Alcaligenes faecalis UA strain, product and application thereof, and method for repairing acid mine environment by acid-resistant urease-producing bacteria

Abstract

The invention discloses an alcaligenes faecalis UA strain, a product and application thereof, and a method for repairing an acid mine environment by acid-resistant urease-producing bacteria, and belongs to the technical fields of industrial microorganisms, microorganism application materials and environment repairing. The UA strain CGMCC No.33976 is found for the first time to produce the acid-resistant urease, the urease is stably produced in the environment with the optimum acidity of pH 4.5-pH 6.5 and the initial pH of 3.5-pH 7.5, and the enzyme production activity is the highest in the environment with the initial pH of 5.5-pH 6.5. The UA strain can effectively reduce acidity and remove metal ions in water in an acid mine wastewater real water sample, the removal rate of aluminum, manganese, zinc and cadmium exceeds 99%, and the removal rate of calcium, iron and lead is 92%-98%. The UA strain is screened from the acid mine environment, has biological safety, has industrial purposes of AMD in-situ direct treatment and acid mine environment in-situ remediation, does not need to add chemical agents to adjust the pH value of the environment, and has ecological safety and environmental sustainability.

Description

Technical Field

[0001] The present invention relates to an Alcaligenes faecalis, in particular to an Alcaligenes faecalis UA strain as an acid-tolerant urease-producing microorganism, and an acid mine environment restoration method using the strain. It belongs to the technical fields of industrial microorganisms, microbial application materials, and environmental restoration technology. Background Art

[0002] Acid mine drainage (AMD) has a series of characteristics such as high acidity (pH 2-3), high sulfate concentration, high heavy metal concentration, large water volume, and a long formation time span, and belongs to the environmental problems left over from mineral extraction worldwide. Due to the relatively complex formation mechanism of acid mine drainage, it is jointly affected by complex and variable factors such as deposit type, mining method, climate conditions, and management technology. Therefore, the effect of source control is limited, and it is necessary to combine more with later treatment. Acid mine drainage is the key link in the acid mine environmental problem chain, and its treatment is a basic technology for mine environment restoration.

[0003] Microbial-induced mineralization is a technology that uses urease produced during the physiological and biochemical processes of microorganisms to convert free heavy metals in the environment into precipitated substances. It belongs to the environmental bioremediation technology and has the technical advantages of industry consensus. It is an ideal technical path for the restoration of extreme or harsh environments such as acid mine environments. The basis for developing a technical solution for applying microbial-induced mineralization technology to acid mine environment restoration is the screening of urease-producing strains. The target strain should possess at least four basic biological characteristics: First, it can survive normally in acid mine drainage, and the optimal growth pH condition is within the acidic range of acid mine drainage; Second, it has stable and high urease activity in acid mine drainage; Third, it can remove harmful heavy metals in acid mine drainage; Fourth, the above three functions can be stably expressed in the real water sample of acid mine drainage, rather than in a simulated solution.

[0004] The prior art "In-situ reduction of the migration risk of heavy metal pollution in abandoned slag piles in Southwest China using the mineralization of microorganisms" (Qiao Suyu, Sichuan University, 2021) discloses two urease-producing strains kp-4 and kp-22, both of which are bacteria of the genus Sarcina ( Sporosarcina sp.), with the characteristic of alkaliphily, and the optimal acidic range of urease activity is between pH 8-9. The treatment of acid mine drainage by the two strains is to first immobilize the bacteria by embedding them in sodium alginate on different materials, and then place them in the wastewater environment. The optimal pH for the reaction of urease in the solid material is 8.0-9.0. The prior art "Experimental study on the synergistic treatment of acid mine drainage by urease-producing bacteria and sulfate-reducing bacteria" (Fan Xiao, Chengdu University of Technology, 2023) discloses a urease-producing strain U-3, which belongs to Paenibacillus chibaensis ( Paenibacillus chibensis). This strain can grow in an environment with an initial pH of 2 - 7, but the optimum is pH 7. Under the laboratory-simulated acidic mine wastewater (initial pH 2 - 4) environment, strain U-3 can grow, but after 14 days of fermentation, the pH value in the solution only increases to 5.3, and the induced precipitation of metal ions mainly focuses on iron elements (total iron, Fe 2+ ), and the removal rate of Mn 2+ is only 45.73%.

[0005] Currently, the reported urease-producing bacteria mainly include the genus Bacillus ( Bacillus ), the genus Sarcina ( Sporoscarcina pasteurii ), the genus Pseudomonas ( Pseudomonas ), the genus Acinetobacter ( Acinetobacter ), etc. The prior art 2019102019024 (CN 109926448 A) discloses an Alcaligenes faecalis FC strain CGMCC No. 16874, which can produce urease and solidify heavy metals in soil, but the fermentation conditions are pH 6.5 - 8.5, and the urease-producing activity is the best at pH 8.0.

[0006] The urease-producing bacteria disclosed in the prior art do not possess the aforementioned four basic biological characteristics, and thus do not have the industrial application prospect of directly treating acidic mine wastewater and environmental remediation. Summary of the Invention

[0007] The purpose of the present invention is to provide a new strain of Alcaligenes faecalis ( Alcaligenes faecalis ). This strain can stably grow in an acidic mine water environment, maintain urease-producing activity, and mineralize various metals including toxic and harmful metals, and has the industrial use of acid-tolerant urease-producing microorganisms.

[0008] To achieve the above purpose, the present invention first provides a new strain of Alcaligenes faecalis, and its technical solution is as follows.

[0009] A strain of Alcaligenes faecalis ( Alcaligenes faecalis ) UA, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit date of March 25, 2025, and the deposit number CGMCC No. 33976.

[0010] The Alcaligenes faecalis UA strain CGMCC No. 33976 was screened from a contaminated soil sample in the mining area of Dexing Copper Mine, Jiangxi Province. It was preliminarily identified as a strain of Alcaligenes faecalis ( Alcaligenes faecalis ), named Alcaligenes faecalis ( Alcaligenes faecalis ) UA strain ( Alcaligenes faecalis UA).

[0011] Experimental data of the present invention prove that the Alcaligenes faecalis UA strain CGMCC No. 33976 has an industrial use of acid-resistant urease production. Accordingly, the present invention first provides a bacterial agent product containing the Alcaligenes faecalis UA strain, and its technical solution is as follows.

[0012] A bacterial agent, comprising the above-mentioned Alcaligenes faecalis ( Alcaligenes faecalis ) UA strain CGMCC No. 33976.

[0013] The present invention also provides the following application-related technical solutions.

[0014] The application of the above-mentioned Alcaligenes faecalis UA strain CGMCC No. 33976 is as an acid-resistant urease-producing bacterium.

[0015] The application of the above-mentioned bacterial agent is as an acid-resistant urease-producing bacterium agent.

[0016] The above application as an acid-resistant urease-producing bacterium or as an acid-resistant urease-producing bacterium agent may specifically be the following solutions.

[0017] The above application is for environmental bioremediation engineering.

[0018] The above application is in the bioremediation of acidic mine environments. Preferably, the application is in the biological treatment of acidic mine wastewater.

[0019] The above application is for metal precipitation and fixation and / or reduction of the acidity of the water environment. Preferred metals include Al 3+ and / or Mn 2+ and / or Zn 2+ and / or Cd 2+ and / or Ca 2+ and / or Fe 3+ and / or Pb 2+ .

[0020] In each of the above application solutions, the suitable culture conditions for the UA strain CGMCC No. 33976 are pH 5.5 to pH 6.5 and a temperature of 35°C. Further optimization of the culture conditions is pH 5.5.

[0021] The present invention also provides a technical solution for the bioremediation of acidic mine environments by acid-resistant urease-producing bacteria. Specifically as follows.

[0022] A method for bioremediating acidic mine environments by acid-resistant urease-producing bacteria, inoculating the Alcaligenes faecalis UA strain CGMCC No. 33976 into acidic mine wastewater, and maintaining the biomass OD of the strain in the culture system 600 = 0.9 to 1.0 during the fermentation process.

[0023] The most common sources of acid mine drainage include ore piles, tailings ponds, and open-pit mines. Different remediation solutions are required for acid mine drainage from different sources. Considering the mine environment, the wastewater generated in the ore dressing process is prone to form acid mine drainage in the form of depressions or water inrush from mine pits due to natural precipitation or industrial water use. The solution for treating acid mine drainage depressions or water inrush from mine pits using UA strains can be as follows: After precipitating the inrush water and filtering out the sediment, introduce it into a microbial sewage treatment tank. Estimate the required biomass of UA strains and urea according to the initial physical and chemical characteristics of the sewage; add urea, stir evenly, and inoculate with UA strains; ferment under the suitable temperature conditions for Alcaligenes faecalis. After sedimentation for a period of time, detect whether the wastewater meets the discharge standards.

[0024] In the method for repairing acid mine environment with acid-tolerant urease-producing bacteria, inoculating UA strains into the acid mine wastewater to be fermented can be inoculating UA strains (containing bacterial liquid) or bacterial agents (containing bacterial agent solution) into the acid mine wastewater; or placing a carrier fixed with Alcaligenes faecalis UA strains or bacterial agents in the acid mine wastewater.

[0025] Compared with the prior art, the beneficial effects of the present invention are: (1) Alcaligenes faecalis ( Alcaligenes faecalis ) UA strain CGMCC No. 33976 is a urease-producing bacterium. The UA strain has adaptability in the entire acidic environment starting from an initial pH of 3.5, and its optimal growth is between pH 4.5 and pH 6.5; the UA strain has good urease-producing ability in the environment with an initial pH of 3.5 to pH 7.5, and its activity is the highest at an initial pH of 5.5 to pH 6.5. The UA strain is an acid-tolerant urease-producing bacterium, which is a new strain different from the existing urease-producing Alcaligenes faecalis. (2) The urease activity of the UA strain is higher than that of the existing urease-producing Alcaligenes faecalis. (3) In the real water sample environment of acid mine drainage, the UA strain can ferment normally and exhibits two performances of effectively reducing the acidity of the water body and removing metals from the water body, and has the industrial uses of in-situ treatment of acid mine drainage and in-situ repair of acid mine environment. The UA strain is a newly disclosed urease-producing strain that simultaneously has acid tolerance, can effectively settle metal ions, and can be directly used for the biological treatment of real water samples of acid mine drainage. (4) The removal rates of four typical toxic and harmful metals, namely Al 3+ 、Mn 2+ 、Zn 2+ 、Cd 2+ in the real water sample of acid mine surface water by the UA strain are all above 99%, and for Ca 2+ 、Fe 3+ 、Pb 2+The removal rates are all between 92% and 98%. (5) The UA strain also has the function of calcium precipitation. (6) The UA strain CGMCC No. 33976 is an acid-tolerant urease-producing bacterium screened from an acidic mine environment and has biological safety. When applied to the in-situ remediation of an acidic mine environment, there is no need to input chemical agents to adjust the environmental pH, and it has ecological safety and environmental sustainability. Description of the Drawings

[0026] Figure 1 It is the growth diagram of the UA strain on an acidic screening plate.

[0027] Figure 2 It is the phylogenetic tree of the Alcaligenes faecalis UA strain (based on 16S rDNA).

[0028] Figure 3 It is the OD of the UA strain CGMCC No. 33976 under different initial pH conditions 600 value and the final pH.

[0029] Figure 4 It is the tolerance effect of the UA strain CGMCC No. 33976 to heavy metals.

[0030] Figure 5 It is the metal ion removal rate of the acidic mine wastewater in the experimental group. (a) Al 3+ , Ca 2+ , Fe 3+ , Mn 2+ , (b) Cu 2+ , Zn 2 + , Cd 2+ , Pb 2+ .

[0031] Figure 6 It is the change of the pH value of the culture system during the cultivation of the UA strain CGMCC No. 33976 in acidic mine wastewater.

[0032] Figure 7 It is the change of the NH4 + concentration of the culture system during the cultivation of the UA strain CGMCC No. 33976 in acidic mine wastewater. Detailed Embodiments

[0033] The technical solutions of the present invention will be further described below in conjunction with the drawings.

[0034] The samples, culture media, and solutions used in the examples are as follows.

[0035] Soil sample: In the polluted soil area of Dexing Copper Mine in Jiangxi Province in late June 2024, a sampling shovel was used to remove the floating soil and weeds on the soil surface, the surface soil was dug open, and samples were collected from the 5 cm - 20 cm layer of the soil. The obtained soil samples were stored in sterilized self-sealing bags, marked with sample information, placed in a foam box with dry ice, and transported back to the laboratory under dark conditions.

[0036] Acid mine drainage (AMD) water sample: In the Dexing Copper Mine mining area in Jiangxi Province, a typical acidic water body (reddish-brown in color and low pH) was selected at the surface water accumulation area, water samples were collected, allowed to stand until the turbid water body was initially separated, the upper suspended particles were filtered off, about 1000 mL of the filtrate was taken, stored in a polyethylene bottle, marked with sample information, placed in a foam box with dry ice, and transported back to the laboratory under dark conditions.

[0037] Urea nutrient broth medium: Peptone 10 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, the pH was adjusted using 1 mol / L H2SO4 solution, after adding the indicator, it was sterilized at 121 °C and 101 kPa in a high-pressure steam sterilizer for 20 min, and after taking it out, 20 g / L of urea was added (the urea solution was separately sterilized by filtration through a 0.22 μm filter membrane); for standby. The indicator was bromocresol green-methyl red mixed indicator, and the addition amount was 2%.

[0038] Acidic screening plate: Nutrient broth medium 18 g / L, agar 25 g / L, the initial pH of the medium was adjusted to 4.0 using 1 mol / L H2SO4 solution, sterilized at high temperature and high pressure for 20 min, after taking it out, the indicator was added, and 20 g / L of urea was added (the urea solution was separately sterilized by filtration through a 0.22 μm filter membrane); for standby. The indicator was bromocresol green-methyl red mixed indicator, and the addition amount was 2%.

[0039] Activation medium for strain UA: Peptone 10 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, the initial pH of the medium was adjusted to 5.0 using 1 mol / L H2SO4 solution, placed in a high-pressure steam sterilizer and sterilized at 121 °C and 101 kPa for 20 min, after taking it out, 20 g / L of urea was added (the urea solution was separately filtered and sterilized using a 0.22 μm filter membrane); for standby.

[0040] Bromocresol green-methyl red mixed indicator: Solution Ⅰ and Solution Ⅱ were mixed evenly at a ratio of 3:1 (V / V), color change range: dark red below pH 5.0, gray-green at pH 5.1, green above pH 5.2.

[0041] Solution Ⅰ: 0.1 g of bromocresol green was dissolved in 95% ethanol and diluted to 100 mL with 95% ethanol.

[0042] Solution II: Dissolve 0.2 g of methyl red in 95% ethanol and dilute it to 100 mL with 95% ethanol.

[0043] Example 1 Enrichment screening and identification of acid-resistant strains.

[0044] 1. Screening of acid-resistant strains 1.1 Soil sample treatment In a laminar flow hood, weigh 10 g of soil sample and add it to a 250 mL conical flask containing 100 mL of sterile water. Cultivate it on a shaker at a rotation speed of 180 rpm / min under natural temperature conditions for 24 h to fully elute the microorganisms from the soil sample and disperse the bacterial cells and spores in the solution. After standing for 30 min, take the supernatant.

[0045] 1.2 Acid enrichment Take 4 mL of the supernatant and place it in a 250 mL conical flask, add 100 mL of urea nutrient broth medium (pH 3.5), and cultivate it on a shaker at a rotation speed of 180 rpm / min and a temperature of 35°C. During the cultivation process, measure the pH of the cultivation system every 24 h. When the pH of the cultivation system is 4.5 ± 0.5, add 1 mol / L H2SO4 solution to adjust the pH to 3.5 ± 0.2, and continue to cultivate until the OD 600 is between 0.6 and 0.8.

[0046] 1.3 Acidic plate screening In a laminar flow hood, take 100 μL of the solution from the cultivation system and dilute it serially to 10 -2 times, then inoculate it into the acidic screening plate with an inoculation amount of 50 μL. Invert the acidic screening plate and cultivate it in a constant temperature incubator at 35°C. The blue colonies that grow are the target colonies, named UA bacteria.

[0047] 1.4 Strain isolation and purification Preparation of UA strain purified solution: Pick the above-mentioned UA bacterial colonies with an inoculation loop and transfer them to the urea nutrient broth medium with an initial pH of 3.5, and cultivate them on a shaker at a rotation speed of 180 rpm / min and a temperature of 35°C until the OD 600 is between 0.8 and 1.0, which is the UA strain purified solution.

[0048] Take 100 μL of the UA strain purified solution in a laminar flow hood and dilute it to 10 -2 times, then inoculate it again into the acidic screening plate with an inoculation amount of 50 μL. Invert the acidic screening plate and cultivate it in a constant temperature incubator at 35°C.

[0049] Figure 1 is the growth diagram of the acidic screening plate of UA strains.

[0050] 2. Strain Identification The isolated and purified strain of UA bacteria has the following colony morphological characteristics: smooth surface, milky white, opaque, irregular edges, and slightly protruding in the middle. The strain is a Gram-negative bacterium. Scanning electron microscopy shows it to be short rod-shaped.

[0051] Extract the DNA of the isolated and purified strain of UA bacteria using the TSINGKE Plant DNA Extraction Kit (universal type), and perform PCR amplification after appropriate dilution (using the bacterial universal 16srDNA primers 27 F and 1492 R). PCR amplification materials: 45 μl of Tsingke 1×TSE101 Gold Mix, 2 μl of 27 F (10P), 2 μl of 1492 R (10P), and 1 μl of DNA template. Amplification program: pre-denaturation at 98°C for 2 min, cycling stage at 98°C for 10 s, 56°C for 10 s, 72°C for 10 s / kb, 35 cycles, extension stage at 72°C for 5 min, and storage stage at 4°C.

[0052] The PCR product was sent to the sequencing department of Beijing Tsingke Biotechnology Co., Ltd. Chengdu Branch for first-generation sequencing. Use the BLAST program on the NCBI website (http: / / www.ncbi.nlm.nih.gov / ) to compare and identify the sequence. The sequence is shown as SEQ ID NO.1, and finally a phylogenetic tree was constructed.

[0053] The isolated and purified strain of UA bacteria was identified as Alcaligenes faecalis ( Alcaligenes faecalis ).

[0054] Figure 2 This is the phylogenetic tree (based on 16S rDNA) of Alcaligenes faecalis UA strain.

[0055] 2.3 Strain Naming and Preservation The above-mentioned strain of Alcaligenes faecalis ( Alcaligenes faecalis ) was named Alcaligenes faecalis UA strain ( Alcaligenes faecalis UA).

[0056] The Alcaligenes faecalis UA strain was deposited in the China General Microbiological Culture Collection Center (CGMCC). The deposit address: Culture Collection Center of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101; The deposit date was March 25, 2025, and the deposit number was CGMCC No. 33976.

[0057] Example 2 The urease-producing ability of Alcaligenes faecalis UA strain CGMCC No. 33976 under different initial pH conditions.

[0058] Preparation of activated bacterial liquid of Alcaligenes faecalis UA strain (hereinafter referred to as UA strain activated liquid): Take 2 mL of purified liquid of UA strain (the same as in Example 1) and inoculate it into 100 mL of UA strain activation medium, and culture it overnight at a temperature of 35°C and a shaker speed of 180 rpm / min, which is the UA strain activated liquid.

[0059] Prepare 100 ml of urea nutrient broth media with different initial pH values (pH 3.5, 4.5, 5.5, 6.5, 7.5) respectively, inoculate 2 mL of UA strain activated liquid into each of them, and after culturing for 24 h at a temperature of 35°C and a shaker speed of 180 rpm / min, measure the urease activity (the urease activity is measured by the conductivity method, referring to Chinese Patent Application 2024102312410, Staphylococcus hominis UPB-1 strain and its products and applications, tailings solidification and repair method) (Table 1).

[0060] Table 1 Urease production ability of UA strain CGMCC No. 33976 under different initial pH conditions

[0061] The experiment shows that UA strain CGMCC No. 33976 has good urease production ability in the environment with an initial pH of 3.5 - pH 7.5. Among them, the urease activity in the environment with an initial pH of 5.5 - pH 6.5 is higher than that in the initial pH range environments on the left and right sides. Especially, the highest urease activity is in the pH 5.5 environment (19.76 mM urea hydrolysed·min -1 、1.976*10 4 U / mL).

[0062] Example 3

[0063] Optimal growth pH of Alcaligenes faecalis UA strain CGMCC No. 33976.

[0064] Prepare urea nutrient broth media with initial pH values of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 respectively, and inoculate 2% of UA strain activated liquid (the same as in Example 2) into each of them. After culturing for 24 h at a shaker speed of 180 rpm / min and a temperature of 35°C, measure the OD 600 and the final pH of each group of bacterial liquids to determine the optimal pH of the strain.

[0065] Figure 3 are the OD 600 values and the final pH of UA strain CGMCC No. 33976 under different initial pH conditions.

[0066] Table 2 OD of UA strain CGMCC No. 33976 under different initial pH conditions 600 value and final pH value

[0067] The experiment shows that starting from the initial pH 3.5 condition, the biomass (OD 600 value) of UA strain CGMCC No. 33976 begins to show a stable increase and remains at a relatively high level until the initial pH 7.0 condition. Starting from the initial pH 3.5 condition, the final pH of the culture medium also increases steadily. This indicates that it has growth adaptability to acidic environments with an initial pH of 3.5 and above. The optimal growth is between pH 4.5 and pH 6.5, and the highest biomass (OD 600 is 2.221) is obtained under the initial pH 5.5 condition.

[0068] Example 4

[0069] Tolerance of Alcaligenes faecalis UA strain CGMCC No. 33976 to heavy metal ions.

[0070] Inoculate the activated solution of UA strain (the same as in Example 2) into the urea nutrient broth medium containing different concentrations of heavy metals (the acidity of the medium is measured as the initial pH 6.0), and the inoculation amount is 2%. The heavy metal contents in each experimental group are shown in Table 3.

[0071] Table 3 Relevant data of heavy metal ion tolerance experiment

[0072] Each group is placed in a shaker at a rotation speed of 180 rpm / min and a temperature of 35 °C for 3 days. After 3 days, measure the OD 600 of the bacterial solution in each group to judge the tolerance of the strain to heavy metals.

[0073] Figure 4 shows the tolerance effect of UA strain CGMCC No. 33976 to heavy metals.

[0074] The experiment shows that UA strain CGMCC No. 33976 has tolerance to heavy metals, especially to Zn 2+ , Pb 2+ with good tolerance. When the concentrations of Zn 2+ , Pb 2+ are 500 mg / L, it still has a relatively high OD 600 value. When the concentrations of Cu 2+ , Cd 2+ are greater than 100 mg / L, the growth activity of Alcaligenes faecalis UA strain begins to decline significantly.

[0075] Example 5

[0076] Treatment of acid mine wastewater by Alcaligenes faecalis UA strain CGMCC No. 33976

[0077] An acid mine wastewater sample was taken for testing, showing a pH of 2.82, and the metal content data are shown in Table 4

[0078] Preparation of concentrated Alcaligenes faecalis UA strain solution (hereinafter referred to as concentrated UA strain solution): Inoculate Alcaligenes faecalis UA strain into the UA strain activation medium and culture it until the logarithmic growth phase (OD 600 = 0.6 - 0.8), centrifuge at 8000 rpm / min and 4 °C, and collect the lower concentrated solution, which is the concentrated UA strain solution

[0079] Experimental group: Take an acid mine wastewater sample and filter it through qualitative filter paper at medium speed. Add the concentrated UA strain solution to the filtrate until OD 600 = 0.5, add analytical pure urea reagent, and the addition amount is 20 g / L

[0080] Control group 1: Take an acid mine wastewater sample and filter it through qualitative filter paper at medium speed. Add the same amount of urea reagent as in the experimental group to the filtrate

[0081] Control group 2: Take an acid mine wastewater sample and filter it through qualitative filter paper at medium speed. Add the concentrated UA strain solution to the filtrate until OD 600 = 0.5, without adding urea

[0082] Both the experimental group and the control groups were placed in a constant temperature shaker at 35 °C with a rotation speed of 180 rpm for cultivation. During the cultivation process, measure the pH and NH4 + concentration of the experimental group solution every 12 h (the method for measuring NH4 + concentration refers to "Water Quality - Determination of Ammonia Nitrogen - Nessler's Reagent Spectrophotometric Method (HJ 535 - 2009)"); after 72 h of cultivation, measure the metal ion concentrations of each group. The relevant data are shown in Table 4 and Table 5 Figures 5 - 7 .

[0083] Table 4 Relevant data of acid mine environment restoration experiment (metal ions)

[0084] Table 5 Relevant data of acid mine environment restoration experiment (pH, NH4 + concentration)

[0085] Figure 5 is the removal rate of metal ions in the acid mine wastewater of the experimental group. (a) Al 3+ , Ca2+ 、 Fe 3+ 、 Mn 2+ ,(b) Cu 2+ 、 Zn 2 + 、 Cd 2+ 、 Pb 2+ . During the cultivation process, Cd 2+ 、 Zn 2+ changed almost synchronously, possibly due to coprecipitation; Figure 6 shows the change of pH value in the culture system during the cultivation of UA strain CGMCC No. 33976 in acid mine wastewater; Figure 7 shows the change of NH4 + concentration in the culture system during the cultivation of UA strain CGMCC No. 33976 in acid mine wastewater.

[0086] Table 4, Table 5 and Figures 5 - 7 The data show that the pH of the acid mine wastewater sample in the experimental group increased significantly, rising from 2.82 to 9.37 after 72 h of remediation, and metal ions were effectively removed. Among them, the removal rates of four typical toxic and harmful metals, Al 3+ 、 Mn 2+ 、 Zn 2+ 、 Cd 2+ were all above 99%, and the removal rates of Ca 2+ 、 Fe 3+ 、 Pb 2+ were all between 92% and 98%. It shows that UA strain has the effect of removing metals in acid mine wastewater and the effect of calcium precipitation. During the cultivation process, both the pH value and NH4 + concentration in the culture system increased significantly with the progress of cultivation. Analysis of the data of the experimental group and the control group showed that the decrease in acidity and the removal of metals in the acid mine wastewater sample were due to the combined action of UA strain CGMCC No. 33976 and urea, and a single action could not have an obvious remediation effect on the acid mine wastewater sample. It shows that the treatment of acid mine wastewater sample by UA strain is due to its urease activity.

[0087] According to experimental observations, during the fermentation and cultivation of the real acid mine wastewater sample, the biomass of UA strain in the culture system should be maintained at OD 600 = 0.9 - 1.0.

[0088] Experiments in Groups 2 to 5 show that: (1) The Alcaligenes faecalis UA strain CGMCC No. 33976 of the present invention has urease-producing activity and is a urease-producing bacterium. (2) The UA strain grows optimally in an environment with an initial pH of 4.5 to 6.5, has stable urease-producing activity in acidic environments with an initial pH of 3.5 or above, and has the highest activity at an initial pH of 5.5 to 6.5, belonging to a typical acid-tolerant urease-producing bacterium. Compared with the prior art urease-producing bacteria, such as the Sarcina sp. strains kp-4 and kp-22, and the Alcaligenes faecalis CGMCC No. 16874, which are suitable for growing in a slightly alkaline environment and have the highest urease-producing activity under alkaline conditions, or the Paenibacillus chibaensis strain U-3, which can tolerate acidity but is suitable for growing in a neutral environment and has poor efficiency in sedimenting metal ions, the UA strain CGMCC No. 33976 has an essential biological difference from them. (3) Compared with the prior art Alcaligenes faecalis CGMCC No. 16874, the highest urease-producing activity is approximately 927 U / mL (the data is combined with the highest urease-producing activity of the strain disclosed in CN 109926448 A, which is 0.53 ± 0.02 ug / h / 10 4 cells and the cell density of 5*10 7 is determined by conversion. The cell density value is 5*10 7 from the empirical data OD summarized from similar cultures of Alcaligenes faecalis during the research process of this technical solution 600 ≈0.8), the urease-producing activity of the UA strain of the present invention is higher than 1.414*10 4 U / mL, and the highest is 1.976*10 4 U / mL, having a significantly higher activity level. (4) Experiments on acid mine surface water samples collected in the field show that the UA strain can be practically applied as an acid-tolerant urease-producing bacterium in the remediation of acid mine surface water and has industrial uses for the bioremediation and biological treatment of acid mine environments.

Claims

1. Alcaligenes faecalis ( Alcaligenes faecalis ) strain UA, deposited in the China General Microbiological Culture Collection Center on March 25, 2025, with the deposit number CGMCC No. 33976.

2. Use of the Alcaligenes faecalis UA strain CGMCC No. 33976 according to claim 1, characterized in that: The application as an acid-resistant urease-producing bacterium 3. Use of the Alcaligenes faecalis UA strain CGMCC No. 33976 according to claim 2, characterized in that: The application in environmental bioremediation engineering 4. Use of the Alcaligenes faecalis UA strain CGMCC No. 33976 according to claim 2, characterized in that: The application in acid mine environment bioremediation 5. Use of the Alcaligenes faecalis UA strain CGMCC No. 33976 according to claim 2, characterized in that: It is used for metal precipitation and fixation and / or reduction of water environment acidity 6. Use of the Alcaligenes faecalis UA strain CGMCC No. 33976 according to claim 5, characterized in that: The metal includes Al 3+ and / or Mn 2+ and / or Zn 2+ and / or Cd 2+ and / or Ca 2+ and / or Fe 3+ and / or Pb 2+ .

7. Bacterial agent, characterized in that: It includes the Alcaligenes faecalis UA strain CGMCC No. 33976 described in claim 1 8. Use of the microbial agent according to claim 7, characterized in that: The application as an acid-resistant urease-producing bacterium agent 9. Method for repairing acid mine environment by acid-tolerant urease-producing bacteria, characterized in that: Inoculate Acidovorax faecalis strain UA (CGMCC No. 33976) into acid mine wastewater, and maintain the biomass OD of strain UA in the culture system at 0.9 - 1.0 during the fermentation process. 600 = 0.9 - 1.

0.

10. The method for repairing acid mine environment by acid-tolerant urease-producing bacteria according to claim 9, wherein: Inoculating the Alcaligenes faecalis UA strain CGMCC No. 33976 into acid mine wastewater means adding the Alcaligenes faecalis UA strain CGMCC No. 33976 or the bacterium agent described in claim 7 into the acid mine wastewater; or placing a carrier fixed with the Alcaligenes faecalis UA strain CGMCC No. 33976 or the bacterium agent described in claim 7 in the acid mine wastewater

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