Mine water pseudomonas and screening method and application thereof in recharge capacity increase

Abstract

The invention discloses mine water pseudomonas as well as a screening method and application thereof in recharge capacity increase, and belongs to the technical field of mine water recharge engineering. A mine water sample is taken back and then filtered through a filter membrane and a vacuum suction filter; after constant-temperature oscillation, inoculating a liquid LB meat extract peptone culture medium for enrichment culture; carrying out strain separation on the bacterium liquid subjected to enrichment culture by adopting a dilution coating plate method; the method comprises the following steps: carrying out streak culture on a single colony on a solid LB culture medium to obtain a pseudomonas colony, the pseudomonas is Pseudomonas sp. B2, the taxonomic name is Pseudomonas sp. B2, the preservation number is GDMCC No.66726, the preservation date is July 22, 2025, and the preservation unit is Guangdong Province Microbial Culture Collection Center, and the screened pseudomonas capable of producing carbonic anhydrase can effectively promote the dissolution of calcite and dolomite, and can be used for producing carbonic anhydrase. The recharge amount is increased, and the recharge efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of mine water reinjection engineering technology, and in particular to a mine water Pseudomonas bacillus and its screening method, and its application in reinjection and capacity expansion. Background Technology

[0002] Coal mining generates large quantities of high-salinity mine water, which typically contains high concentrations of inorganic salts such as sulfate and sodium ions, as well as pollutants like suspended solids and heavy metals. Furthermore, the retention of mine water underground leads to water accumulation in tunnels, equipment corrosion, and an increased risk of water inrush accidents, severely impacting coal mine safety and operational efficiency. Currently, the large volume of mine water presents a challenge due to its low utilization rate. Direct discharge or improper treatment not only wastes water resources but also causes serious pollution to surrounding groundwater and the ecological environment. Therefore, effective treatment of mine water is a crucial measure for achieving resource recycling and ensuring safe coal mine production.

[0003] Currently, there are two main methods for treating mine water: recycling and reinjection. Mine water reinjection refers to the use of engineering facilities to guide or inject mine water into underground aquifers. Existing reinjection technologies largely rely on hydraulic fracturing, but this technology cannot solve the blockage problem caused by calcium and magnesium ion precipitation in mine water, leading to a decrease in reinjection efficiency. In existing research literature and findings, no scholars have yet applied microorganisms to the field of reinjection. This presents a novel approach to mine water treatment: microbial-enhanced reinjection.

[0004] Carbonic anhydrase-producing Pseudomonas bacteria can catalyze the reaction of carbon dioxide with water to produce bicarbonate and hydrogen ions, thereby accelerating the dissolution of carbonate minerals. This aligns perfectly with the characteristic that the Ordovician limestone karst fissure aquifers are primarily composed of carbonate rocks. Furthermore, there are currently no studies applying carbonic anhydrase-producing Pseudomonas bacteria to mine water reinjection. Therefore, carbonic anhydrase-producing Pseudomonas bacteria have enormous application prospects and potential in promoting the volume and permeability enhancement of mine water reinjection. However, how to efficiently screen highly active carbonic anhydrase-producing Pseudomonas bacteria from mine water and apply them to mine water reinjection for volume and permeability enhancement remains a pressing technical challenge.

[0005] Therefore, developing a simple, low-cost, and efficient method for screening Pseudomonas bacteria and applying it to mine water reinjection to improve reinjection efficiency and water quality is of great practical significance and application value. Summary of the Invention

[0006] Technical problems to be solved:

[0007] To address the technical problems of existing technologies, such as large total volume but low utilization rate, blockage caused by calcium and magnesium ion precipitation in mine water, and decreased reinjection efficiency, this invention provides a method for screening Pseudomonas aeruginosa in mine water and its application in reinjection volume enhancement. The first objective of this invention is to provide a simple and low-cost method for artificially screening carbonic anhydrase-producing Pseudomonas aeruginosa from mine water. The second objective is to provide an application of carbonic anhydrase-producing Pseudomonas aeruginosa to promote mine water reinjection volume enhancement and permeability improvement. Carbonic anhydrase-producing Pseudomonas aeruginosa can accelerate mineral dissolution, increase the fracture network, improve permeability, increase the mine water reinjection volume, and improve reinjection efficiency. Furthermore, this method can improve mine water quality and reduce pollution to the groundwater reinjection area. Before reinjection, the mine water... Inoculating water with carbonic anhydrase-producing Pseudomonas aeruginosa can effectively reduce sulfate and sodium ions in the water through adsorption, thereby reducing groundwater pollution in the recharge area. The carbonic anhydrase-producing Pseudomonas aeruginosa accelerates the dissolution of calcite and dolomite minerals in the Ordovician limestone layer, expanding primary fissures and increasing permeability. The Ordovician limestone contains a certain amount of organic matter, which provides usable nitrogen and carbon sources for microorganisms. These three factors work synergistically to effectively promote the recharge of mine water into the Ordovician limestone aquifer. This invention extracts carbonic anhydrase-producing Pseudomonas aeruginosa from primary mine water, and then inoculates the primary mine water with the extracted bacteria. This allows Pseudomonas aeruginosa containing the carbonic anhydrase-producing Pseudomonas aeruginosa gene to gradually become the dominant community, increasing the reversible hydrolysis reaction of water and CO2 catalyzed by microorganisms and enhancing the dissolution of minerals.

[0008] Technical solution:

[0009] To achieve the above objectives, this application provides the following technical solution:

[0010] A method for screening Pseudomonas aeruginosa in mine water, comprising the following steps:

[0011] Step 1: After the mine water sample is collected, it is filtered through a filter membrane and a vacuum filter.

[0012] Step 2: After constant temperature shaking, inoculate with liquid LB meat extract peptone medium for enrichment culture;

[0013] Step 3: After enrichment culture, the bacterial solution is diluted and spread on agar plates for bacterial strain isolation.

[0014] Step 4: After streaking the single colony onto solid LB medium, Pseudomonas colonies are obtained.

[0015] Furthermore, in the first step, the mine water is the surface mine water of Lilou Coal Mine that has undergone pretreatment by filtration through a membrane filter and a vacuum filter.

[0016] Furthermore, the mine water in the first step is SO4-Na type water.

[0017] Furthermore, in the second step, the mine water, after being subjected to isothermal oscillation, was inoculated into three bottles of liquid LB medium at a ratio of 1:350 and cultured for 3 days.

[0018] Furthermore, the liquid LB meat extract peptone medium in the second step consists of: yeast extract, 5g; peptone, 10g; NaCl, 5g; pH value, 6.8–7.0.

[0019] Furthermore, the second step of liquid LB meat extract peptone medium also contains 0.0029 g / L ZnSO4·7H2O. Before use, the medium is sterilized in an autoclave at 120°C for 20 min.

[0020] Furthermore, the OD of the enriched cultured samples in the second step 600 The highest-performing group continued to be inoculated with liquid LB medium at a ratio of 1:350.

[0021] Furthermore, the bacterial culture enriched in the second step is inoculated with fresh liquid LB medium every 24 hours, and this process is repeated three times to obtain a high-purity Pseudomonas bacterial culture.

[0022] Furthermore, in the third step, the enriched bacterial culture is prepared according to a 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 The bacterial culture was serially diluted, and the diluted bacterial culture was spread on solid LB medium using the dilution plating method and incubated in a 25°C incubator for 3 days.

[0023] Furthermore, in the fourth step, single colonies in the solid LB medium are picked up with a sterile inoculation loop and inoculated into liquid LB medium for 3 days. The bacterial culture in the liquid LB medium is then streaked onto the solid LB medium with a sterile inoculation loop. After repeating the streaking 5 to 6 times, a highly pure carbonic anhydrase-producing Pseudomonas aeruginosa is obtained.

[0024] Furthermore, the solid LB medium has the same composition as the liquid LB medium, with the addition of 1.5% agar.

[0025] The Pseudomonas bacillus obtained from mine water by any of the above screening methods is Pseudomonas sp. B2, with taxonomic name Pseudomonas sp., accession number GDMCC No. 66726, accession date July 22, 2025, and depositary institution Guangdong Provincial Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0026] This application also discloses the application of *Pseudomonas aeruginosa* in mine water in promoting the volume increase and infiltration enhancement of mine water reinjection, the steps of which are as follows:

[0027] S1: Mine water is pumped from the underground water tank to the surface. The mine water is then passed through a coupled coagulation and sedimentation reaction tank to remove impurities and microorganisms.

[0028] S2: Continuously add bacterial solution at the connection between the recycled water tank and the reinjection pipeline to reduce Na in the mine water. + and SO4 2- The content of the bacteria was determined by adding 4 mg of bacterial solution. 3 / h, improves mine water quality;

[0029] S3: Injecting Ordovician limestone karst fissure aquifers through reinjection wells;

[0030] S4: Regularly collect reinjection water samples for monitoring and observe the control effect of scale monitoring devices installed in reinjection wells and pipelines.

[0031] Explanation of the principle: Using the hanging plate method, hanging plates are placed inside the reinjection well and in the reinjection pipeline. These plates are periodically removed, and the amount of scale is quantified by weighing or observing the thickness of the scale. Comparing the scale amount before and after adding microorganisms, as well as at different addition levels, a significant reduction in scale amount indicates a significant effect of microbial regulation.

[0032] Beneficial effects:

[0033] This application provides a method for screening Pseudomonas aeruginosa in mine water and its application in reinjection and volume expansion, which has the following beneficial effects:

[0034] 1. In this invention, Pseudomonas sp. B2 is the dominant species in mine water with a relatively high abundance. This is the first time that carbonic anhydrase-producing Pseudomonas has been used in mine water reinjection technology.

[0035] 2. The permeability of the Ordovician limestone karst fissure aquifer of this invention decreases from 1.2 × 10⁻⁶ within 0-5 days. -3 μm 2 Increased to 3.5×10 -3 μm 2 ;

[0036] 3. After the Ordovician limestone karst fissure aquifer of this invention has been stably expanded, the permeability remains at 2.8 × 10⁻⁶. -3 μm 2 ;

[0037] 4. Carbonic anhydrase-producing Pseudomonas aeruginosa accelerates the dissolution of dolomite and calcite, preventing precipitation, scaling and blockage during reinjection, increasing the original fracture network of the Ordovician limestone aquifer, increasing the reinjection volume and improving reinjection efficiency.

[0038] 5. The carbonic anhydrase-producing Pseudomonas bacilli screened in this invention can effectively remove sulfate and sodium ions from mine water through adsorption before reinjection, thereby improving the quality of reinjection water and reducing pollution to the reinjection area.

[0039] 6. This invention screens carbonic anhydrase-producing Pseudomonas aeruginosa from the water in the Lilou mine. The raw materials are widely available and inexpensive.

[0040] 7. The microbial screening process is simple, requires little investment, does not require special equipment, and is easy to control. Attached Figure Description

[0041] Figure 1 The images show three bottles of liquid LB medium containing mine water at a ratio of 1:350, enriched for 3 days. The left image is the first enrichment image on day 0, and the right image is the first enrichment image on day 3.

[0042] Figure 2 Photographs showing the colonies isolated after streak plating in this application;

[0043] Figure 3 For the Ca in groups A, B, C, and D of this application, over 60 days 2 Concentration line graph;

[0044] Figure 4 For Groups E and F of this application Ca 2+ Concentration change graph over 30 days;

[0045] Figure 5 This is a process diagram of on-site reinjection for this application.

[0046] Explanation of reference numerals in the attached diagram: 5-1, Downhole water tank; 5-2, Water quality monitoring system A; 5-3, Water pump A; 5-4, Coagulation sedimentation reagent tank; 5-5, Magnetic separation + high-density pool coupling device; 5-6, Flocculation sedimentation tank; 5-7, Discharge device; 5-8, Water quality monitoring system B; 5-9, Reclaimed water tank; 5-10, Primary culture tank; 5-11, Secondary culture tank; 5-12, Bacterial solution storage tank; 5-13, Water pump B; 5-14, Electromagnetic metering pump; 5-15, Recharge pipeline; 5-16, Water quality monitoring device; 5-17, Booster pump; 5-18, Recharge well; 5-19, Coal seam; 5-20, Ordovician limestone aquifer; 5-21, Ordovician limestone strata rock salt dissolution zone; 5-22, Recharge water mound. Detailed Implementation

[0047] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0048] The Ordovician limestone karst fissure aquifers in this application are mainly carbonate rocks filled with calcite and dolomite.

[0049] The components of liquid LB meat extract peptone medium are: yeast extract, 5g; peptone, 10g; NaCl, 5g; ZnSO4·7H2O 0.0029g / L, pH value: 6.8~7.0. Before use, the medium should be sterilized in an autoclave at 121℃ for 20min.

[0050] The solid LB medium has the same composition as the liquid LB medium, with the addition of 1.5% agar.

[0051] Example 1:

[0052] A screening method for carbonic anhydrase-producing Pseudomonas aeruginosa, comprising the following steps:

[0053] Step 1: The SO4-Na type mine water on the surface of Lilou Coal Mine is pretreated by filtration through a membrane filter and a vacuum filter.

[0054] Step 2: As Figure 1 As shown, after isothermal shaking, mine water was inoculated into liquid LB meat extract peptone medium at a ratio of 1:350 for enrichment culture for 3 days. The OD of the enriched samples was... 600 The highest-quality group was inoculated with liquid LB medium at a ratio of 1:350. The enriched bacterial culture was inoculated with fresh liquid LB medium every 24 hours, and the process was repeated three times to obtain a high-purity Pseudomonas bacterial culture.

[0055] Step 3: Accumulate the enriched bacterial culture at a ratio of 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 The bacterial culture was serially diluted, and the diluted bacterial culture was spread on solid LB medium using the dilution plating method and incubated in a 25°C incubator for 3 days.

[0056] Step 4: As Figure 2 As shown, single colonies from the two plates with the highest dilution were picked up with a sterile inoculation loop and inoculated into liquid LB medium and cultured for 3 days. Then, they were streaked on solid LB medium with a sterile inoculation loop. After repeating the streaking 5 to 6 times, Pseudomonas aeruginosa with high purity was obtained.

[0057] Example 2:

[0058] The application of a type of Pseudomonas aeruginosa in promoting the volume increase and infiltration enhancement of mine water reinjection, the steps are as follows:

[0059] S1: Mine water is pumped from the underground water tank to the surface. The mine water is then passed through a coupled coagulation and sedimentation reaction tank to remove impurities and microorganisms.

[0060] S2: Continuously add bacterial solution at the connection between the recycled water tank and the reinjection pipeline to reduce Na in the mine water. + and SO4 2- The content of the bacteria was determined by adding 4 mg of bacterial solution. 3 / h, improves mine water quality;

[0061] S3: Injecting Ordovician limestone karst fissure aquifers through reinjection wells;

[0062] S4: Regularly collect reinjection water samples for monitoring and observe the control effect of scale monitoring devices installed in reinjection wells and pipelines.

[0063] Example 3:

[0064] The application of a carbonic anhydrase-producing Pseudomonas aeruginosa to promote the volume increase and permeability enhancement of mine water reinjection, the specific steps are as follows:

[0065] Rock samples from the Lilou recharge target stratum were crushed and ground, and then sieved through a 60-mesh sieve. 180g of the ground Ordovician limestone powder was added to 900ml of pretreated mine water from the Lilou surface, along with four groups of bacterial solutions selected in Example 1, with two parallel samples prepared for each group. The solutions were sealed with breathable sealing film and placed in a constant temperature incubator. The temperature was set at 25℃ for the first 30 days, and then adjusted to 50℃ for the next 30 days without further disturbance. Nine sampling points were set up, and samples were taken at 0, 5, 10, 20, 30, 35, 40, 50, and 60 days. The concentration of cations was determined using an inductively coupled plasma atomic emission spectrometer (ICP-OES 720), and the concentration of anions was determined using a Metrohm ion chromatograph and potentiometric titrator.

[0066] Table 1 shows the experimental conditions.

[0067] Table 1

[0068]

[0069]

[0070] In the first 5 days of the experiment, group D dissolved 169% more Ca than group A. 2+ and 22% Mg 2+ In the later stages of the experiment (5–60 days), Ca 2 + Concentration decreased (up to 65%). Ca 2+ The changes in concentration over 60 days are shown in the attached figure. Figure 3 As shown. SO4 2- During days 0–20, group D showed a decrease of approximately 800 mg / L, while group A showed a decrease of approximately 400 mg / L. Group D's Na... + The Na level in group A decreased by 300 mg / L within 0–20 days.+ It decreased by approximately 250 mg / L.

[0071] Example 4:

[0072] Experimental groups E and F were set up, with two replicates in each group. In group E, 20 ml of culture medium was added to the experimental system every 10 days to provide additional carbon and nitrogen sources for the microorganisms. In group F, 20 ml of bacterial culture was added every 10 days to directly increase the number of microorganisms. The supplementary experiment lasted for 30 days, with the samples being artificially disturbed once a day. The temperature was always set at 25℃, and five sampling points were set up, taking samples at days 0, 5, 10, 20, and 30.

[0073] The experimental conditions are shown in Table 2 below.

[0074] Table 2

[0075]

[0076] Dissolve Ca in the first 5 days 2+ Group E's concentration increased from 11.87 mg / L to 62.85 mg / L, and Group F's concentration also increased from 11.87 mg / L to 62.85 mg / L. (The remaining text appears to be incomplete and contains tyrosine phosphate.) 2+ Group E: 1.76 mg / L → 19.74 mg / L; Group F: 1.76 mg / L → 19.74 mg / L. Ca 2+ Mg 2+ The concentration change graph over 30 days is attached. Figure 4 As shown.

[0077] Example 5:

[0078] like Figure 5 As shown, when mine water is drawn from the underground water tank 5-1, it passes through the water quality monitoring system A5-2 and is then transported by the water pump A5-3 to the magnetic separation + high-density pool coupling device 5-5. Coagulant, magnetic powder and flocculant are pre-added to the coagulation and sedimentation reaction agent tank 5-4 to remove sludge and impurities. The sludge and impurities are flocculated and precipitated in the flocculation sedimentation tank 5-6 and then discharged by the discharge device 5-7.

[0079] Pretreated mine water is collected in recycled water tank 5-9 and monitored in real time by water quality monitoring system B (5-8). The bacterial solution is enriched and cultured in primary culture tank 5-10, then transferred to secondary culture tank 5-11 for a new round of enrichment and culture. After cultivation, the bacterial solution is transferred to bacterial solution storage tank 5-12 for later use. Mine water in recycled water tank 5-9 is pumped into reinjection pipeline 5-15 by pump B (5-13). At the pipeline inlet, electromagnetic metering pump 5-14 draws water at a reinjection flow rate of 200 m³ / h, matching the bacterial solution concentration. 3 2% of / h (i.e., 4m) 3 The bacterial solution is injected into the pipeline at a rate of / h, and the pretreated mine water is fully mixed in the pipeline to form a mixture of mine water and carbon anhydrase microorganisms.

[0080] A water quality monitoring device 5-16 is placed at the outlet of the reinjection pipeline to monitor the water quality of the mixed liquor. The mixed liquor is then pumped by a booster pump 5-17 (flow rate 200 m³ / h). 3 (With a pumping speed of 32m and a pressure of 1-2MPa), the solution is pressurized and transported to reinjection well 5-18. The reinjection well then penetrates the coal seam 5-19 and injects the solution at a uniform rate into the Ordovician limestone aquifer 5-20, forming a reinjection water mound at 5-22. The continuously added bacterial solution remains active in the formation, and the carbonic anhydrase secreted by the microorganisms continuously catalyzes the dissolution of calcite, continuously expanding formation fractures in the rock salt dissolution zone of the Ordovician limestone formation at 5-21. Within 0-5 days, the permeability increases from 1.2 × 10⁻⁶. -3 μm 2 Increased to 3.5×10 -3 μm 2 The bacterial solution was continuously added throughout the entire recharge cycle, and the recharge efficiency remained stable at 200m³. 3 / h, the blockage rate decreased from 15% / month to 3%, the expanded pore structure stabilized, and the permeability remained at 2.8×10 -3 μm 2 There was no obvious blockage.

[0081] The embodiments proposed in this invention are preferred embodiments, but are not limited to the content described above. Those skilled in the art can easily replicate the above embodiments and further extend and modify them, but as long as they do not depart from the spirit of this invention, they are all within the protection scope of this invention.

Claims

1. A method for screening Pseudomonas aeruginosa in mine water, characterized in that, The specific steps are as follows: Step 1: After the mine water sample is collected, it is filtered through a filter membrane and a vacuum filter. Step 2: After constant temperature shaking, inoculate with liquid LB meat extract peptone medium for enrichment culture; Step 3: After enrichment culture, the bacterial solution is diluted and spread on agar plates for bacterial strain isolation. Step 4: After streaking the single colony onto solid LB medium, Pseudomonas colonies are obtained.

2. The method for screening Pseudomonas aeruginosa in mine water according to claim 1, characterized in that: In the first step, the mine water is the surface mine water of Lilou Coal Mine, which has been pretreated by filtration through a membrane filter and a vacuum filter. The mine water is of the SO4-Na type.

3. The method for screening Pseudomonas aeruginosa in mine water according to claim 1, characterized in that: In the second step, after constant temperature shaking, the mine water was inoculated into three bottles of liquid LB medium at a ratio of 1:350 and cultured for 3 days.

4. The method for screening Pseudomonas aeruginosa in mine water according to claim 1, characterized in that: The components of the liquid LB meat extract peptone medium in the second step are: yeast extract, 5g; peptone, 10g; NaCl, 5g; pH value, 6.8-7.0; the liquid LB meat extract peptone medium in the second step also contains ZnSO4·7H2O 0.0029g / L. Before use, the medium is placed in an autoclave for sterilization at 120℃ for 20min.

5. The method for screening Pseudomonas aeruginosa in mine water according to claim 1, characterized in that: The OD of the enriched cultured samples in the second step 600 The highest-quality group was inoculated with liquid LB medium at a ratio of 1:

350. The bacterial culture enriched in the second step was inoculated with fresh liquid LB medium every 24 hours, and this process was repeated three times to obtain a high-purity Pseudomonas bacterial culture.

6. The method for screening Pseudomonas aeruginosa in mine water according to claim 1, characterized in that: In the third step, the bacterial culture solution after enrichment is prepared at a ratio of 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 The bacterial culture was serially diluted, and the diluted bacterial culture was spread on solid LB medium using the dilution plating method and incubated in a 25°C incubator for 3 days.

7. The method for screening Pseudomonas aeruginosa in mine water according to claim 1, characterized in that: In the fourth step, single colonies in the solid LB medium are picked up with a sterile inoculation loop and inoculated into liquid LB medium for 3 days. The bacterial culture in the liquid LB medium is then streaked onto the solid LB medium with a sterile inoculation loop. After repeating the streaking 5 to 6 times, a highly pure carbonic anhydrase-producing Pseudomonas aeruginosa is obtained.

8. The method for screening Pseudomonas aeruginosa in mine water according to claim 1, characterized in that: The solid LB medium has the same composition as the liquid LB medium, with the addition of 1.5% agar.

9. A *Pseudomonas aeruginosa* strain obtained by the screening method according to any one of claims 1-8, characterized in that: The Pseudomonas species described is Pseudomonas sp. B2, with taxonomic name Pseudomonas sp., accession number GDMCC No. 66726, accession date July 22, 2025, and depositary institution Guangdong Provincial Microbial Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

10. The application of *Pseudomonas aeruginosa* as described in claim 9 in promoting the volume increase and permeability enhancement of mine water reinjection, characterized in that... The steps are as follows: S1: Mine water is pumped from the underground water tank to the surface. The mine water is then passed through a coupled coagulation and sedimentation reaction tank to remove impurities and microorganisms. S2: Continuously add bacterial solution at the connection between the recycled water tank and the reinjection pipeline to reduce Na in the mine water. + and SO4 2- The content of the bacteria was determined by adding 4 mg of bacterial solution. 3 / h, improves mine water quality; S3: Injecting Ordovician limestone karst fissure aquifers through reinjection wells; S4: Regularly collect reinjection water samples for monitoring and observe the control effect of scale monitoring devices installed in reinjection wells and pipelines.

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