Method and kit for synchronously removing manganese and ammonia by utilizing composite flora

Abstract

One or more embodiments of the invention relate to the technical field of environmental governance, in particular to a method and a kit for synchronously removing manganese and ammonia by using composite flora. The method comprises the following steps: taking a to-be-extracted sample in a contaminated site, carrying out primary treatment, extracting microorganisms in the to-be-extracted sample, and preparing a bacterial solution; domesticating and enriching the microorganisms extracted from the bacterial liquid to obtain a composite flora; wherein the composite flora comprises high-efficiency manganese oxidizing bacteria and ammonia nitrogen removing bacteria, the high-efficiency manganese oxidizing bacteria are pseudomonas, and the ammonia nitrogen removing bacteria are bacillus; inoculating the composite flora into a reactor, wherein the reactor contains a to-be-treated water body containing manganese and ammonia nitrogen pollutants; and under anaerobic or aerobic conditions, culturing the composite flora in the reactor to a plurality of periods so as to synchronously remove manganese and ammonia nitrogen pollutants in the water body to be treated.

Description

Technical Field

[0001] This specification relates to the field of environmental remediation technology, specifically to a method and kit for simultaneous manganese and ammonia removal using a complex microbial community. Background Technology

[0002] Currently, manganese and ammonia nitrogen pollution in water bodies is an increasingly serious environmental problem worldwide, especially in regions with rapid industrialization and urbanization.

[0003] Manganese is a common metallic element widely used in industries such as steel production and battery manufacturing. Manganese typically exists in water in two forms: dissolved and precipitated. Divalent manganese in water has low toxicity to humans, livestock, and aquatic organisms, but low concentrations can affect the color, odor, and taste of water. Manganese pollution in water bodies mainly originates from the following sources: industrial emissions (steel smelting, mining, and other industrial activities may discharge manganese and its compounds into water bodies); agricultural activities (manganese-containing fertilizers and pesticides used in agriculture may also enter water bodies); and natural sources (manganese from the Earth's crust may enter water bodies through geological processes). The main problem with manganese pollution is that excessively high levels of manganese in water bodies can affect water quality and pose a serious threat to aquatic life. High concentrations of manganese can be toxic to the growth and reproduction of aquatic organisms. Furthermore, manganese can increase water color, affecting the visual appeal of the water and consequently impacting the safety of drinking water.

[0004] Ammonia (NH3) is a nitrogen compound, which is usually found in water as nitrate nitrogen (NH3). - -N), nitrite nitrogen (NH2) - -N) and ammonium nitrogen (NH4) + Ammonia exists in the form of nitrogen (N-). The main sources of ammonia pollution include: agricultural non-point source pollution, where nitrogen-rich substances such as fertilizers and manure used in agriculture enter water bodies through rainwater runoff; industrial wastewater discharge, with ammonia present in wastewater from fertilizer production, dyeing, and pulp and paper manufacturing industries; and domestic sewage, where urban sewage also contains high concentrations of ammonia, especially when wastewater treatment is incomplete. Ammonia pollution has a significant impact on aquatic ecosystems, particularly during eutrophication. Excessive ammonia concentrations lead to a decrease in dissolved oxygen levels in water bodies, thus affecting the survival of aquatic organisms. Furthermore, ammonia itself is toxic; high concentrations in water can cause direct physiological harm to fish and other aquatic life, even leading to death.

[0005] Manganese and ammonia pollution not only affects water quality but also poses a threat to ecosystems and human health. Therefore, comprehensive management is needed, including effective pollution source control, wastewater treatment, and ecological restoration measures, to reduce the impact of such pollution. Summary of the Invention

[0006] This specification describes a method and kit for simultaneous manganese and ammonia removal using a complex bacterial community, which can simultaneously and efficiently remove divalent manganese and ammonia nitrogen from water.

[0007] In a first aspect, embodiments of this specification provide a method for simultaneous manganese and ammonia removal using a composite microbial community, the method comprising the following steps:

[0008] After preliminary treatment of the samples to be extracted from the contaminated site, microorganisms are extracted from the samples and prepared into a bacterial solution. The microorganisms extracted from the bacterial solution are domesticated and enriched to obtain a complex microbial community. The complex microbial community includes highly efficient manganese oxidizing bacteria and ammonia nitrogen removing bacteria. The highly efficient manganese oxidizing bacteria is Pseudomonas, and the ammonia nitrogen removing bacteria is Bacillus. The complex microbial community is inoculated into a reactor containing water pollutants of manganese and ammonia nitrogen. Under anaerobic or aerobic conditions, the complex microbial community in the reactor is cultured for multiple cycles to simultaneously remove manganese and ammonia nitrogen pollutants from the water to be treated.

[0009] This technical solution aims to remove manganese (Mn) from water through the synergistic effect of a complex bacterial community. 2+ ) and ammonium nitrogen (NH4) + -N). Specifically, specific microbial communities (complex microbial communities) are utilized to remove pollutants through different biological processes under varying physiological conditions. The highly efficient manganese-oxidizing bacteria (Pseudomonas) within the complex microbial community possess a strong manganese oxidation capacity, capable of removing divalent manganese (Mn) from water. 2+ ) oxidized to tetravalent manganese (Mn) 4+ The latter, usually in an insoluble form, can precipitate and be removed from water. This process helps reduce the concentration of manganese in the water and effectively prevents manganese from affecting water quality, such as color and toxicity to aquatic organisms. Ammonia nitrogen removal bacteria (Bacillus) can effectively degrade ammonia nitrogen in water. Ammonia nitrogen removal usually occurs through two pathways: the biological conversion of ammonia into nitrite (NO2). - ) and nitrates (NO3) - Bacillus bacteria can absorb ammonia nitrogen, either through direct assimilation, or by reducing its concentration in water. Bacillus plays a crucial role in ammonia nitrogen removal, especially under both anaerobic and aerobic conditions.

[0010] In the implementation process, microorganisms from the contaminated site are first extracted and prepared into a bacterial solution. For example, the microorganisms in this method are derived from sediments (e.g., bottom mud) and surface / groundwater of the water body to be treated. These are indigenous microorganisms, avoiding the introduction of alien species and posing no ecological risk. Then, through appropriate domestication and enrichment processes, highly efficient manganese-oxidizing bacteria and ammonia nitrogen-removing bacteria are selected to ensure that they can fully exert their role in removing pollutants in the reactor.

[0011] The enriched microbial community is inoculated into the reactor, which contains manganese and ammonia nitrogen pollutants to be treated. The environmental conditions in the reactor (such as pH and temperature) can be adjusted to conditions suitable for microbial growth and pollutant removal, typically including anaerobic or aerobic conditions.

[0012] By cultivating a complex microbial community in the reactor for multiple cycles, manganese and ammonia nitrogen are simultaneously removed through the growth and metabolism of the microbial community. This simultaneous removal process significantly improves the removal efficiency of pollutants.

[0013] In one embodiment, the sample to be extracted includes upstream soil, downstream river sediment, surface water, and groundwater.

[0014] In one embodiment, the sample to be extracted includes upstream soil and downstream river sediments.

[0015] In one embodiment, after preliminary treatment of the sample to be extracted from the contaminated site, the microorganisms in the sample to be extracted are extracted and prepared into a bacterial solution, including:

[0016] Microorganisms were extracted from upstream soil and downstream river sediments using a two-step centrifugation method. Specifically, downstream river sediments were mixed with deionized water at a mass ratio of 1:9, glass beads were added, and the mixture was shaken for 2 hours. Then, the mixture was centrifuged at a differential speed to remove impurities from the downstream river sediment suspension while retaining the microorganisms. The resulting bacterial suspension was stored at 4°C for subsequent inoculation.

[0017] In one embodiment, after preliminary treatment of the sample to be extracted from the contaminated site, the microorganisms in the sample to be extracted are extracted and prepared into a bacterial solution, including:

[0018] Microorganisms were extracted from surface water and groundwater using a filtration oscillation method. Specifically, the method involved: using a filter membrane with a pore size of 0.22 μm to retain microorganisms in surface water and groundwater, then breaking the filter membrane and placing it in deionized water, using a vortex shaker to peel off the microorganisms from the filter membrane, using differential centrifugation to remove the broken filter membrane and collect the microorganisms, and preparing a bacterial solution, which was then stored at 4°C for subsequent inoculation.

[0019] In one embodiment, the domestication and enrichment of the indigenous microorganisms extracted from the bacterial solution includes:

[0020] The initial Mn(II) concentration was set at 55 mg / L, and the NH4+ concentration was set at 50 mg / L. + -N concentration was used to prepare artificially simulated groundwater, and extracted microorganisms were inoculated under aerobic and anaerobic conditions respectively. A reactor was then established to domesticate and enrich the microorganisms.

[0021] In one embodiment, the concentration ratio of Pseudomonas to Bacillus is 1:1.

[0022] In one embodiment, the complex microbial community also includes Nitrosospira and Nitrosomonas.

[0023] In one embodiment, the complex microbial community in the reactor is cultured under aerobic conditions for at least three cycles to simultaneously remove manganese and ammonia nitrogen pollutants from the water to be treated; wherein each cycle is at least four days.

[0024] Secondly, embodiments of this application provide a kit for removing bioavailable vanadium from soil, comprising: a complex microbial community, wherein the complex microbial community includes highly efficient manganese oxidizing bacteria and ammonia nitrogen removing bacteria, wherein the highly efficient manganese oxidizing bacteria is Pseudomonas, and the ammonia nitrogen removing bacteria is Bacillus, and the concentration ratio of Pseudomonas to Bacillus is 1:1.

[0025] In one embodiment, the complex microbial community also includes Nitrosospira and Nitrosomonas.

[0026] The solutions provided in this specification achieve simultaneous manganese and ammonia removal by enriching functional microorganisms from the environment and then constructing a composite microbial community. Traditional removal technologies typically require separate treatment of manganese and ammonia nitrogen pollution, while this technology can remove both pollutants from water simultaneously, reducing treatment time and costs and improving overall water quality remediation efficiency. Using microorganisms to degrade pollutants is more environmentally friendly than traditional chemical treatment methods. The composite microbial community degrades pollutants through biological metabolic pathways, avoiding the use of chemical agents and thus reducing the negative environmental impact of chemicals. Compared to traditional physical and chemical methods (such as flocculation, adsorption, and reverse osmosis), the composite microbial community technology has lower operating costs because the cost of microbial cultivation and management is relatively low. Microorganisms can proliferate naturally, reducing the need for artificial chemical additives. This technology is applicable to various water quality conditions (such as pH changes and temperature fluctuations) and can effectively function in different polluted environments. By adjusting the environmental conditions within the reactor, the metabolic processes of the microorganisms can be optimized, thereby improving pollutant removal efficiency. The use of microorganisms enables long-term stable pollution control; the composite microbial community can maintain its activity and removal efficiency through multiple cycles, avoiding secondary pollution problems that may occur with conventional chemical methods. This technical solution can not only remove pollutants from water bodies, but also improve the ecological environment of water bodies through the metabolic activities of microorganisms, restore the habitat of aquatic organisms, and enhance the stability of the aquatic ecosystem. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 A summary bar chart of the physicochemical properties of sediment samples from the contaminated site is shown.

[0029] Figure 2 A line graph showing the removal efficiency of microbial Mn(II) from sediments is shown.

[0030] Figure 3 The changes in Mn(II) concentration in aquatic microorganisms were shown;

[0031] Figure 4 The changes in NH4+-N concentration in sediment microorganisms are shown; where a: changes in ammonia nitrogen concentration in a reactor with only ammonia nitrogen added; b: changes in ammonia nitrogen concentration in a reactor with both manganese and ammonia nitrogen added.

[0032] Figure 5The changes in the concentration of ammonia nitrogen oxidation byproducts in sediment microorganisms are shown; where a: changes in nitrate nitrogen concentration in a reactor with only ammonia nitrogen added; b: changes in nitrate nitrogen concentration in a reactor with both ammonia nitrogen and manganese added; c: changes in nitrite nitrogen concentration in a reactor with ammonia nitrogen added; d: changes in nitrite nitrogen concentration in a reactor with both ammonia nitrogen and manganese added.

[0033] Figure 6 The graph shows the changes in the concentrations of three nitrogen species in aquatic microorganisms; where a: a line graph showing the changes in ammonia nitrogen concentration; b: a line graph showing the changes in nitrate nitrogen concentration; c: a line graph showing the changes in nitrite nitrogen concentration.

[0034] Figure 7 A horizontal bar chart of microbial phyla is shown;

[0035] Figure 8 A heatmap at the genus level for microorganisms is shown;

[0036] Figure 9 The graph shows the changes in pollutant concentrations in the microbial agent; where a: changes in divalent manganese concentration in reactors with only manganese added; b: changes in divalent manganese concentration in reactors with both manganese and ammonia nitrogen added; c: changes in ammonia nitrogen concentration in reactors with only ammonia nitrogen added; d: changes in ammonia nitrogen concentration in reactors with both manganese and ammonia nitrogen added. Detailed Implementation

[0037] It should be understood that the scope of protection of this application is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of this application is for describing specific embodiments and not for limiting the scope of protection of this application; in the specification and claims of this application, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0038] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description in this application, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this application may be used to implement this application.

[0039] Example 1

[0040] Domestication and enrichment of microorganisms

[0041] (1) Experimental setup

[0042] Microorganisms were extracted from soil and river sediments using a two-step centrifugation method. Specifically, sediments and deionized water were mixed at a mass ratio of 1:9, glass beads were added, and the mixture was shaken for 2 hours. Then, differential centrifugation was used to remove impurities from the sediment suspension while retaining the microorganisms, creating a bacterial suspension which was stored at 4°C for subsequent inoculation. Alternatively, microorganisms were extracted from water samples using a filtration-oscillation method. Specifically, a 0.22 μm pore size filter membrane was used to trap microorganisms from the water sample. The membrane was then broken up and placed in deionized water. A vortex mixer was used to peel off the microorganisms from the membrane. Differential centrifugation was then used to remove the broken membrane and collect the microorganisms, creating a bacterial suspension which was stored at 4°C for subsequent inoculation.

[0043] In the batch experiment, an initial Mn(II) concentration of 55 mg / L and an NH4+ concentration of 50 mg / L were set. + -N concentration was used to prepare artificially simulated groundwater (detailed composition shown in Table 1). Sediment microorganisms were inoculated under aerobic and anaerobic conditions to establish reactors for microbial acclimatization and enrichment, and to explore the optimal conditions for pollutant removal. Furthermore, to enrich functional microorganisms in the contaminated site water, an initial Mn(II) concentration of 55 mg / L and an NH4+ concentration of 50 mg / L were set. + -N concentration was used to prepare artificially simulated groundwater, and aquatic microorganisms were inoculated under optimal pollutant removal conditions (reactor setup details are shown in Table 2). Samples were taken every 24 hours during all microbial cultivation processes to monitor Mn(II) and NH4+ concentrations in the reactor. + -N, NO2 - -N and NO3 - -N concentration.

[0044] Table 1. Composition of artificially simulated groundwater

[0045]

[0046]

[0047] Table 2. Microbial Acclimation Reactor Setup

[0048]

[0049]

[0050] (2) Sample characterization and determination

[0051] ① Determination of divalent manganese

[0052] Mn(II) was determined spectrophotometrically. Specifically, 25 mL of water sample filtered through a 0.22 μm filter, 10 mL of potassium pyrophosphate-sodium acetate buffer, and 3 mL of potassium periodate solution were added to a 50 mL colorimetric tube. The solution was then diluted to the mark with water, shaken well, and allowed to develop color for 10 min. Using a zero-concentration blank as a reference, the absorbance of the solution was measured at 525 nm using a 50 mm cuvette. The Mn(II) concentration was calculated by substituting the values ​​into a standard curve.

[0053] ② Determination of trinitrogen

[0054] Ammonia nitrogen was determined using the Nessler's reagent spectrophotometric method. Specifically, 2 mL of water sample filtered through a 0.22 μm filter was added to a 50 mL colorimetric tube and diluted to the 50 mL mark. Then, 1.0 mL of sodium potassium tartrate solution and 1.5 mL of Nessler's reagent were added and mixed thoroughly. The spectrophotometer was adjusted to a wavelength of 420 nm, and after 15 minutes of development, the absorbance was measured using a zero-concentration blank as a reference. The ammonia nitrogen concentration was calculated by substituting the values ​​into the standard curve.

[0055] Nitrite nitrogen was determined using ultraviolet spectrophotometry. Specifically, 0.4 mL of water sample filtered through a 0.22 μm filter was added to a 50 mL colorimetric tube, and diluted with water to the 50 mL mark. Then, 1 mL of colorimetric reagent was added and mixed thoroughly. After 20 minutes of development, the spectrophotometer wavelength was adjusted to 540 nm, and the absorbance was measured using a zero-concentration blank as a reference. The nitrite nitrogen concentration was calculated by substituting the values ​​into a standard curve.

[0056] Nitrate nitrogen was determined using ultraviolet spectrophotometry. Specifically, 2 mL of water sample filtered through a 0.22 μm filter was added to a 50 mL colorimetric tube and diluted to the 50 μm mark with water. Then, 1 mL of 1 mol / L hydrochloric acid solution and 0.1 mL of 0.8% sulfamic acid solution were added and mixed thoroughly. The spectrophotometer was adjusted to wavelengths of 220 nm and 275 nm, respectively. Using a zero-concentration blank as a reference, the absorbance was measured. The absorbance was corrected using the following formula: A = A 220 -2A 275 The nitrate nitrogen concentration was obtained by substituting the calculated absorbance into the standard curve.

[0057] Example 2

[0058] Results and Discussion

[0059] (1) Selection of sediments

[0060] Through the measurement of the physicochemical properties of sediment samples from contaminated sites (see details) Figure 1 Taking into account the representativeness and pollution level of each site, upstream soil and downstream river sediments were ultimately selected to prepare bacterial solutions for subsequent experiments.

[0061] (2) Oxidation of divalent manganese

[0062] Under aerobic and anaerobic conditions, reactors were established using upstream soil sediment microorganisms and downstream river sediment microorganisms as inoculations, with divalent manganese and / or ammonia nitrogen as pollutants. The optimal conditions for the removal of divalent manganese pollutants were explored while simultaneously acclimating the microorganisms. Figure 2 By the end of the third cycle, the reactor using upstream soil microorganisms as inoculum showed the highest removal efficiency of divalent manganese under aerobic conditions, reaching 88.8% with only divalent manganese added. When both ammonia nitrogen and divalent manganese were added simultaneously, the removal efficiency reached 95.1%. Downstream river sediment microorganisms were the next most effective, with a removal efficiency of approximately 84.64% when only divalent manganese was added, and approximately 82.3% when both ammonia nitrogen and divalent manganese were added.

[0063] In comparison, the removal efficiency of divalent manganese under anaerobic conditions is much lower than that under aerobic conditions. Figure 2 By the end of the third cycle, under anaerobic conditions, the removal efficiency of divalent manganese (DMC) in the reactor using upstream soil microorganisms as inoculum and adding only DMC was 69.3%, while the removal efficiency in the reactor adding both ammonia nitrogen and DMC was 57.9%. Using downstream river sediment microorganisms as inoculum, the removal efficiency of DMC in the reactor using only DMC was 62.05%, while the removal efficiency in the reactor adding both ammonia nitrogen and DMC was 57.2%. Therefore, considering all experimental results, the removal efficiency of DMC is optimal under aerobic conditions.

[0064] Furthermore, to acclimatize and enrich the microorganisms in the water, a reactor was established under optimal conditions for manganese divalent removal, using surface water and groundwater microorganisms from the polluted area as inoculum and manganese divalent and / or ammonia nitrogen as pollutants. By the end of the third cycle, under the condition of adding only manganese divalent, the removal efficiency of manganese divalent from surface water microorganisms was 72.5%, and the removal efficiency from groundwater microorganisms was 36.2%; under the condition of simultaneously adding manganese divalent and ammonia nitrogen, the removal efficiency of manganese divalent from surface water microorganisms was 82.5%, and the removal efficiency from groundwater microorganisms was 35.5%. Figure 3 ).

[0065] (3) Removal of ammonia nitrogen

[0066] Under aerobic and anaerobic conditions, reactors were established using upstream soil sediment microorganisms and downstream river sediment microorganisms as inoculations, with divalent manganese and / or ammonia nitrogen as pollutants. The optimal conditions for ammonia nitrogen pollutant removal were explored while simultaneously acclimating the microorganisms. Figure 4By the end of the third cycle, the reactor using upstream soil microorganisms as inoculum showed the highest ammonia nitrogen removal efficiency under aerobic conditions, reaching 73.4% with only ammonia nitrogen added, and 78.9% with both ammonia nitrogen and divalent manganese added. Downstream river sediment microorganisms were the next most effective, with a removal efficiency of approximately 65.6% with only divalent manganese added, and approximately 67.6% with both ammonia nitrogen and divalent manganese added.

[0067] In comparison, the removal efficiency of ammonia nitrogen under anaerobic conditions is slightly lower than that under aerobic conditions, but the difference is not significant. Figure 4 By the end of the third cycle, under anaerobic conditions, the ammonia nitrogen removal efficiency was 62.0% in the reactor using upstream soil microorganisms as inoculum and adding only ammonia nitrogen pollutants, and 59.3% in the reactor adding both ammonia nitrogen and divalent manganese. Using downstream river sediment microorganisms as inoculum, the ammonia nitrogen removal efficiency was 62.5% in the reactor using only ammonia nitrogen pollutants, and 52.2% in the reactor adding both ammonia nitrogen and divalent manganese. Therefore, considering all experimental results, ammonia nitrogen removal is most effective under aerobic conditions.

[0068] During the process of removing ammonia nitrogen by oxidizing it into nitrogen gas, nitrate nitrogen and nitrite nitrogen are generated as byproducts. Figure 5 No nitrate nitrogen formation was detected in the first cycle, but from the third week onwards, nitrate nitrogen showed a trend of first increasing and then decreasing. Nitrite nitrogen accumulation was consistently observed during the acclimatization period, and from the third cycle onwards, nitrite nitrogen showed a trend of first increasing and then decreasing.

[0069] Furthermore, to acclimatize and enrich the microorganisms in the water, a reactor was established under optimal ammonia nitrogen removal conditions, using surface water and groundwater microorganisms from the polluted area as inoculum and divalent manganese and / or ammonia nitrogen as pollutants. By the end of the third cycle, under the condition of adding only ammonia nitrogen, the ammonia nitrogen removal efficiency of surface water microorganisms was 63.8%, and that of groundwater microorganisms was 59.5%; under the condition of simultaneously adding divalent manganese and ammonia nitrogen, the ammonia nitrogen removal efficiency of surface water microorganisms was 65.7%, and that of groundwater microorganisms was 62.5%. The variation patterns of nitrate nitrogen and nitrite nitrogen in the aquatic microbial reactor were the same as those in soil and river sediment microbial reactors. Figure 6 ).

[0070] (4) Experimental Conclusion

[0071] Through a series of batch experiments, we found that regardless of the inoculum used, the removal efficiency of divalent manganese pollutants was significantly better under aerobic conditions than under anaerobic conditions, with upstream soil microorganisms showing the best performance. For ammonia nitrogen pollutant removal, although the removal efficiency was similar between aerobic and anaerobic conditions, the removal efficiency under aerobic conditions was slightly higher. Therefore, considering all factors, the microorganisms acclimated in the experiment showed the best removal efficiency for both divalent manganese and ammonia nitrogen pollutants under aerobic conditions.

[0072] Example 3

[0073] Constructing a composite functional microbial community

[0074] (1) Experimental setup

[0075] Destructive sampling was performed on a sediment reactor acclimated to optimal pollutant removal conditions. Experimental products were removed and microorganisms harvested by differential centrifugation. 16S rRNA sequencing was conducted to determine the community composition and functional microorganisms. Highly efficient manganese-oxidizing bacteria *Pseudomonas* and ammonia-removing bacteria *Bacillus* were screened using the streak plating method. These two bacteria were used as inoculum, with an initial Mn(II) concentration of 55 mg / L and an NH4+ concentration of 50 mg / L. + -N concentration was used to prepare artificially simulated groundwater (detailed composition shown in Table 1). A reactor was established under optimal pollutant removal conditions (reactor setup detailed in Table 3) to verify its function. Furthermore, functional microorganisms were mixed in specific proportions to construct a composite functional microbial community, and a reactor was established to determine its pollutant removal capacity.

[0076] Table 3. Reactor setup for pure culture performance verification

[0077]

[0078]

[0079] (2) Sample characterization and determination

[0080] ① For the determination methods of divalent manganese and trinitrogen, please refer to (2) Sample characterization and determination in the above implementation 1.

[0081] ② Microbial sequencing

[0082] Total genomic DNA was extracted from the microbial community according to the kit instructions. The quality of the extracted genomic DNA was assessed using 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using NanoDrop2000. Using the extracted DNA as a template, PCR amplification was performed on the V3-V4 variable region of the 16S rRNA gene. The PCR products were recovered using 2% agarose gel electrophoresis, and purified using a DNA gel purification kit. The purified amplicon was sent to Shanghai Meiji Biotechnology Co., Ltd. for 16S rRNA high-throughput sequencing.

[0083] Example 4

[0084] Results and Discussion

[0085] (1) Microbial diversity analysis and identification of functional microorganisms

[0086] The phylum-level composition of microorganisms in each reactor under optimal pollutant removal conditions is as follows: Figure 7 As shown, in reactors where only divalent manganese was added as a pollutant, Proteobacteria had the highest abundance (85.17% in soil sediment reactors and 78.12% in river sediment reactors); in reactors where only ammonia nitrogen was added as a pollutant, Bacteroidota had the highest abundance (39.91% in soil sediment reactors and 53.63% in river sediment reactors); and in reactors where both divalent manganese and ammonia nitrogen were added as pollutants, Proteobacteria had the highest abundance (68.28% in soil sediment reactors and 67.99% in river sediment reactors).

[0087] The composition of microorganisms at the genus level in each reactor under optimal pollutant removal conditions is as follows: Figure 8 As shown in the figure, the top ten most abundant microorganisms include Pseudomonas, Nitrosospira, Nitrosomonas, and Bacillus. Among them, Nitrosomonas and Bacillus were enriched for removing ammonia nitrogen; Pseudomonas was enriched for heavy metal resistance. After comprehensive consideration, Bacillus and Pseudomonas were ultimately selected, and their ability to remove pollutants by individual bacteria was verified to construct a composite functional microbial community.

[0088] (2) Validation of single-strain function

[0089] Under optimal pollutant removal conditions, reactors were established using Pseudomonas and Bacillus as inoculants, respectively, and divalent manganese and / or ammonia nitrogen as pollutants, to verify their removal capabilities for divalent manganese and ammonia nitrogen. Figure 9By the end of the third cycle, the removal efficiency of Pseudomonas aeruginosa for divalent manganese was approximately 65.1% with only divalent manganese added, and approximately 63.8% with both divalent manganese and ammonia nitrogen added. The removal efficiency of Bacillus aureus for divalent manganese was approximately 59.7% with only divalent manganese added, and approximately 59.3% with both divalent manganese and ammonia nitrogen added. Comparatively, Pseudomonas aeruginosa demonstrated a stronger removal capacity for divalent manganese.

[0090] To verify the removal capabilities of the two microorganisms for ammonia nitrogen pollutants, reactors were established using ammonia nitrogen and / or divalent manganese as pollutants. Figure 9 By the end of the third cycle, the removal efficiency of ammonia nitrogen by *Pseudomonas* was approximately 46.7% with only ammonia nitrogen added, and approximately 43.5% with both divalent manganese and ammonia nitrogen added. The removal efficiency of ammonia nitrogen by *Bacillus* was approximately 48.9% with only ammonia nitrogen added, and approximately 47.8% with both divalent manganese and ammonia nitrogen added. Comparatively, *Bacillus* demonstrated a stronger ability to remove ammonia nitrogen.

[0091] (3) Establishment and functional verification of complex functional microbial communities

[0092] To further enhance the ability of microorganisms to remove pollutants, functionally validated Pseudomonas and Bacillus were mixed in a 1:1 ratio to prepare a composite functional microbial community. To verify the pollutant removal capacity of the microbial agent, a reactor was constructed using the prepared agent as inoculum and divalent manganese and / or ammonia nitrogen as pollutants. Figure 9 By the end of the third cycle, the removal efficiency of divalent manganese was approximately 68.3% with only divalent manganese added, and approximately 65.8% with the simultaneous addition of divalent manganese and ammonia nitrogen; the removal efficiency of ammonia nitrogen was approximately 53.2% with only ammonia nitrogen added, and approximately 52.0% with the simultaneous addition of divalent manganese and ammonia nitrogen. In addition, some nitrate and nitrite nitrogen were generated as byproducts during the ammonia nitrogen removal process, and their concentration variation patterns were consistent with those of the sediment microbial reactor.

[0093] Based on the above-described solution, embodiments of this invention provide a method for simultaneous manganese and ammonia removal using a composite microbial community, the method comprising the following steps:

[0094] After preliminary treatment of the samples to be extracted from the contaminated site, microorganisms are extracted from the samples and prepared into a bacterial solution. The microorganisms extracted from the bacterial solution are domesticated and enriched to obtain a complex microbial community. The complex microbial community includes highly efficient manganese oxidizing bacteria and ammonia nitrogen removing bacteria. The highly efficient manganese oxidizing bacteria is Pseudomonas, and the ammonia nitrogen removing bacteria is Bacillus. The complex microbial community is inoculated into a reactor containing water pollutants of manganese and ammonia nitrogen. Under anaerobic or aerobic conditions, the complex microbial community in the reactor is cultured for multiple cycles to simultaneously remove manganese and ammonia nitrogen pollutants from the water to be treated.

[0095] In one embodiment, the sample to be extracted includes upstream soil, downstream river sediment, surface water, and groundwater.

[0096] In one embodiment, the sample to be extracted includes upstream soil and downstream river sediments.

[0097] In one embodiment, after preliminary treatment of the sample to be extracted from the contaminated site, the microorganisms in the sample to be extracted are extracted and prepared into a bacterial solution, including:

[0098] Microorganisms were extracted from upstream soil and downstream river sediments using a two-step centrifugation method. Specifically, downstream river sediments were mixed with deionized water at a mass ratio of 1:9, glass beads were added, and the mixture was shaken for 2 hours. Then, the mixture was centrifuged at a differential speed to remove impurities from the downstream river sediment suspension while retaining the microorganisms. The resulting bacterial suspension was stored at 4°C for subsequent inoculation.

[0099] In one embodiment, after preliminary treatment of the sample to be extracted from the contaminated site, the microorganisms in the sample to be extracted are extracted and prepared into a bacterial solution, including:

[0100] Microorganisms were extracted from surface water and groundwater using a filtration oscillation method. Specifically, the method involved: using a filter membrane with a pore size of 0.22 μm to retain microorganisms in surface water and groundwater, then breaking the filter membrane and placing it in deionized water, using a vortex shaker to peel off the microorganisms from the filter membrane, using differential centrifugation to remove the broken filter membrane and collect the microorganisms, and preparing a bacterial solution, which was then stored at 4°C for subsequent inoculation.

[0101] In one embodiment, the domestication and enrichment of the indigenous microorganisms extracted from the bacterial solution includes:

[0102] The initial Mn(II) concentration was set at 55 mg / L, and the NH4+ concentration was set at 50 mg / L. + -N concentration was used to prepare artificially simulated groundwater, and extracted microorganisms were inoculated under aerobic and anaerobic conditions respectively. A reactor was then established to domesticate and enrich the microorganisms.

[0103] In one embodiment, the concentration ratio of Pseudomonas to Bacillus is 1:1.

[0104] In one embodiment, the complex microbial community also includes Nitrosospira and Nitrosomonas.

[0105] In one embodiment, the complex microbial community in the reactor is cultured under aerobic conditions for at least three cycles to simultaneously remove manganese and ammonia nitrogen pollutants from the water to be treated; wherein each cycle is at least four days.

[0106] Secondly, embodiments of this application provide a kit for removing bioavailable vanadium from soil, comprising: a complex microbial community, wherein the complex microbial community includes highly efficient manganese oxidizing bacteria and ammonia nitrogen removing bacteria, wherein the highly efficient manganese oxidizing bacteria is Pseudomonas, and the ammonia nitrogen removing bacteria is Bacillus, and the concentration ratio of Pseudomonas to Bacillus is 1:1.

[0107] In one embodiment, the complex microbial community also includes Nitrosospira and Nitrosomonas.

[0108] In summary, the method provided by this invention achieves a manganese removal efficiency of approximately 68.3% when only manganese is added, and approximately 65.8% when both manganese and ammonia nitrogen are added simultaneously. Similarly, the ammonia nitrogen removal efficiency is approximately 53.2% when only ammonia nitrogen is added, and approximately 52.0% when both manganese and ammonia nitrogen are added simultaneously. This effectively reduces the levels of manganese and ammonia nitrogen in water bodies and mitigates ecological and health risks. Therefore, this technical solution combines a complex microbial community of highly efficient manganese-oxidizing bacteria and ammonia nitrogen-removing bacteria to simultaneously remove manganese and ammonia nitrogen pollutants from water bodies under different environmental conditions. This method not only offers advantages such as high efficiency, environmental friendliness, and low cost, but also adapts to various water pollution conditions, providing a sustainable water remediation solution with broad application prospects.

[0109] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for simultaneous manganese and ammonia removal using a composite microbial community, characterized in that, The method includes the following steps: After preliminary treatment of the samples to be extracted from the contaminated site, microorganisms are extracted from the samples and prepared into bacterial solutions. The microorganisms extracted from the bacterial solution are domesticated and enriched to obtain a complex microbial community; wherein, the complex microbial community includes highly efficient manganese oxidizing bacteria and ammonia nitrogen removing bacteria, wherein the highly efficient manganese oxidizing bacteria is Pseudomonas and the ammonia nitrogen removing bacteria is Bacillus. The composite microbial community is inoculated into a reactor containing water to be treated containing manganese and ammonia nitrogen pollutants; Under anaerobic or aerobic conditions, the complex microbial community in the reactor is cultured for multiple cycles to simultaneously remove manganese and ammonia nitrogen pollutants from the water to be treated.

2. The method according to claim 1, characterized in that, The samples to be extracted include upstream soil, downstream river sediments, surface water, and groundwater.

3. The method according to claim 2, characterized in that, The samples to be extracted include upstream soil and downstream river sediments.

4. The method according to claim 2, characterized in that, After preliminary treatment of the samples to be extracted from the contaminated site, microorganisms are extracted from the samples and prepared into a bacterial solution, including: Microorganisms were extracted from upstream soil and downstream river sediments using a two-step centrifugation method. Specifically, downstream river sediments were mixed with deionized water at a mass ratio of 1:9, glass beads were added, and the mixture was shaken for 2 hours. Then, the mixture was centrifuged at a differential speed to remove impurities from the downstream river sediment suspension while retaining the microorganisms. The resulting bacterial suspension was stored at 4°C for subsequent inoculation.

5. The method according to claim 2, characterized in that, After preliminary treatment of the samples to be extracted from the contaminated site, microorganisms are extracted from the samples and prepared into a bacterial solution, including: Microorganisms were extracted from surface water and groundwater using a filtration oscillation method. Specifically, the method involved: using a filter membrane with a pore size of 0.22 μm to retain microorganisms in surface water and groundwater, then breaking the filter membrane and placing it in deionized water, using a vortex shaker to peel off the microorganisms from the filter membrane, using differential centrifugation to remove the broken filter membrane and collect the microorganisms, and preparing a bacterial solution, which was then stored at 4°C for subsequent inoculation.

6. The method according to any one of claims 1-5, characterized in that, The process of domesticating and enriching the indigenous microorganisms extracted from the bacterial solution includes: The initial Mn(II) concentration was set at 55 mg / L, and the NH4+ concentration was set at 50 mg / L. + -N concentration was used to prepare artificially simulated groundwater, and extracted microorganisms were inoculated under aerobic and anaerobic conditions respectively. A reactor was then established to domesticate and enrich the microorganisms.

7. The method according to claim 6, characterized in that, The concentration ratio of Pseudomonas to Bacillus was 1:

1.

8. The method according to claim 6, characterized in that, The complex microbial community also includes Nitrosospira and Nitrosomonas.

9. The method according to any one of claims 1-5, characterized in that, Under aerobic conditions, the complex microbial community in the reactor is cultivated for at least three cycles to simultaneously remove manganese and ammonia nitrogen pollutants from the water to be treated; wherein each cycle is at least four days.

10. A kit for simultaneous manganese and ammonia removal using a composite microbial community, characterized in that, include: The complex microbial community includes highly efficient manganese oxidizing bacteria and ammonia nitrogen removing bacteria. The highly efficient manganese oxidizing bacteria is Pseudomonas, and the ammonia nitrogen removing bacteria is Bacillus. The concentration ratio of Pseudomonas to Bacillus is 1:1.

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