification process, a control experiment using sterilized soil was designed. In this experiment, sufficient dissolved ferrous iron was added to the sterilized soil to simulate its key role in supporting microorganisms during gas-liquid interface film formation. The amount of ferrous iron added was calculated based on the amount of iron dissolved and released after the previous ethanol + RF reaction, including both dissolved iron and iron on the membrane, totaling 616 mg / L. After a 21-day reaction period, from... Figure 17 A small amount of metal film deposition was observed at the gas-liquid interface. The ferrous concentration in the post-reaction solution was measured, revealing a residual ferrous concentration of approximately 327.8 mg / L. This indicates that the initial amount of added ferrous iron was sufficient to meet the requirements of the entire reaction process.

[0082] Further analysis of the composition of the gas-liquid interface film after the reaction, Figure 17 Fe in b and c 2+ The results showed that the iron content on the membrane was approximately 630.9 µg, while the arsenic content was only 0.52 µg. These values ​​were significantly lower than those of the ethanol + RF group, which dissolved the same amount of iron. This indicates that under sterilization conditions, the absence of microorganisms had a significant impact on the formation of the gas-liquid interface membrane and the accumulation of arsenic and iron, thus verifying the key role of microorganisms in the natural purification mechanism.

[0083] Example 7: Soil column experiment verification I. Experimental Methods Use a transparent 500 mL plastic graduated cylinder for the soil column experiment, cutting off the portion above 400 mL to ensure the reaction liquid surface is in full contact with oxygen. Figure 18 a). Soil columns were set up at a depth of 15 cm (approximately 260 mL) in the soil and 5 cm (approximately 360 mL) in the water cover. Using these soil columns, a blank control group was established with only water and no other substances added, and an experimental group was established with the system concentration (final concentrations of ethanol and RF in the system) of 34 mM ethanol + 100 μM RF, with 5 replicates for each group. A 21-day settling period constituted one cycle. After one cycle, a custom-designed calibrated sampler was used. Figure 18 b) Sampling was performed, and the soil samples were separated into 3cm increments for preservation. The arsenic concentration of each layer was then extracted. After sampling, the control group was replenished with fresh deionized water. The treatment group was supplemented with ethanol and riboflavin at concentrations of 34 mM and 100 μM, respectively, and allowed to stand for another cycle to observe the long-term effects.

[0084] II. Experimental Results The initial soil arsenic concentration was 215 mg / kg. Natural removal was achieved through five cycles, each lasting 21 days. After each cycle, the top layer of water was discarded, and water, ethanol, and riboflavin were added again before the cycle was repeated. A total of five cycles were performed. The results after the fifth cycle were as follows: Figure 19As shown, the total arsenic content in the topsoil at 3 cm depth decreased by 127.4 mg / kg after five natural purification cycles (arsenic removal rate reached 59%).

Claims

1. A method for the natural removal of arsenic pollution from soil, characterized in that, Carbon source is added to the water body overlying the flooded soil contaminated with microorganisms and iron and arsenic, forming a microbial-iron-arsenic composite film at the water-air interface. The microbial-iron-arsenic composite film is then retrieved, thus achieving the natural removal of arsenic pollution from the soil.

2. The method according to claim 1, characterized in that, The final concentration of the added carbon source is 8–35 mM.

3. The method according to claim 1 or 2, characterized in that, The carbon source is one or more of ethanol, acetic acid, lactic acid, or glucose.

4. The method according to claim 1, characterized in that, The method also includes adding an iron reduction catalyst.

5. The method according to claim 4, characterized in that, The iron reduction catalyst is an electron shuttle.

6. The method according to claim 5, characterized in that, The electron shuttle is selected from one or more of riboflavin, phenazine-1-carboxylic acid, anthraquinone 2,6-disulfonic acid disodium, 1-hydroxyphenazine, 2-hydroxy-1,4-naphthoquinone, anthraquinone 2-sulfonic acid sodium, phenazine, or flavonoids.

7. The method according to claim 6, characterized in that, The final concentration of the added electron shuttle is 10–500 μM.

8. The method according to claim 1, characterized in that, The microorganisms include arsenic-resistant bacteria and iron-reducing bacteria.

9. The method according to claim 1, characterized in that, It also includes removing the water, adding a carbon source, forming a bacteria-iron-arsenic composite film at the water-air interface, and then retrieval the bacteria-iron-arsenic composite film, repeating the above steps.

10. The method according to claim 1, characterized in that, The flooded soil is a flooded paddy field.

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