Alcaligenes faecalis UA strain, its products and applications, and methods for repairing acidic mine environments using acid-resistant urease-producing bacteria

Abstract

The present invention discloses the UA strain of Alcaligenes faecalis, its products and applications, and a method for remediating acidic mine environments using acid-resistant urease-producing bacteria. These strains belong to the fields of industrial microorganisms, microbial application materials, and environmental remediation. The UA strain, CGMCC No. 33976, is the first acid-resistant urease-producing bacteria discovered. Its optimal acidity lies between pH 4.5 and pH 6.5, with stable urease production at an initial pH of 3.5 to pH 7.5 and highest enzyme activity at an initial pH of 5.5 to pH 6.5. In real samples of acidic mine wastewater, the UA strain effectively reduces acidity and removes metal ions, achieving removal rates exceeding 99% for aluminum, manganese, zinc, and cadmium, and 92% to 98% for calcium, iron, and lead. The UA strain, screened from acidic mine environments, exhibits biosafety and is suitable for in-situ direct treatment of AMD and in-situ remediation of acidic mine environments. It eliminates the need for chemical pH adjustment, ensuring ecological safety and environmental sustainability.

Description

Technical Field

[0001] The present invention relates to Alcaligenes faecalis, in particular to an acid-resistant urease-producing strain of Alcaligenes faecalis UA, and a method for remediating an acidic mine environment using the strain. The invention belongs to the technical fields of industrial microorganisms, microbial application materials, and environmental remediation. Background Art

[0002] Acid mine drainage (AMD), characterized by high acidity (pH 2-3), high sulfate and heavy metal concentrations, large water volumes, and a long history of formation, is a global environmental problem left over from mineral mining. Due to its complex formation mechanism, influenced by diverse factors such as ore deposit type, mining methods, climate conditions, and management techniques, AMD's effectiveness at source control is limited, requiring extensive integration with post-processing treatment. AMD is a key link in the chain of acid mine environmental problems, and its management is a fundamental technology for mine environmental remediation.

[0003] Microbial induced mineralization is a technology that uses urease produced by microbial physiological and biochemical processes to convert free heavy metals in the environment into precipitates. It is an environmental bioremediation technology with industry-wide technical advantages. It is currently the ideal technical path for the remediation of extreme or harsh environments such as acidic mine environments. The basis for applying microbial induced mineralization technology to the development of technical solutions for the remediation of acidic mine environments is the screening of urease-producing strains. The target strain should have at least four basic biological characteristics: First, it can survive normally in acidic mine wastewater, and the optimal growth pH conditions are within the acidic range of acidic mine wastewater; second, it has stable and high urease production activity in acidic mine wastewater; third, it can remove harmful heavy metals in acidic mine wastewater; and fourth, the above three functions can be stably performed in real water samples of acidic mine wastewater, rather than simulated solutions.

[0004] The existing technology "Using microbial mineralization to reduce the risk of heavy metal pollution migration from abandoned slag piles in southwest China" (Qiao Suyu, Sichuan University, 2021) discloses two urease-producing strains kp-4 and kp-22, both of the genus Sarcinia ( Sporosarcina sp.) bacteria, which are alkaliphilic, and the optimal acidic range for urease production activity is between pH 8 and 9. The two strains treat acidic mine wastewater by first embedding the bacteria on different materials with sodium alginate for fixation, and then placing them in the wastewater environment. The urease of the solid material reacts best at pH 8.0-9.0. The prior art "Experimental Study on the Cooperative Treatment of Acidic Mine Wastewater by Urease-Producing Bacteria and Sulfate-Reducing Bacteria" (Fan Xiao, Chengdu University of Technology, 2023) discloses a urease-producing bacteria U-3 strain, which belongs to Paenibacillus millefolius ( Paenibacillus chibensisThe strain can grow in an initial pH of 2 to 7, but the optimum pH is 7. In the laboratory simulated acid mine wastewater (initial pH 2 to 4) environment, the U-3 strain can grow, but the pH value of the solution only increased to 5.3 after 14 days of fermentation, and the induced precipitation of metal ions is mainly concentrated in iron (total iron, Fe 2+ ), for Mn 2+ The removal rate is only 45.73%.

[0005] The urease-producing bacteria reported so far mainly include Bacillus ( Bacillus ), Sporosarcina ( Sporoscarcina pasteurii ), Pseudomonas spp. ( Pseudomonas ), Acinetobacter spp. ( Acinetobacter ) etc. Prior art 2019102019024 (CN 109926448 A) discloses a strain of Alcaligenes faecalis FC CGMCC No. 16874, which can produce urease and fix heavy metals in soil. However, the fermentation conditions are pH 6.5-8.5, and the urease activity is optimal at pH 8.0.

[0006] The urease-producing bacteria disclosed in the prior art do not possess the aforementioned four basic biological characteristics, and therefore have no industrial application prospects for direct treatment of acid mine wastewater and environmental remediation. Summary of the Invention

[0007] The purpose of the present invention is to provide a new alcaligenes faecalis ( Alcaligenes faecalis ) strain. This strain can grow stably in acidic mine water environments, maintain urease production activity, and mineralize a variety of metals, including toxic and hazardous metals, and has industrial applications as an acid-resistant urease-producing microorganism.

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

[0009] A bacillus alcaligenes faecalis ( Alcaligenes faecalis ) UA strain, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with a deposit date of March 25, 2025, and a deposit number of CGMCC No. 33976.

[0010] Alcaligenes faecalis strain UA CGMCC No. 33976 was isolated from contaminated soil samples in the Dexing copper mining area in Jiangxi Province. It was initially identified as a strain of Alcaligenes faecalis ( Alcaligenes faecalis ) strain, named Alcaligenes faecalis ( Alcaligenes faecalis )UA strain ( Alcaligenes faecalis UA).

[0011] Experimental data from the present invention demonstrates that Alcaligenes faecalis UA strain CGMCC No. 33976 has acid-resistant urease production for industrial applications. Accordingly, the present invention first provides a microbial agent product comprising the Alcaligenes faecalis UA strain, the technical solution of which 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 bacterial agent.

[0016] The above application as acid-resistant urease-producing bacteria or application as acid-resistant urease-producing bacteria agent may be specifically as follows.

[0017] The above applications are environmental bioremediation engineering applications.

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

[0019] The above applications are for metal precipitation fixation and / or reduction of acidity in 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 the above application schemes, the suitable culture conditions for UA strain CGMCC No. 33976 are pH 5.5 to pH 6.5 and temperature 35°C. The culture conditions are further optimized to pH 5.5.

[0021] The present invention also provides a technical solution for repairing acidic mine environments using acid-resistant urease-producing bacteria, as detailed below.

[0022] A method for remediating acidic mine environments with acid-resistant urease-producing bacteria is to inoculate Alcaligenes faecalis UA strain CGMCC No. 33976 into acidic mine wastewater and maintain the strain biomass OD during the fermentation process. 600 =0.9~1.0.

[0023] The most common sources of AMD include ore piles, tailings ponds, and open-pit mines. Different remediation strategies are required for AMD from different sources. Considering that in mining environments, wastewater generated during the beneficiation process can easily form depressions or gushing water from mines and tunnels due to natural precipitation or industrial water use, the UA bacterial strain can be used to treat AMD depressions or gushing water from mines and tunnels. The following scheme can be used to treat AMD depressions or gushing water from mines and tunnels: After settling and filtering the gushing water, the water is introduced into a microbial wastewater treatment tank. Based on the initial physical and chemical characteristics of the wastewater, the required UA bacterial strain biomass and urea are estimated; urea is added, mixed, and inoculated with the UA bacterial strain; and fermentation is carried out at a temperature suitable for the growth of Alcaligenes faecalis. After a period of sedimentation, the wastewater is tested to ensure that it meets discharge standards.

[0024] In the method for repairing the acidic mine environment with acid-resistant urease-producing bacteria, the UA strain is inoculated into the acidic mine wastewater to be fermented. The method can be to inoculate the UA strain (containing bacterial liquid) or bacterial agent (containing bacterial agent solution) into the acidic mine wastewater; or a carrier fixed with the UA strain or bacterial agent of Alcaligenes faecalis is placed in the acidic mine wastewater.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) the present invention can generate the Alcaligenes faecalis ( Alcaligenes faecalis UA strain CGMCC No. 33976 is a urease-producing bacterium. UA strain is adaptable to the entire acidic environment starting from an initial pH of 3.5, and grows optimally between pH 4.5 and pH 6.5. UA strain has good urease-producing ability in an initial pH of 3.5 to pH 7.5, with the highest activity at an initial pH of 5.5 to pH 6.5. UA strain is an acid-resistant urease-producing bacterium and is a new strain different from the existing urease-producing Bacillus faecalis. (2) The urease-producing activity of UA strain is higher than that of the existing urease-producing Bacillus faecalis. (3) In the real water sample environment of acidic mine wastewater, UA strain can ferment normally and show the performance of effectively reducing water acidity and removing water metals. It has industrial uses for in-situ treatment of acidic mine wastewater and in-situ remediation of acidic mine environments. The UA strain is the first to be publicly disclosed as a new urease-producing strain that is both acid-resistant and effectively precipitates metal ions and can be directly used for biological treatment of real water samples of acid mine wastewater. (4) The UA strain is effective in treating Al in real water samples of acid mine surface water. 3+ 、Mn 2+ 、Zn 2+ 、Cd 2+ The removal rates of four typical toxic and harmful metals are all above 99%, and the removal rate of Ca 2+ 、Fe 3+ , Pb 2+The removal rates were between 92% and 98%. (5) The UA strain also has a calcium precipitation effect. (6) The UA strain CGMCC No. 33976 is an acid-resistant urease-producing bacterium selected from an acidic mine environment and is biosafe. When used in the field remediation of acidic mine environments, it does not require the use of chemical agents to adjust the environmental pH, and is ecologically safe and environmentally sustainable. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph of the growth of UA strain on an acidic screening plate.

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

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

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

[0030] Figure 5 is the metal ion removal rate of acid mine drainage 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 The pH value changes of the culture system during the cultivation of acid mine wastewater UA strain CGMCC No. 33976.

[0032] Figure 7 The NH4 in the culture system during the culture of acid mine wastewater UA strain CGMCC No. 33976 + Concentration changes. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

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

[0035] Soil samples: In late June 2024, samples were collected from the contaminated soil area of ​​the Dexing copper mining area in Jiangxi Province. Surface soil and weeds were removed using a sampling shovel, and the surface soil was dug up to collect samples from the 5-20 cm layer. The obtained soil samples were stored in sterile, self-sealing bags, labeled with sample information, placed in a foam box filled with dry ice, and transported back to the laboratory in the dark.

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

[0037] Urea nutrient broth: 10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride. Adjust the pH with 1 mol / L H₂SO₄ solution. After adding an indicator, sterilize in an autoclave at 121°C, 101 kPa for 20 min. Remove the broth and add 20 g / L urea (sterilize the urea solution separately by filtration through a 0.22 μm filter). Set aside. A 2% bromocresol green-methyl red mixed indicator is used.

[0038] Acid screening plates: 18 g / L nutrient broth, 25 g / L agar. Adjust the initial pH of the medium to 4.0 using 1 mol / L H₂SO₄ solution. Sterilize the medium under high temperature and high pressure for 20 min. Add the indicator, 20 g / L urea (sterilize the urea solution separately by filtration through a 0.22 μm filter), and set aside. Use a 2% bromocresol green-methyl red mixed indicator.

[0039] Strain UA activation medium: 10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride. Use 1 mol / L H2SO4 solution to adjust the initial pH of the medium to 5.0. Place the medium in a high-pressure steam sterilizer at 121°C, 101 kPa for 20 min, remove it, and add 20 g / L urea (the urea solution is sterilized separately by filtration using a 0.22 μm filter membrane). Set aside.

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

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

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

[0043] Example 1

[0044] Enrichment, screening and identification of acid-resistant strains.

[0045] 1. Screening of acid-resistant strains

[0046] 1.1 Soil sample preparation

[0047] In a laminar flow hood, weigh 10 g of soil sample and add it to a 250 mL Erlenmeyer flask containing 100 mL of sterile water. Incubate the sample on a shaker at 180 rpm / min at ambient temperature for 24 hours to allow the microorganisms to be fully eluted from the soil and the bacteria and spores to disperse in the solution. After 30 minutes, collect the supernatant.

[0048] 1.2 Acidic enrichment

[0049] Transfer 4 mL of the supernatant to a 250 mL conical flask and add 100 mL of urea nutrient broth (pH 3.5). Incubate on a shaker at 180 rpm / min and 35°C. Measure the pH of the culture system every 24 h during the incubation process. When the pH of the culture system is 4.5 ± 0.5, add 1 mol / L H2SO4 solution to adjust the pH to 3.5 ± 0.2. Continue incubating until the OD value of the culture system reaches 0. 600 Between 0.6 and 0.8.

[0050] 1.3 Acidic plate screening

[0051] In a clean bench, 100 μL of solution was taken from the culture system and diluted to 10 -2 Then, inoculate 50 μL of the culture medium onto an acidic screening plate. Invert the acidic screening plate and incubate it in a 35°C incubator. The blue colonies that grow are the target colonies and are named UA bacteria.

[0052] 1.4 Strain isolation and purification

[0053] Preparation of UA strain purified solution: The above UA bacterial colonies were picked up with an inoculating loop and placed in urea nutrient broth with an initial pH of 3.5. The culture was carried out on a shaker at 180 rpm / min and a temperature of 35°C until the OD 600 If the concentration is between 0.8 and 1.0, it is the purified liquid of UA strain.

[0054] In the clean bench, take 100 μL of UA strain purified solution and dilute it to 10 -2After that, the second inoculation was carried out on the acidic screening plate with an inoculum volume of 50 μL. The acidic screening plate was inverted and cultured in a constant temperature incubator at 35°C.

[0055] Figure 1 This is a graph of the growth of UA strain on an acidic screening plate.

[0056] 2. Strain identification

[0057] The isolated and purified UA strain exhibits a smooth, milky white, opaque colony surface with irregular edges and a slight central protrusion. It is a Gram-negative bacterium and appears as a short rod under scanning electron microscopy.

[0058] DNA was extracted from the isolated and purified UA strain using the TSINGKE Plant DNA Extraction Kit (Universal). After appropriate dilution, PCR amplification was performed using universal bacterial 16s rDNA primers 27F and 1492R. PCR amplification materials included 45µl of 1× TSE101 Gold Mix, 2µl of 27F (10P), 2µl of 1492R (10P), and 1µl of DNA template. The amplification protocol was as follows: pre-strain at 98°C for 2 minutes, followed by a cycling phase of 98°C for 10 seconds, 56°C for 10 seconds, and 72°C for 10 seconds / kb for 35 cycles, an extension phase at 72°C for 5 minutes, and a storage phase at 4°C.

[0059] The PCR product was sent to the sequencing department of the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. for first-generation sequencing. The BLAST program from the NCBI website (http: / / www.ncbi.nlm.nih.gov / ) was used to compare and identify the sequences (shown as SEQ ID NO. 1). Finally, a phylogenetic tree was constructed.

[0060] The UA bacteria were isolated and purified and identified as Alcaligenes faecalis ( Alcaligenes faecalis ).

[0061] Figure 2 is the phylogenetic tree of Alcaligenes faecalis UA strains (based on 16S rDNA).

[0062] 2.3 Strain naming and preservation

[0063] The above-mentioned Alcaligenes faecalis ( Alcaligenes faecalis ) strain, named Alcaligenes faecalis UA strain ( Alcaligenes faecalis UA).

[0064] The strain UA of Alcaligenes faecalis was deposited in the General Microbiology Center of China Culture Collection (CGMCC), China General Microbiology Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101, China. The deposit date is March 25, 2025, and the deposit number is CGMCC No. 33976.

[0065] Example 2

[0066] Urease production capacity of Alcaligenes faecalis UA strain CGMCC No. 33976 under different initial pH conditions.

[0067] Preparation of Alcaligenes faecalis UA strain activated bacterial solution (referred to as UA strain activated solution): 2 mL of UA strain purified solution (same as in Example 1) was inoculated into 100 mL of strain UA activation medium and cultured overnight at 35°C and a shaker speed of 180 rpm / min to obtain the UA strain activated solution.

[0068] 100 ml of urea nutrient broth culture medium with different initial pH values ​​(pH 3.5, 4.5, 5.5, 6.5, and 7.5) was prepared and inoculated with 2 ml of UA strain activation solution. After incubation at 35°C and a shaker speed of 180 rpm / min for 24 h, the urease activity was determined (the urease activity was determined by the conductivity method, referring to Chinese patent application 2024102312410 Staphylococcus aureus UPB-1 strain, its products and applications, and tailings solidification and remediation method) (Table 1).

[0069] Table 1 Urease production capacity of UA strain CGMCC No. 33976 under different initial pH conditions

[0070]

[0071] The results showed that UA strain CGMCC No. 33976 had good urease production ability in the initial pH range of 3.5 to 7.5. The urease activity in the initial pH range of 5.5 to 6.5 was higher than that in the initial pH ranges on both sides. In particular, the highest urease activity was found in the pH 5.5 environment (19.76 mM urea hydrolyzed·min -1 , 1.976*10 4 U / mL).

[0072] Example 3

[0073] Optimal growth pH of Alcaligenes faecalis UA strain CGMCC No. 33976.

[0074] Prepare urea nutrient broth medium with initial pH of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7, inoculate 2% of UA strain activation solution (same as in Example 2), and culture for 24 h at a shaking speed of 180 rpm / min and a temperature of 35°C. Measure the OD of each bacterial solution. 600 The final pH was used to determine the optimal pH for the strain.

[0075] Figure 3 is the OD of UA strain CGMCC No. 33976 under different initial pH conditions 600 value and final pH.

[0076] Table 2 OD of UA strain CGMCC No. 33976 under different initial pH conditions 600 Value and final pH value

[0077]

[0078] The experiment showed that: from the initial pH 3.5 condition, the biomass (OD 600 The pH value of the culture medium (pH 7.0) began to increase steadily and remained at a high level until the initial pH 7.0. From the initial pH 3.5, the final pH of the culture medium also increased steadily. This shows that it has adaptability to acidic environments with an initial pH of 3.5 and above. It grows best between pH 4.5 and pH 6.5, and has the highest biomass (OD) at an initial pH of 5.5. 600 is 2.221).

[0079] Example 4

[0080] Tolerance of Alcaligenes faecalis UA strain CGMCC No. 33976 to heavy metal ions.

[0081] The activated UA strain (same as in Example 2) was inoculated into urea nutrient broth containing various heavy metal concentrations (the medium acidity was determined to be an initial pH of 6.0), at an inoculum size of 2%. The heavy metal content in each experimental group is shown in Table 3.

[0082] Table 3 Data related to heavy metal ion tolerance test

[0083]

[0084] Each group was cultured at a shaker speed of 180 rpm / min and a temperature of 35°C for 3 days. The OD of the bacterial solution of each group was measured after 3 days. 600 , to determine the strain's tolerance to heavy metals.

[0085] Figure 4This is the tolerance effect of UA strain CGMCC No. 33976 to heavy metals.

[0086] Experiments show that UA strain CGMCC No. 33976 has tolerance to heavy metals, especially Zn 2+ , Pb 2+ Well tolerated, in Zn 2+ , Pb 2+ When the concentration is 500 mg / L, it still has a high OD 600 When Cu 2+ 、Cd 2+ When the concentration was greater than 100 mg / L, the growth activity of the UA strain of Alcaligenes faecalis decreased significantly.

[0087] Example 5

[0088] Treatment of acid mine drainage by Alcaligenes faecalis UA strain CGMCC No. 33976.

[0089] A sample of acid mine drainage was taken for testing, showing a pH of 2.82. The metal content data is shown in Table 4.

[0090] Preparation of Alcaligenes faecalis UA strain concentrate (abbreviated as UA strain concentrate): inoculate the UA strain of Alcaligenes faecalis into the UA strain activation medium and culture it to the logarithmic growth phase (OD 600 =0.6-0.8), centrifuged at 8000 rpm / min, 4°C, and collected the lower layer of concentrate, which was the UA strain concentrate.

[0091] Experimental group: Acidic mine wastewater samples were filtered through qualitative filter paper at a medium speed, and concentrated bacterial solution of UA strain was added to the filtrate until the OD 600 =0.5, add analytical grade urea reagent, the addition amount is 20 g / L.

[0092] Control group 1: Take the acid mine wastewater sample and filter it at medium speed through qualitative filter paper, and add the same amount of urea reagent as that of the experimental group to the filtrate.

[0093] Control group 2: Acid mine wastewater samples were filtered through qualitative filter paper at a medium speed, and concentrated bacterial solution of UA strain was added to the filtrate until the OD 600 =0.5, without adding urea.

[0094] The experimental group and the control group were cultured in a 35℃ constant temperature shaker at a speed of 180 rpm / min. During the culture process, the pH and NH4 + Concentration (NH4 +The concentration determination method refers to the "Water Quality - Determination of Ammonia Nitrogen - Nessler's Reagent Spectrophotometric Method (HJ 535-2009)"); after 72 hours of incubation, the metal ion concentrations of each group were measured. Relevant data are shown in Tables 4 and 5. Figures 5 to 7 .

[0095] Table 4 Data related to acid mine environment remediation experiment (metal ions)

[0096]

[0097] Table 5 Data related to acid mine environment remediation experiment (pH, NH4 + concentration)

[0098]

[0099] Figure 5 is the metal ion removal rate of acid mine drainage in the experimental group, (a) Al 3+ , Ca 2+ 、Fe 3+ 、Mn 2+ , (b) Cu 2+ 、Zn 2 + 、Cd 2+ , Pb 2+ During the cultivation process, Cd 2+ 、Zn 2+ The changes were almost synchronous, probably due to the occurrence of coprecipitation; Figure 6 It is the change of pH value in the culture system during the cultivation of acid mine drainage UA strain CGMCC No. 33976; Figure 7 The NH4 in the culture system during the culture of acid mine wastewater UA strain CGMCC No. 33976 + Concentration changes.

[0100] Table 4, Table 5 and Figures 5 to 7 The data showed that the pH of the acid mine drainage samples in the experimental group increased significantly from 2.82 to 9.37 after 72 hours of remediation, and the metal ions were effectively removed. 3+ 、Mn 2+ 、Zn 2+ 、Cd 2+ The removal rates of four typical toxic and harmful metals are all above 99%, including Ca 2+ 、Fe 3+ , Pb 2+ The removal rates were all between 92% and 98%. This indicates that the UA strain has the ability to remove metals from acidic mine wastewater and to precipitate calcium. During the culture process, the pH value of the culture system was affected by the influence of NH4 +The concentrations increased significantly over the course of the incubation period. Analysis of the data from the experimental and control groups revealed that the reduction in acidity and metal removal in the AMD samples was due to the combined action of UA strain CGMCC No. 33976 and urea; either alone did not significantly remediate the AMD samples. This suggests that the UA strain's ability to remediate AMD samples is due to its urease production.

[0101] According to experimental observations, during the fermentation of real acid mine drainage water samples, the biomass of UA strains in the culture system was maintained at OD 600 =0.9~1.0 is appropriate.

[0102] The results of experiments from groups 2 to 5 showed that: (1) the present invention's Alcaligenes faecalis UA strain CGMCC No. 33976 has urease-producing activity and is a urease-producing bacterium. (2) The UA strain is best grown in an initial pH 4.5 to pH 6.5 environment, and has stable urease-producing activity in an acidic environment with an initial pH of 3.5 or above, with the highest activity at an initial pH of 5.5 to pH 6.5, making it a typical acid-resistant urease-producing bacterium. Compared with the existing urease-producing bacteria, such as the spore-forming bacteria KP-4 and KP-22 of the sarginicus strains and the Alcaligenes faecalis CGMCC No. 16874, which are suitable for alkaline environments and have the highest urease production activity under alkaline conditions, or the Bacillus millefolius strain U-3, which is acid-resistant but suitable for neutral environments and has poor metal ion precipitation efficiency, the UA strain CGMCC No. 33976 has essential biological differences from them. (3) Compared with the prior art, the highest urease activity of Alcaligenes faecalis CGMCC No.16874 is about 927 U / mL (the data is combined with the highest urease activity of 0.53±0.02 ug / h / 10 4 cell and cell density 5*10 7 The cell density is 5*10 7 The OD data is derived from the empirical data of similar culture of Alcaligenes faecalis carried out during the research of this technical solution. 600 ≈0.8), the urease activity of the UA strain of the present invention is higher than 1.414*10 4 U / mL, the highest is 1.976*10 4 U / mL, with significantly higher activity levels. (4) Field sampling experiments on acidic mine surface water showed that the UA strain can be used as an acid-resistant urease-producing bacterium in the remediation of acidic mine surface water, and has industrial applications in bioremediation and bioremediation of acidic mine environments.

Claims

1. Alcaligenes faecalis ( Alcaligenes faecalis ) UA strain, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with a deposit date of March 25, 2025, and a deposit number of CGMCC No. 33976.

2. The use of the Alcaligenes faecalis UA strain CGMCC No. 33976 according to claim 1, characterized in that: It is an application for producing urease in an acidic environment.

3. The use of Alcaligenes faecalis UA strain CGMCC No. 33976 according to claim 2, characterized in that: It is an application of environmental bioremediation engineering.

4. The use of Alcaligenes faecalis UA strain CGMCC No. 33976 according to claim 2, characterized in that: It is an application in bioremediation of acidic mining environments.

5. The use of Alcaligenes faecalis UA strain CGMCC No. 33976 according to claim 2, characterized in that: It is used for metal precipitation fixation and / or reduction of acidity in water environments.

6. The use of 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. A microbial agent, characterized in that: It comprises the Alcaligenes faecalis UA strain CGMCC No. 33976 described in claim 1.

8. The use of the microbial agent according to claim 7, characterized in that: It is an application for producing urease in an acidic environment.

9. A method for repairing an acidic mine environment using acid-resistant urease-producing bacteria, characterized by: Acidic mine wastewater was inoculated with Alcaligenes faecalis UA strain CGMCC No. 33976. During the fermentation process, the UA strain biomass OD in the culture system was maintained. 600 =0.9~1.

0.

10. The method for repairing an acidic mine environment using acid-resistant urease-producing bacteria according to claim 9, characterized in that: The inoculation of the acid mine wastewater with Alcaligenes faecalis UA strain CGMCC No. 33976 comprises adding the Alcaligenes faecalis UA strain CGMCC No. 33976 or the bacterial agent described in claim 7 to the acid mine wastewater; or placing a carrier immobilized with the Alcaligenes faecalis UA strain CGMCC No. 33976 or the bacterial agent described in claim 7 into the acid mine wastewater.

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